tobacco shoot regeneration from calli in temporary ... · tobacco shoot regeneration from calli in...
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Tobacco shoot regeneration from calli in temporary
immersion culture for biosynthesis of heterologous
biopharmaceuticals
Sherwin Savio Barretto
Thesis submitted for the Degree of Doctor of Philosophy PhD
Imperial College London
Department of Life Sciences
Faculty of Natural Sciences
Imperial College London
2014
1
Declaration of Originality
I hereby declare that this thesis, submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy of Imperial College London, represents my own work and has not been
previously submitted to this or any other institute for any degree, diploma or other
qualification.
Sherwin Savio Barretto
2
Copyright Declaration
The copyright of this thesis rests with the author and is made available under a Creative
Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy,
distribute or transmit the thesis on the condition that they attribute it, that they do not use it
for commercial purposes and that they do not alter, transform or build upon it. For any reuse
or redistribution, researchers must make clear to others the licence terms of this work.
3
Abstract
‘Molecular farming’, the use of transgenic plants to produce biopharmaceutical proteins is
emerging as a new biotechnological paradigm. Transgenic plants offer several advantages
over conventional microbial and mammalian cell host technologies. In particular,
transplastomic plants, with transformed plastid genomes, are capable of massive expression
of foreign proteins and represent a promising platform for biopharmaceutical synthesis.
The main theme of this PhD thesis is the investigation of in vitro regeneration of tobacco
(Nicotiana tabacum) shoots from callus tissue in temporary immersion (TI) culture for
heterologous biopharmaceutical synthesis. There is special emphasis on subunit vaccine
expression in transplastomic tobacco, in which foreign protein accumulation is correlated
with chloroplast number and development during the organogenesis process.
Studies using transplastomic N. tabacum expressing TetC (tetanus toxin fragment C)
investigated the influence of several culture parameters on biomass regeneration and
recombinant protein expression. The parameters investigated include medium nitrogen source
ratio, sucrose concentration and hydrodynamics. These studies highlight the sensitivity of
transplastomic protein yields to the culture microenvironment, and provide a starting point
for further optimisation. Further studies demonstrated the feasibility of TI culture for
biosynthesis of proteolytically-unstable transplastomic subunit vaccines, p24 (HIV antigen)
and VP6 (rotavirus antigen). TI culture is also demonstrated as a means for nuclear
expression of functional Guy’s 13 monoclonal antibody. Finally, the use of TI culture as the
basis of novel technological innovations is investigated. This includes the demonstration of
transplastomic protein expression in a prototype large-scale mechanical temporary immersion
bioreactor. Encapsulation of callus aggregates in an alginate matrix for long-term germplasm
preservation was trialled, prior to temporary immersion regeneration.
Overall, this work presents a novel in vitro propagation method for the contained large-scale
biosynthesis of biopharmaceutical proteins, as a potential alternative to conventional plant
propagation platforms based on agricultural cultivation or cell suspension culture.
4
Acknowledgements
I gratefully acknowledge all the individuals who have provided the assistance and support I
needed to complete this complex assignment.
First of all, I would like to express my sincere gratitude and deep appreciation to my
supervisor, Prof. Peter Nixon, for providing me with the opportunity to pursue this exciting
and challenging PhD project, and for all his guidance and support. I am equally grateful to
my co-supervisor Prof. Klaus Hellgardt whose guidance and invaluable technical ‘know-
how’ has helped me tremendously. I would like to give my deepest thanks to Dr Franck
Michoux, the post-doc who started this project and who in many ways has acted as my
‘unofficial supervisor’. I am extremely grateful for his assistance, support, time and patience.
I would like to thank all members of the 7th floor of the Ernst Chain Building, past and
present, for their cooperation, encouragement, support and friendship. I would especially like
to thank Hussain Haji Taha, Dr Jianfeng Yu, Shengxi Shao, Dr Niaz Ahmad, Dr Steven
Burgess, Dr Marko Boehm, Dr Charlotte Ward, Dr Agripina Banda, Sana Asghar, Xu Zhao,
Alexandros Papagiannakis, Jiyao Gan, Chi Zeng, Zheng-Yi Wei, Jayasudha Nagarajan,
Marin Sawa, Charlie Cotton, Jeffery Douglass, Sven Dc, Dr Tanai Cardona Londono, Dr
Karim Maghlaoui, Dr Alison Telfer, Katharina Brinkert, Dr Wojciech Bialek, Dr Andreas
Fantuzzi, Dr Gillian Young, Ruiqiong An, Dr Masooma Rasheed, Ewelina Krysztofinska,
Mostafa Jamshidiha, Dr Lisa Hale, Dr Justin Yeoman, Dr James MacDonald, Bhavish Patel,
Dr Christian Richard, Amanda Koslovaite and all other lab-fellows, past and present, whom I
have had the greatest pleasure crossing paths with. In particular, I will always remember Prof.
Bill Rutherford for his carefree attitude, advice and chats.
I would like to thank all my collaborators, for their help, guidance, materials and equipment. I
would like to thank Prof. John Gray, University of Cambridge for providing me with the
transplastomic seeds for the expression of p24 and VP6 and the accompanying antibodies. I
would like to thank Prof. Julian Ma and Pascal Drake for providing me with the transformant
seeds for the expression of Guy’s 13 monoclonal antibody. These collaborative efforts have
helped in the advancement of the scientific endeavour.
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I would like to thank the Biotechnology and Biological Sciences Research Council (BBSRC)
Targeted Priority Studentships initiative for funding this work.
I am deeply indebted to my family for their love, support, encouragement and prayers for my
success.
Finally, I would like to give thanks to Almighty God, for the innumerable graces and
blessings bestowed upon me to enable me to undertake this pursuit.
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Table of Contents
Table of Contents .................................................................................................................................... 6
List of Figures ........................................................................................................................................ 14
List of Tables ......................................................................................................................................... 17
List of Abbreviations ............................................................................................................................. 18
Chapter 1. Introduction .................................................................................................................. 23
1.1 The Biotechnology revolution and recombinant biopharmaceuticals ................................. 23
1.2 Transgenic plants .................................................................................................................. 25
1.2.1 The scope of transgenic plants as an alternative host technology ............................... 25
1.2.2 The benefits of transgenic plant host systems relative to conventional platforms ..... 27
1.2.3 Bioprocessing of plant-derived biopharmaceuticals .................................................... 29
1.2.3.1 Choice of localisation target in transgenic plant host systems ................................. 29
1.2.3.2 The choice of in vitro or soil-based cultivation for growth of transgenic plants ...... 30
1.2.3.3 The use of bioreactors in cell and tissue cultures ..................................................... 32
1.2.3.4 The temporary immersion culture format ................................................................ 33
1.2.3.5 Downstream processing ........................................................................................... 37
1.2.4 Chloroplast transformation ........................................................................................... 38
1.2.4.1 An overview of plant genetic transformation strategies .......................................... 38
1.2.4.2 The plastid genome as a target for genetic transformation ..................................... 39
1.2.4.2.1 Plastids organelles in higher plants .................................................................... 39
1.2.4.2.2 Features of the plastid genome .......................................................................... 40
1.2.4.2.3 The plastid genome as a novel target for genetic engineering .......................... 42
1.2.4.2.4 Methods for transformation of the chloroplast ................................................ 42
1.2.4.2.5 Benefits of plastid transformation ...................................................................... 44
1.2.4.2.5.1 Hyperexpression of transgenic proteins in plastids ..................................... 44
7
1.2.4.2.5.2 Post-transcriptional regulation of plastid protein synthesis and implications
for transformation vectors ................................................................................................ 45
1.2.4.2.5.3 Polycistronic transcription of plastid genes and opportunities for metabolic
pathway engineering ........................................................................................................ 46
1.2.4.2.5.4 Maternal inheritance of the chloroplast genome and implications for
biosafety 47
1.2.4.2.6 Applications of plastid genetic engineering ........................................................ 48
1.2.4.2.6.1 Plastid engineering for improvement of agronomic traits and Rubisco
activity 48
1.2.4.2.6.2 Molecular farming of protein biopharmaceuticals ...................................... 49
1.2.4.2.6.3 Expression of fusion proteins with affinity tags........................................... 50
1.3 Background to PhD Project ................................................................................................... 51
1.3.1 The PhD project in the context of previous studies ...................................................... 51
1.3.2 Aims of Study ................................................................................................................ 52
Chapter 2. Materials and Methods ................................................................................................. 54
2.1 Stock Solutions ...................................................................................................................... 54
2.1.1 Standard solutions and buffers ..................................................................................... 54
2.1.2 Antibodies ..................................................................................................................... 54
2.2 Cultivation of transgenic Nicotiana tabacum ....................................................................... 56
2.2.1 Nicotiana tabacum growth conditions ......................................................................... 56
2.2.2 In vitro micropropagation of transgenic N. tabacum ................................................... 57
2.2.2.1 In vitro germination of sterile seedlings .................................................................. 57
2.2.2.2 Callus induction and proliferation, and suspension cultures ................................... 57
2.2.2.3 Temporary immersion regeneration of shoots from callus ..................................... 58
2.2.2.4 Temporary immersion organogenesis from encapsulated calli (modified procedure)
58
2.2.2.5 Regeneration of shoots in a large-scale hydraulic bioreactor ................................. 59
2.2.2.5.1 Construction of large-scale bioreactor ............................................................... 59
2.2.2.5.2 Operation of large-scale bioreactor .................................................................... 59
8
2.2.2.6 Harvest of regenerated shoots, and fresh and dry weight determination .............. 60
2.3 Protein Analysis ..................................................................................................................... 61
2.3.1 Total Soluble Protein Extraction ................................................................................... 61
2.3.2 Determinination of protein concentration ................................................................... 61
2.3.3 SDS-PAGE ...................................................................................................................... 62
2.3.4 Staining of polyacrylamide gels .................................................................................... 62
2.3.5 Immunoblotting and Enhanced Chemiluminescence (ECL) Detection ......................... 62
2.3.6 Indirect ELISA to assess functional activity of plant-expressed monoclonal antibodies
63
2.4 Analysis of in vitro regenerated plant biomass ..................................................................... 64
2.4.1 Viability assay of in vitro regenerated shoots ............................................................... 64
2.4.2 Chlorophyll Fluorometry ............................................................................................... 65
Chapter 3. Parameters affecting the dynamics of biomass growth and transplastomic protein
accumulation in temporary immersion culture .................................................................................... 66
3.1 Introduction .......................................................................................................................... 66
3.1.1 In vitro differentiated plant tissues for molecular farming .......................................... 66
3.1.2 Nt-pJST12 as a rational model host system for investigating the impact of the culture
environment on recombinant protein expression ........................................................................ 67
3.2 In vitro shoot regeneration via organogenesis of N. tabacum callus and the influence on
transplastomic protein expression ................................................................................................... 68
3.2.1 Callus organogenesis as a developmental pathway for in vitro plantlet regeneration 68
3.2.2 In vitro morphogenesis dynamics during temporary immersion culture ..................... 69
3.2.2.1 Design of experiment .................................................................................................... 69
3.2.2.2 Results and Discussion .................................................................................................. 69
3.2.2.2.1 Dynamics of biomass growth and morphogenesis ............................................. 69
3.2.2.2.2 Differential expression of TetC during in vitro organogenesis ........................... 72
3.3 Impact of Hyperhydricity on Expression of TetC .................................................................. 75
3.3.1 The hyperhydricity phenomenon in in vitro micropropagation ................................... 75
3.3.2 TetC accumulation in vitrified and non-vitrified leaves ................................................ 76
9
3.3.3 Discussion on the influence of hyperhydricity on transplastomic protein expression . 78
3.4 The Impact of Tissue Culture Media on Morphogenesis and TetC yield .............................. 79
3.4.1 The impact of ammonium : nitrate ratio on TetC yield ................................................ 79
3.4.1.1 The nitrogen requirements of in vitro plant growth ............................................... 79
3.4.1.2 Design of experiment ................................................................................................ 80
3.4.1.3 Results ...................................................................................................................... 80
3.4.1.3.1 Influence of nitrogen source ratio on growth and TetC expression ................... 80
3.4.1.3.1.1 Influence of nitrogen source ratio on biomass accumulation and
morphogenesis .................................................................................................................. 80
3.4.1.3.1.2 Influence of nitrogen source ratio on yield of TetC ..................................... 83
3.4.1.3.2 pH shift of media during temporary immersion culture..................................... 85
3.4.1.4 Discussion on the influence of nitrogen pool on in vitro regeneration and
transplastomic protein expression ........................................................................................... 86
3.4.2 Effect of initial media pH on biomass growth and TetC expression ............................. 88
3.4.2.1 Design of experiment ............................................................................................... 88
3.4.2.2 Results and Discussion ............................................................................................. 89
3.4.2.2.1 The influence of initial medium pH on biomass growth and TetC expression ... 89
3.4.3 The Impact of Varying Sucrose and Irradiance for Photomixotrophic Propagation
Regimes 91
3.4.3.1 The importance of exogenous saccharides and irradiance in in vitro plant tissue
culture 91
3.4.3.2 Design of experiment ............................................................................................ 92
3.4.3.3 Results and Discussion .......................................................................................... 92
3.4.3.3.1 Effect of sucrose and irradiance on biomass accumulation and TetC expression
92
3.4.3.3.2 Effect of sucrose and irradiance on TetC expression .......................................... 94
3.4.3.3.3 Pulse amplitude modulation (PAM) fluorometry to assess photosynthetic
activity of in vitro regenerated shoots .................................................................................. 97
3.4.3.4 Implications of these findings on transplastomic molecular farming ................... 98
10
3.4.4 The influence of altered MS media strength on biomass accumulation and yield ...... 99
3.4.4.1 Design of experiment ............................................................................................... 99
3.4.4.2 Results and Discussion ............................................................................................. 99
3.4.4.2.1 Influence of altered MS media strength on biomass accumulation and TetC
expression 99
3.5 Effect of Temporary Immersion Culture Hydrodynamics on Viability, Growth and
Transplastomic Protein Expression in early-stage Callus Morphogenesis ...................................... 103
3.5.1 The importance of hydrodynamics in plant cell and tissue cultures ................................. 103
3.5.2 Investigation of the effects of fluid hydrodynamics on callus morphogenesis, viability and
heterologous protein turnover during pneumatic immersion ................................................... 104
3.5.2.1 Aims of experiment ................................................................................................. 104
3.5.3 Characterisation of key parameters............................................................................ 105
3.5.3.1 Characterisation of the rheological and hydrodynamic properties of the pneumatic
submersion of plant biomass .................................................................................................. 105
3.5.3.2 Characterisation of the rheological properties of tissue culture media ................ 105
3.5.3.3 Parameters for characterising the hydrodynamic flow field ................................. 106
3.5.3.4 Estimation of average shear rate ........................................................................... 106
3.5.3.5 Estimation of specific power input ........................................................................ 107
3.5.3.6 Cumulative Energy Dissipation .............................................................................. 107
3.5.4 Design of experiment ................................................................................................. 107
3.5.5 Results ......................................................................................................................... 108
3.5.5.1 Effect of shear rate and power dissipation on biomass accumulation .................. 108
3.5.5.2 Effect of shear rate and gassed power input on mitochondrial activity ................ 109
3.5.5.3 Effect of shear rate and gassed power input on TetC expression ......................... 110
3.6 Discussion on in vitro morphogenesis of callus in TIBs and transplastomic protein
expression, and the various parameters affecting these ............................................................... 114
Chapter 4. Expression and assembly of Guy’s 13 monoclonal antibody via temporary immersion
shoot regeneration ............................................................................................................................. 117
4.1 Introduction : Monoclonal antibody production in transgenic plants................................ 117
11
4.1.1 Monoclonal antibodies as biopharmaceuticals .......................................................... 117
4.1.2 Plant systems for antibody production ....................................................................... 119
4.1.3 Guy’s 13 monoclonal antibody as a topical immunotherapy agent for prevention of
dental caries ................................................................................................................................ 120
4.2 Expression and assembly of Guy’s 13 mAb by temporary immersion regeneration of N.
tabacum cv. Xanthii ........................................................................................................................ 121
4.2.1 Design of experiment .................................................................................................. 121
4.2.2 Results ......................................................................................................................... 121
4.2.2.1 Non-reducing Western Immunoblotting to confirm expression and assembly of
Guy’s 13 IgG1 in transgenic tobacco ....................................................................................... 121
4.2.2.2 Antigen binding assay for functional studies of expressed Guy’s 13 monoclonal
antibody 123
4.2.3 Discussion ................................................................................................................... 126
4.2.3.1 Plants possess the relevant machinery for expression of functional antibodies... 126
4.2.3.2 The impact of hyperhydricity on functional Guy’s 13 mAb titre in temporary
immersion regeneration ......................................................................................................... 127
4.2.3.3 Demonstration of mAb production in in vitro shoot regeneration via temporary
immersion culture ................................................................................................................... 128
Chapter 5 Expression of transplastomic proteolytically unstable proteins via temporary
immersion shoot regeneration ........................................................................................................... 130
5.1 Introduction ........................................................................................................................ 130
5.1.1 In planta proteolysis of recombinant proteins ........................................................... 130
5.1.2 Transplastomic expression of vaccine subunits susceptible to proteolytic degradation
131
5.1.2.1 VP6 as a potential subunit vaccine against rotavirus infection ............................. 132
5.1.2.2 p24 as a subunit vaccines against HIV.................................................................... 132
5.2 Expression of transplastomic proteins susceptible to degradation via temporary immersion
shoot regeneration ......................................................................................................................... 133
5.2.1 Accumulation of plastid-expressed rotavirus VP6 via temporary immersion shoot
regeneration and comparison to soil-grown seedlings .............................................................. 133
12
5.2.1.1 Design of experiment ............................................................................................. 133
5.2.1.2 Results .................................................................................................................... 134
5.2.1.2.1 VP6 stability in soil-grown tobacco leaves ........................................................ 134
5.2.1.2.2 Expression of VP6 in in vitro temporary immersion-regenerated biomass ...... 135
5.2.2 Accumulation of HIV-1 p24 antigen via temporary immersion shoot regeneration and
comparison to soil-grown seedlings ........................................................................................... 136
5.2.2.1 Design of experiment ............................................................................................. 136
5.2.2.2 Results .................................................................................................................... 137
5.2.2.2.1 p24 stability in soil-grown tobacco leaves ........................................................ 137
5.2.2.2.2 Expression of p24 in temporary immersion-regenerated shoots ..................... 138
5.2.3 Discussion on the expression of proteolytically unstable transplastomic proteins via TI
regeneration ............................................................................................................................... 140
Chapter 6. Developing new tools for in vitro molecular farming ................................................. 143
6.1 Development of large-scale mechanical bioreactor ........................................................... 143
6.1.1 The need for scale-up of in vitro organogenesis for molecular farming purposes ..... 143
6.1.2 Design of experiment .................................................................................................. 144
6.1.3 Results ......................................................................................................................... 145
6.1.3.1 Biomass Accumulation and Organogenesis ........................................................... 145
6.1.3.2 Comparison of biomass accumulation between the mechanical bioreactor and RITA
147
5.1.3.3 Comparative analysis of TetC expression in the mechanical bioreactor ............... 148
6.1.3.4 Discussion on the development of a large-scale bioreactor ............................... 150
6.2 The influence of pre-culture preservation of encapsulated callus on temporary immersion
morphogenic potential and TetC expression .................................................................................. 152
6.2.1 Synthetic seed technology .......................................................................................... 152
6.2.2 Design of experiment .................................................................................................. 153
6.2.3 Results ......................................................................................................................... 153
6.2.3.1 Influence of duration and temperature of encapsulated callus preservation on
growth and morphogenesis in temporary immersion culture ............................................... 153
13
6.2.3.2 Influence of Influence of duration and temperature of encapsulated callus
preservation on TetC expression in regenerated shoots ........................................................ 155
6.2.3.3 Discussion on the influence of alginate encapsulation on temporary immersion
regeneration and TetC expression .......................................................................................... 155
6.3 Discussion on described studies and how they relate to new developments in in vitro
molecular farming ........................................................................................................................... 156
Chapter 7 Summary and future directions ...................................................................................... 158
7.1 In vitro plant tissue culture as an alternative platform for biosynthesis of
biopharmaceuticals ......................................................................................................................... 158
7.2 The influence of temporary immersion shoot regeneration on biosynthesis of
transplastomic proteins .................................................................................................................. 162
7.3 Scale-up of callus-to-shoot regeneration for biopharmaceutical expression .................... 164
7.4 Biosynthesis and assembly of functional monoclonal antibodies in temporary immersion
shoot regeneration ......................................................................................................................... 165
7.5 Implementation of robust bioprocesses for biopharmaceutical synthesis ........................ 167
Bibliography ........................................................................................................................................ 169
14
List of Figures
Figure 1.1 Technological design and operational principle of Twin‐Flask system .............................. 35
Figure 1.2 Technological design and operational principle of RITA® system ..................................... 36
Figure 1.3 Technological design and operational principle of bioreactor of immersion by bubbles
system ................................................................................................................................................. 36
Figure 1.4 Downstream processing routes for seed and leaf crops ................................................... 38
Figure 1.5 Gene map of plastid genome from tobacco (N. tabacum). ............................................... 41
Figure 1.6 Schematic representation of the chloroplast expression cassette .................................... 46
Figure 2.1 Operation of large-scale mechanical bioreactor ............................................................... 60
Figure 3.1 Callus-meristemoid transition and shoot bud formation. ................................................. 71
Figure 3.2 Logistic increase of fresh and dry biomass accumulation during in vitro organogenesis in
RITA® TIBs .......................................................................................................................................... 72
Figure 3.3 SDS-PAGE and immunoblot showing differential expression of TetC. .............................. 74
Figure 3.4 Increase in TetC volumetric yield and fresh biomass. ....................................................... 74
Figure 3.5 Visual comparison between non-vitrified and vitrified shoots .......................................... 76
Figure 3.6 Investigation of hyperhydricity on TetC accumulation at different time intervals of
temporary immersion culture, by SDS-PAGE and immunoblot. ......................................................... 77
Figure 3.7 Effect of hyperhydricity on TetC accumulation at various sucrose concentrations and
irradiances. .......................................................................................................................................... 78
Figure 3.8 Effect of NO3-: NH4
+ ratio on developmental status of N. tabacum regenerated shoots
after 40-day temporary immersion culture. ....................................................................................... 82
Figure 3.9 Influence of NO3-: NH4
+ ratio on fresh and dry biomass accumulation. ............................ 83
Figure 3.10 SDS-PAGE and immunoblot analysis of lysates to assess TetC expression under various
NO3-: NH4
+ ratios. ................................................................................................................................ 84
Figure 3.11 Densitometric quantification of TetC intrinsic yields and volumetric yields under various
NO3-: NH4
+ ratios, from immunoblot data in Figure 3.10. ................................................................... 84
Figure 3.12 Shift in medium pH over temporary immersion culture period.. .................................... 85
Figure 3.13 Influence of media pH on fresh and dry biomass accumulation ..................................... 90
Figure 3.14 SDS PAGE and immunoblot analysis of of media pH effects on TetC expression. ........... 90
Figure 3.15 Visual demonstration of the effect of sucrose concentration on shoot morphogenesis
after 40-day temporary immersion culture ....................................................................................... 93
Figure 3.16 Effect of sucrose concentration and irradiance on fresh and dry biomass accumulation.
… ......................................................................................................................................................... 94
15
Figure 3.17 SDS-PAGE and immunoblot showing the effect of sucrose and light on Tetc expression.
…. ......................................................................................................................................................... 95
Figure 3.18 Influence of sucrose and light levels on TetC intrinsic yield (ng TetC per µg total soluble
protein (TSP)), determined densitometrically. ................................................................................... 96
Figure 3.19 Influence of sucrose and light levels on estimated absolute TetC yield (µg TetC per
volume of bioreactor) ......................................................................................................................... 96
Figure 3.20 Effect of MS media strength on fresh and dry biomass accumulation. ......................... 101
Figure 3.21 TIB cultures at 40 days (prior to harvest) at 100%, 50%, 25% MS medium strength .... 101
Figure 3.22 Visual comparison of 40-day temporary immersion cultures at 100% and 200% MS
medium strength .............................................................................................................................. 102
Figure 3.23 SDS-PAGE and immunoblot showing the effect of MS media strength on TetC
expression. ........................................................................................................................................ 102
Figure 3.24 Plots showing the influence of average shear rate and energy dissipation rate on fresh
and dry biomass accumulation, after 3, 20 and 40-day cultures ...................................................... 109
Figure 3.25 Plots showing the influence of average shear rate, energy dissipation rate and total
energy dissipation (after 20 days culture only) on mitochondrial respiratory activity after 0, 3, 20
and 40-day cultures .......................................................................................................................... 110
Figure 3.26 SDS-PAGE and immunoblots showing the effect of air flow rate on TetC expression. .......
. ....................................................................................................................................................... 111
Figure 3.27 Impact of hydrodynamics on TetC intrinsic yield (ng/µg) after 40-day culture………………..
. ....................................................................................................................................................... 112
Figure 3.28 Absolute recombinant protein yield depends on both intrinsic yield and biomass growth
... ....................................................................................................................................................... 116
Figure 4.1 Structure of immunoglobulin G (IgG)............................................................................... 119
Figure 4.2 SDS-PAGE and Western Immunoblot of lysates of in vitro and soil-cultivated biomass
under non-reducing conditions. ....................................................................................................... 123
Figure 4.3 Lysate titration curve showing the binding of Guy’s 13 mAb to the purified SWCF
fragment of SA I/II. ............................................................................................................................ 124
Figure 5.1 SDS-PAGE and immunoblot showing VP6 accumulation in the leaves of an 8-week old
soil-grown plant. ............................................................................................................................... 135
Figure 5.2 SDS-PAGE and Western immunoblot for demonstration of the expression of VP6 in TIB-
grown biomass ................................................................................................................................. .136
Figure 5.3 SDS-PAGE and immunoblot showing p24 accumulation in the leaves of an 8-week old
soil-grown plant. ............................................................................................................................... 138
16
Figure 5.4 SDS-PAGE and Western immunoblot for demonstration of the expression of p24 in TIB-
grown biomass. ................................................................................................................................. 139
Figure 6.1 Large 60 l mechanical bioreactor in operation ................................................................ 144
Figure 6.2 Schematic showing operation of large bioreactor. .......................................................... 145
Figure 6.3 Visual demonstration of shoot morphogenesis in large- and small-scale temporary
immersion bioreactors. ..................................................................................................................... 146
Figure 6.4 Increase in fresh and dry biomass accumulation in the mechanical temporary immersion
bioreactor after 50 and 80 days culture ........................................................................................... 147
Figure 6.5 Comparison of fresh and dry biomass accumulation in large bioreactor and RITA® culture
vessels. .............................................................................................................................................. 148
Figure 6.6 SDS-PAGE and immunoblot demonstrating TetC expression in large mechanical TIB ..........
. ....................................................................................................................................................... 149
Figure 6.7 Comparison of TetC yield in large bioreactor and RITA® culture vessels. ....................... 150
Figure 6.8 Callus aggregates encapsulated in a sodium alginate matrix. ......................................... 153
Figure 6.9 Effect of callus encapsulation duration and temperature on fresh and dry biomass
accumulation in temporary immersion shoot regeneration cultures. ............................................. 154
Figure 6.10 SDS-PAGE and immunoblot demonstrating TetC expression in shoot biomass
regenerated from encapsulated callus stored for various durations and temperatures. ................ 155
17
List of Tables
Table 2.1 List of antibodies used. ....................................................................................................... 55
Table 3.1 Variable fluorescence / maximal fluorescence (Fv/Fm) measurements for leaves
regenerated by temporary immersion culture of Nt-pJST12 under different photomixotrophic
treatments .......................................................................................................................................... 98
Table 4.1 EC50 titres and EC50 dilutions of plant lysates with standard errors, derived from 4-
parameter logistic curve fitting to titration curves ........................................................................... 125
18
List of Abbreviations
% (v/v) % volume / volume
% (w/v) % weight / volume
3’-UTR 3’ untranslated region
5’-UTR 5’ untranslated region
µmol.m-2.s-1 Micromoles of photons per square metre, per second (units of
photosynthetic photon flux)
aadA gene encoding aminoglycoside 3’’-adenylyl transferase for
spectinomycin resistance (in plastid transformation cassettes)
AMV Alfalfa Mosaic Virus
APS Ammonium persulfate
ATP Adenosine triphosphate
atpB Plastidial gene encoding beta subunit of ATP synthase
BIB® Bioreactor of immersion by bubbles
BiP Binding protein (molecular chaperone)
BIT® Twin flasks bioreactor system
BSA Bovine serum albumin
CCD Charge-coupled device (digital imaging system)
CPMV Cowpea Mosaic Virus
dH2O Distilled water
DNA Deoxyribonucleic acid
DTP diphtheria–tetanus–pertussis (vaccine)
DW Dry weight
cGMP ‘current good manufacturing practice’
CHO Chinese hamster ovary cells
CIM Callus induction medium
CTB cholera toxin B subunit
E. coli Escherichia coli
DSP Downstream processing
DTT Dithiothreitol
EBA Expanded bed adsorption
EC50 Half maximal effective concentration
19
ECL Enhanced Chemiluminescence
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme-linked immunosorbent assay
ER Endoplasmic reticulum
FDA United States Food and Drug Administration
FFD fractional factorial design
Fc crystallizable fragment
Fd ferredoxin
Fv Variable fragment (of immunoglobulin)
Fv/Fm Maximum PSII quantum yield
FW Fresh weight
GFP+ Variant of green fluorescent protein
GM Genetically modified
GMO Genetically modified organism
GMP ‘Good manufacturing practice’
GOGAT glutamine 2-oxoglutarate amino transferase (or glutamate synthase)
GS glutamine synthetase
GST glutathione-S-transferase
H Heavy chain (of antibody)
H2L2 fully assembled antibody composed of two heavy and two light chains
Hc Heavy chain (of TetC)
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIC Hydrophobic interaction chromatography
His6-MBP His-tagged derivative of the maltose binding protein
HRP Horseradish peroxidase
HSA Human serum albumin
hST Human somatotropin
Ig Immunoglobulin
IGF Insulin-like growth factor
IgG Immunoglobulin G
IMAC Immobilized metal ion affinity chromatography
IR Inverted repeat (region of plastome)
IVIG intravenous immunoglobulin
kb Kilobase
20
kDa Kilodalton
L Light chain
Lc Light chain (of TetC)
LSC Large single-copy (region of plastome)
M Molar / moles per litre; unit of concentration
MBP Maltose binding protein
MES 2-(N-morpholino)ethanesulfonic acid
mRNA Messenger RNA
MS medium Murashige & Skoog medium
N. tabacum Nicotiana tabacum
NAA 1-napthaleneacetic acid
NME new molecular entity
NMR Nuclear magnetic resonance
ORF Open reading frame
OspA Outer surface protein A
p24 HIV surface antigen
PAM Pulse amplitude modulated (fluorometry)
PBS Phosphate buffered saline
PE Polyethylene
PEB-A Protein extraction buffer
PEG Polyethylene glycol
PGI Plastid-genome incompatibility
PHB Polyhydroxybutyric acid
PPF Photosynthetic photon flux [units: µmol photons m-2s-1]
PPM ™ Plant Preservative Mixture, a broad-spectrum biocide supplied by Plant
Cell Technology, Inc.
Prrn Promoter of plastid-encoded ribosomal RNA gene
PsbA Plastid gene encoding D1 subunit of photosystem II
ptDNA Plastid DNA
PSII Photosystem II
PTOX plastid terminal oxidase
PVDF polyvinylidene fluoride
PVX Potato Virus X
ORF Open reading frame
21
rbcL Plastidial gene encoding Rubisco large sububit
RBS Ribosomal binding site
RER rough endoplasmic reticulum
RITA® Recipient for Automated Temporary Immersion (translated from
French), lab-scale TIB supplied by CIRAD.
RNA Ribonucleic acid
RO Reverse osmosis
ROS Reactive oxygen species
Rpm Revolutions per minute; frequency of rotation
rrn Plastidial gene encoding ribosomal RNA
RSM Response surface methodology
Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase
SA I/II Streptococcal antigen
SBB Slug Bubble Bioreactor
scFv Single-chain Fv antibody fragment
SD Standard deviation
SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis
SE Somatic embryogenesis
spp. species
SSC Small single-copy (region of plastome)
T7G10 Gene 10 from bacteriophage T7
TBS Tris-buffered saline
TBSV Tomato Bushy Stunt Virus
TDZ Thidiazuron
TEMED N,N,N,N-tetramethylenediamine
TeNT Tetanospasmin (also known as TeTx)
TetC Tetanus toxin fragment C
TF triphenylformazan
TI Temporary immersion
TIB Temporary immersion bioreactor
TIS Temporary immersion system
TMB 3,3′,5,5′-Tetramethylbenzidine
TMV Tobacco Mosaic Virus
TRI bioreactor Temporary root zone immersion bioreactor
22
TSP Total soluble protein
TTC 2,3,5-triphenyltetrazolium chloride
UTR Untranslated region
VP6 Rotavirus surface antigen
WUB Wave and Undertow Bioreactor
WT Wild-type
23
Chapter 1. Introduction
1.1 The Biotechnology revolution and recombinant biopharmaceuticals
The etymology of ‘biotechnology’ is a fusion of ‘biology’ and ‘technology’, and true to its
name, biotechnology is concerned with the exploitation and industrialisation of biological
agents for useful ends. The term ‘biotechnology’ was coined in 1919 by a Hungarian
engineer, Karl Ereky, who envisioned a great ‘biochemical age’, analogous to the stone and
iron ages (Kennedy, 1991; Webb and Atkinson, 1992). Biotechnology encompasses a broad
and divergent array of technologies with various levels of sophistication, which makes a
precise definition of the field rather difficult (Gavrilescu and Chisti, 2005). Biotechnology
may be defined as the “controlled and deliberate application of simple biological agents –
living or dead cells or cell components – in technically useful operations, either of productive
manufacture or as service operations” (Campbell, 1992). According to the European
Federation of Biotechnology, biotechnology can be considered “the integrated use of
biochemistry, microbiology and engineering sciences in order to achieve technological
application of the capabilities of microorganisms, cultured tissues / cells and parts thereof.”
(Klingenberg, 1984). Whichever definition is invoked, the common theme is the utilisation of
biological systems for some useful end, such as producing valuable products (such as
biopharmaceuticals) or performing a useful service (such as bioremediation).
Biotechnology can be divided into four major market segments: biopharmaceutical,
agricultural, environmental and industrial (Colwell, 2002). Although thought of as one of the
hallmarks of the modern era, biotechnology can be considered to be one of the oldest of
human activities. Primitive microbial and processing technologies have been used for
millennia, in activities such as grain milling, brewing, cheese-making, baking, food
preservation and traditional folk medicine, which over time became highly sophisticated
semi-empirical industries (Hulse, 2004). These so-called ‘old biotechnology’ applications are
the precursors to the modern biotechnology revolution, which has emerged and proliferated
over the last 40 years, through a convergence of four distinct fields of activity, namely
genetic engineering, protein engineering, metabolic pathway engineering and biochemical
(bioprocess) engineering (Gavrilescu and Chisti, 2005). The modern biotechnology field was
24
arguably born in 1973, when the first recombinant DNA was produced by cloning a gene into
a bacterial plasmid (Cohen et al., 1973). What distinguishes the ‘old’ from the ‘new’
biotechnology paradigms is that the former focuses on the production of low- to medium-
value products in bulk quantities, whereas the latter is usually geared towards extremely high-
value products that are required in minute quantities for pharmaceutical or diagnostic
purposes (Webb and Atkinson, 1992). Many countries are realising the socio-economic
benefits of biotechnology, and implementing integrated programmes for developing their
own native biotechnology industries, for the purpose of industrial regeneration, job creation,
social progress, sustainability and enhanced quality of life for their citizens through novel
medical and commercial products (Gavrilescu and Chisti, 2005).
Pharmaceuticals are defined as substances that are used for diagnosis, treatment, cure or
prevention of diseases or substances used to enhance physical or mental well-being (Nagels
et al., 2012), and may be broadly categorised as small molecule chemical therapeutics and
protein biopharmaceuticals. Protein biopharmaceuticals may be categorised into four major
groups (Leader et al., 2008; Strohl and Knight, 2009): (1) protein therapeutics with enzymatic
or regulatory activity (e.g. insulin, growth hormone, factor IX replacement therapies); (2)
proteins with specific targeted activity (e.g. monoclonal antibodies and immunoadhesins); (3)
vaccines (such as fragment C of tetanus toxin); and (4) protein diagnostics for biomarkers
such as glucagon, and imaging agents such as technetium-labeled antibodies. Of all the
market segments for recombinant proteins, the biomedical segment is the one which is
growing most rapidly (Colwell, 2002). Recombinant biopharmaceuticals represent a
modernisation of an old technology. Prior to the advent of recombinant DNA technology,
biotherapeutics were derived in very small amounts from largely unsafe human or animal
sources. The use of variolation with infectious smallpox scab material for centuries (Colwell,
2002), and anti-dipthera animal sera since at least 1895 (Strohl and Knight, 2009) were
primitive attempts at vaccination. In the early 20th century therapeutics were derived from
human or animal sources, such as blood clotting factors, human serum albumin from plasma,
insulin from porcine or bovine pancreas, and glucocerebrosidase from placenta (Sethuraman
and Stadheim, 2006). United States Food and Drug Administration (FDA) approval of the
world’s first commercial biopharmaceutical products, a monoclonal antibody-based
diagnostic kit in 1981, soon followed by Genentech’s bacterially produced insulin in 1983
(Johnson, 1983), set the pace for an exponential increase in biopharmaceuticals to enter the
market in the coming decades.
25
The three major industrial host platforms for producing recombinant proteins are bacteria
(mainly Escherichia coli), yeast and mammalian cells (Sabalza et al., 2014). Mammalian
cells are commonly used for producing biopharmaceuticals for which the glycosylation
pattern is of some relevance to the molecule’s intrinsic activity (Sodoyer, 2004). The large-
scale production of recombinant pharmaceutical proteins is often hampered by the poor
expression of their mature, active forms in prokaryotic hosts such as E. coli and by the high
costs and the limited scalability of traditional fermenter-based platforms using mammalian
cells (Merlin et al., 2014). An emerging alternative to these incumbent expression systems are
transgenic plants, which may address these technical issues (Sharma and Sharma, 2009).
1.2 Transgenic plants
1.2.1 The scope of transgenic plants as an alternative host technology
The genetic transformation of plants was first demonstrated in the early 1980s (Fraley et al.,
1983), and since then, an intense programme of research and development has been
undertaken, involving several industry and academic parties, to exploit transgenic plant host
systems. The original focus of transgenic plant biotechnology was improvement of crop
plants, either agronomic traits or improving their nutritional quality (Clarke and Daniell,
2011; Uncu et al., 2013). Although these are still active fields of research, the same molecular
technologies are being applied to the biosynthesis of high-value biopharmaceuticals. Until
recently, mammalian cells have commanded a near monopoly on production of complex
biopharmaceutical proteins, especially those requiring post-translational modifications
(Kuystermans et al., 2007) and antibodies (Birch and Racher, 2006; Zhang and Shen, 2012).
However, in recent decades, transgenic plants have emerged as a promising platform for the
efficient production of biopharmaceutical proteins (Rybicki, 2010). Plants have been used to
express a diverse range of mammalian proteins, including monoclonal antibodies, therapeutic
enzymes, blood proteins, cytokines, growth factors and growth hormones (Davies, 2010; Ko
et al., 2009; Xu et al., 2012b). Plants, being eukaryotic, are capable of post-translational
processing required for bioactivity, such as protein folding, disulfide bond formation, subunit
assembly, proteolytic cleavage, and glycosylation (Xu et al., 2012b). The use of plants to
26
produce recombinant pharmaceutical proteins has been termed ‘molecular farming’ (Spök et
al., 2008a).
Molecular farming can be rightly considered a ‘disruptive technology’ paradigm as it seeks to
take market share away from conventional biotechnologies, not through incremental
improvements, but by changing the game plan (Fischer et al., 2013). The field emerged and
gained momentum in the early 1990s, when Prodigene started producing avidin commercially
in maize seeds (Hood et al., 1997), followed by a host of other analytical proteins, including
β-glucuronidase, laccase and trypsin (Twyman et al., 2013; Witcher et al., 1998). However,
several roadblocks, including early technological inefficiencies, initially low yields,
regulatory barriers, hyped expectations, public recalcitrance to genetically-modified crops
and high-profile environmental transgressions caused the original molecular farming bubble
to burst (Fischer et al., 2013; Sabalza et al., 2014). The demand for recombinant biologics has
instead been met by incremental innovations in mammalian cell technology (Stoger et al.,
2014; Zhang and Shen, 2012). However, this bubble did not define the industry, but rather
represented early ‘teething’ problems intrinsic in every novel technological platform and
presented an opportunity to re-evaluate and reposition molecular farming in the context of the
biopharmaceutical landscape. The molecular farming paradigm has since re-emerged in the
2000s, with the convergence of transplastomics, a plethora of various in vitro and agricultural
cultivation platforms, bioprocess design, improved molecular biology strategies and yield
improvements providing solutions to the aforementioned problems.
The prospect of plants as competitive and commercially viable biopharmaceutical platform
has recently been realised, after decades of research and incremental improvements. In 2006,
the US Department of Agriculture approved a poultry vaccine against Newcastle disease
made by Dow Agrosciences (IN, USA), manufactured in tobacco cell suspensions, which was
the first license ever issued to a veterinary vaccine produced in plant cells (Katsnelson et al.,
2006). Although the vaccine was never commercialised because the company withdrew from
animal vaccine research, this represents a regulatory milestone in the acceptance of plants as
a manufacturing platform (Ritala et al., 2014). In 2012, the first recombinant plant-based
biopharmaceutical was approved for human use, by the FDA (Anon., 2012). Taliglucerase
alfa (ELELYSO™) is a carrot cell-expressed human β-glucocerebrosidase used as an enzyme
replacement therapy for Gaucher disease, produced by the Israeli company Protalix
BioTherapeutics (Zimran et al., 2011). The utility of plant-made vaccines in pandemic
27
situations was recently demonstrated when the experimental passive vaccine ZMapp, was
rolled out as treatment during the 2014 Ebola virus outbreak in West Africa, despite not
having gone through clinical trials. ZMapp (Kentucky BioProcessing), a combination of three
monocolonal antibodies produced in Nicotiana benthamiana, was found to be effective in
reversion of advanced Ebola virus disease in nonhuman primates (Qiu et al., 2014). A
number of clinical trials of plant-derived biopharmaceuticals are currently underway. A Phase
I clinical trial was recently completed in January 2014, for a personalised plant-derived
vaccine for the treatment of non-Hodgkin’s lymphoma, a product jointly developed by Icon
Genetics GmbH (Halle, Germany) and Bayer Innovation GmbH (Düsseldorf, Germany)
(Ritala et al., 2014).
As well as high-value pharmaceutical synthesis, transgenic plants are seen as a cost-
competitive source of low-value products required in moderate or bulk quantities, such as
industrial enzymes (Hood et al., 2007; Howard et al., 2011), analytical proteins (Hood and
Woodard, 2005), nutraceuticals (Ajjawi and Shintani, 2004; Zhao, 2007), chemical
feedstocks (Lynd et al., 1999) and biodegradable polymers (Mooney, 2009) with the potential
to displace petroleum-derived sources. The sustainability credentials of plant host systems are
often cited (Mooney, 2009). Plants are presented as carbon-neutral solar-powered systems
that, as photosynthetic organisms require little in the way of energy or material inputs
(Mooney, 2009; Sharma and Sharma, 2009). This is only partially true, as plant growth
approaches (both micropropagation and agricultural growth) are still very intensive
operations, in terms of fertiliser / media supply, mechanisation, downstream processing,
storage and supply chaining and the many ancillary operations surrounding cultivation.
However, the establishment of plant-derived chemicals would represent a step towards a
sustainable bio-economy and an improvement to the current petroleum-based system, which
is characterised by increasing feedstock prices and greenhouse gas emissions (Ragauskas et
al., 2006).
1.2.2 The benefits of transgenic plant host systems relative to conventional platforms
Transgenic plants have a number of benefits that set them apart from conventional bacterial,
yeast and mammalian cell-based host systems. A major advantage of molecular farming is
that plant cultivation does not require a high degree of technological input, and basic
28
agricultural techniques can be used (Ma et al., 2013). Some species can grow in almost any
soil or climate (Koprowski, 2005). Moreover, if field cultivation is not pursued,
micropropagative techniques are well-established and straightforward compared to equivalent
microbial or mammalian cell fermentation plants. The microeconomics of transgenic plant
cultivation enables them to be cost-effective host systems, offering order-of-magnitude cost
savings compared to conventional microbial systems (Twyman et al., 2003). It has been
suggested that agricultural production of recombinant protein could be 10-50 times cheaper
than E. coli fermentation (Kusnadi et al., 1997). The elimination of fermenters and other
sophisticated intensive equipment associated with standard ‘stainless steel’ biotechnology
facilities reduces capital expenditure and running costs (Twyman et al., 2003). This is even
true for micropropagation, as plant tissue culture vessels have fewer energy and resource
demands than fermenters. Although set-up costs for cultivation are high, further maintenance
costs are minimal, as only standard agricultural or micropropagation practices are involved
(Stoger et al., 2002). Scalability is an important consideration for any host system. In
principle, plant cultivation is infinitely scalable (Rybicki, 2009), limited only by light, space
and nutrients. In vitro micropropagation allows a ‘scale-out’ approach, through simple
multiplication of culture vessels. In comparison, fermentation systems and transgenic animals
have limits to capacity increase. Moreover, the speed and flexibility of scale-up is important.
It may take several years to achieve ten-fold scale-up of a herd of transgenic sheep through
natural breeding cycles, but transgenic plants can be scaled up 100-1000 fold in a single
generation through soil-based or in vitro regeneration from seeds, callus or explants
(Schillberg et al., 2002; Twyman et al., 2003). If scale-down is required, surplus transgenic
animals must be sacrificed or maintained at a loss, whereas the capacity devoted to
agriculture or micropropagation can be adjusted as required (Twyman et al., 2003). In terms
of regulatory fulfilment, biopharmaceutical production in planta for administration to humans
and animals provides an additional margin of safety as compared to biopharmaceuticals
produced in animal tissues (Koprowski, 2005). Importantly, plant cells do not harbour any
known human pathogens (Xu et al., 2011). This is a significant limitation of mammalian cell
culture and was brought to light in 2009, when Genzyme halted their CHO cell production of
their Gaucher disease therapeutic Cerezyme® after infection with calicivirus (Rybicki, 2010).
29
1.2.3 Bioprocessing of plant-derived biopharmaceuticals
1.2.3.1 Choice of localisation target in transgenic plant host systems
An advantage of transgenic plants as multicellular differentiated host systems is the
possibility of localising foreign protein to almost any organ or cellular compartment,
including leaves, stems, seeds, hairy roots, cell suspension cultures, the apoplast, transformed
chloroplasts and seed protein storage vacuoles (Jiang and Sun, 2002; Rybicki, 2009). Two
plant organs that are emerging as the target host organs of choice are seeds and leaves (Lau
and Sun, 2009; Xu et al., 2012b). Leaves as host organs are amenable to transplastomic and
transient expression (Xu et al., 2012b). However, leaves suffer one important disadvantage
that affects their storage and distribution; leaf proteins are generally unstable without further
processing (Jamal et al., 2009). Seeds are suitable tissues for expressing and storing foreign
proteins, as they have intrinsically high levels of protein accumulation during seed
development (Bewley, 1997; Lau and Sun, 2009; Wilken and Nikolov, 2012). The plant’s
intrinsic capability to partition proteins into specific tissue sinks (Hood and Woodard, 2005)
can be exploited to effectively concentrate heterologous proteins. For example in transgenic
maize kernels, trypsin expression in the embryos is over 100-fold greater than in the
endosperm, and the embryo makes up less than 10% of the kernel dry weight, so separation
of the embryo from the endosperm greatly concentrates the protein (Hood and Woodard,
2005). Compared to leafy biomass, protein localisation in seed crops will result in lower
yields, though increased protein stability in dessicant storage organelles devoid of proteases
allows for long-term storage. Recombinant hirudin expressed as an oleosin fusion in canola
was stable and unmodified for at least three years (Parmenter et al., 1996). Single-chain Fv
(scFv) antibodies expressed in tobacco seeds were found to retain their functionality after
storage at room temperature for 18 months (Ramírez et al., 2001). The degree of protein
stability in seeds presents a decoupling point between cultivation and downstream processing
as biomass does not need to be processed immediately after harvesting (Boothe et al., 2010;
Nikolov and Hammes, 2002; Wilken and Nikolov, 2012). For vaccines, the degree of stability
provided by seeds can circumvent the need for cold-chain refrigeration (Boothe et al., 2010).
30
1.2.3.2 The choice of in vitro or soil-based cultivation for growth of transgenic plants
In contrast to microbial or mammalian cell expression systems, there is a large plethora of
plant expression systems that can be exploited for foreign protein production. There are two
main options for commercial transgenic plant biomass production: soil-based cultivation of
whole plants or in vitro culture, which include micropropagation (plant tissue culture), hairy
root and cell suspension cultures (Xu et al., 2012b).
Soil-based cultivation is the most straightforward and scalable method of cultivation as
standard agricultural techniques can be used. Agricultural soil-based cultivation of transgenic
plants is an extremely low-cost platform, and on the basis of process economics alone, can
compete with established bacterial and mammalian cell fermentation systems (Doran, 2000).
With soil-based cultivation, there are two options: field or greenhouse cultivation. Field
cultivation provides the best process economy, though this is balanced against risks posed by
pests, parasites, anthropogenic pollution and environmental variation which can reduce batch-
to-batch consistency and product quality (Fischer et al., 2012). Greenhouse cultivation is
considerably more expensive than field growth but conditions can be more easily controlled
for greater reproducibility (Doran, 2000; Fischer et al., 2012). It is estimated that a
monoclonal antibody expressed in greenhouse-grown alfalfa will cost US$500–600 g-1 based
on operations in a 250 m2 greenhouse including heating, labour and consumables for protein
extraction and purification (Khoudi et al., 1999). Tobacco and Arabidopsis are being firmly
established model organisms for molecular farming (Budzianowski, 2010; Demeyer, 2011).
However, they are annual crops, completing their life cycle within one year (Craufurd and
Wheeler, 2009; Håkansson, 2003; Wheeler et al., 2000). This may limit the potential for
large-scale agricultural cultivation, as only one harvest a year is allowed. The establishment
of transformation schemes for perennial plant species has been advocated, as these remain
viable for long periods can be cultivated perpetually and the reduced number of breeding
cycles can mitigate genetic instability in transformant lines (D'Aoust et al., 2004; Doran,
2000; Xu et al., 2012b). The perennial legume forage crop, Alfalfa (Medicago Sativa) has
been successfully transformed (Busse et al., 2002; Khoudi et al., 1999; Vlahova et al., 2005).
One potential limitation of soil-based cultivation is gene segregation associated with sexual
reproduction, which can cause variation in protein yields and product quality. This is
especially true with the production of multimeric proteins such as monoclonal antibodies, in
31
which large variations of the fully assembled product have been reported over successive
generations (De Neve et al., 1998; Khoudi et al., 1999).
In comparison with soil-grown plants, in vitro approaches, such as cell suspension and
micropropagation, allow propagation in sterile culture vessels under defined conditions,
giving greater process consistency and reduced batch-to-batch variability (Xu et al., 2011). In
vitro plant and tissue cultures have been used for over 50 years for the synthesis of a wide
range of secondary metabolites, with important roles in pharmaceuticals, cosmetics,
perfumeries, dyeing, and flavour industries (Eibl and Eibl, 2008; Steingroewer et al., 2013).
Commercially successful products include ginseng saponins, shikonin, berberine and
paclitaxel (‘Taxol’) (Fujita, 1988; Kim et al., 1991; Zhang and Zhong, 2004; Zhong, 2002).
In contrast, the use of in vitro cultures for recombinant protein expression is still in its
infancy (Eibl and Eibl, 2008), although a wide diversity of foreign proteins have been
produced, including monoclonal antibodies (Boivin et al., 2010; Holland et al., 2010; Sharp
and Doran, 2001a; Vasilev et al., 2013) and subunit vaccines (Lai and Chen, 2012; Michoux
et al., 2013; Michoux et al., 2011). Carrot cell suspension cultures are used by Protalix
(Israel) for the production of their key product, Gaucher disease therapeutic ELELYSO™
(Zimran et al., 2011). Both suspension and micropropagative cultures circumvent the ‘genetic
instability’ problem, as they are based on asexual (vegetative) reproduction. Plant cell
suspension culture involves agitation of friable callus tissue in bioreactors or shake flasks to
form small aggregates and single cells (Hellwig et al., 2004; Xu et al., 2011).
Micropropagation is defined as the culture of somatic cells, tissues or organs under controlled
in vitro conditions for the generation of clonal progeny plants, in a relatively short time
(Dubranszki and da Silva, 2010). ‘Micropropagation’ and ‘plant tissue culture’ are often used
interchangeably, though in the literature, ‘micropropagation’ is usually used in reference to
generating elite progeny plantlets in vitro for ex vitro transfer or because the plants
themselves are a commercial product (Abbasin et al., 2010; da Silva et al., 2007; Dubranszki
and da Silva, 2010; Santana-Buzzy et al., 2007; Zych et al., 2005). Micropropagative tissue
culture hinges on two intrinsic properties of plant tissues, totipotency, the ability of somatic
cells to divide and regenerate whole plants, and plasticity to generate one type of tissue from
another (Georgiev et al., 2009). Regeneration can follow two morphogenic pathways:
organogenesis or somatic embryogenesis (Ziv, 2000). There are five main types of cultured
tissues: seedlings, isolated embryos, organs, explants (tissue or callus cultures), and isolated
cells or small aggregates in liquid suspension (suspension culture) (Sajc et al., 2000).
32
1.2.3.3 The use of bioreactors in cell and tissue cultures
The use of in vitro suspension culture and micropropagation for biomass production
facilitates the utilisation of bioreactor technologies, allowing the tightly controlled growth
conditions for enhanced target protein expression. Compared to traditional protocols,
bioreactor technologies are time and labour-saving, scalable, allow improved nutrient and
oxygen transfer, and facilitate enhanced growth and multiplication (Akin-Idowu et al., 2009).
Originally, stainless steel stirred tank, bubble column and airlift reactors used for microbial
fermentation were employed, after minor modifications, for plant cell suspension cultures
(Eibl and Eibl, 2008). At high biomass concentrations exceeding 30 g dry weight l−1, poor
oxygen transfer and heterogeneous biomass distribution is observed in airlift bioreactors and
bubble column reactors (Eibl and Eibl, 2008; Tanaka, 1981). Therefore stirred tanks are
preferable for cell suspension cultures at high cell densities. Variations on these conventional
reactors as well as non-traditional designs have become more common in recent years,
especially for micropropagation and root culture. The issues associated with impeded oxygen
transfer and high shear has led to the development of liquid-dispersed and gas-phase
bioreactors, also known as ‘spray’ and ‘mist’ bioreactors, which are used mainly in hairy root
culture (Towler et al., 2006; Weathers et al., 2008; Weathers et al., 2010). In particular, the
‘balloon’ type bubble bioreactor has been adopted by several Korean companies for ginseng
root culture at the 10,000–20,000 l scale, and can also be operated in ‘ebb-and-flow’ mode
for micropropagation (Choi et al., 2006; Weathers et al., 2010). Rotating drum bioreactors
have been conventionally used for fermentations, though they have been adapted to plant
suspension and tissue culture (Weathers et al., 2010).
One of the most momentous changes in plant cell and tissue bioprocessing is the shift
towards disposable single-use reactors, often based on plastic bags (Eibl and Eibl, 2008; Eibl
et al., 2010; Weathers et al., 2010). Capital costs of disposable culture systems are far less
than for the usual stainless steel tanks. The stringent good manufacturing practice (GMP)
requirements for therapeutic compounds necessitate dedicated vessels or costly cleaning
operations between runs (Weathers et al., 2010). Although disposable reactors may have
limited scalability, their lower costs allow multiple units to be used (Weathers et al., 2010).
33
Mechanically-driven bag bioreactors, characterised by wave-induced motion, include the
BioWave, AppliFlex, Tsunami-Bioreactor, Optima and OrbiCell (Eibl and Eibl, 2006). The
BioWave was found to be effective for cultivating tobacco, grape, apple and yew cells up to
10 l culture volume, achieving a 40g fresh weight l-1 day-1 maximum productivity (Eibl and
Eibl, 2008). The Wave and Undertow Bioreactor (WUB) operates by a wave-and-undertow
mechanism, whereas agitation and aeration in a Slug Bubble Bioreactor (SBB) occurs by
movement of large-diameter ‘Taylor-like’ or slug bubbles through a column; both were
successfully used to express isoflavones in tobacco and soya suspension cells (Terrier et al.,
2007).
Bioreactors have allowed enormous scale-up of cell suspension cultures, which often
mitigates against low yields. The 200 l Orbshake device was used for the 100 l culture of
tobacco BY-2 suspensions, giving cell growth and recombinant protein yields comparable to
shake flasks, hence allowing over 100-fold scaling without loss of productivity (Ritala et al.,
2014; Schillberg et al., 2013). Culture volumes of up to 70 m3 were achievable when
microbial fermenters were adapted for plant cells (Eibl and Eibl, 2008). Phyton Biotech in
Germany produces taxanes and paclitaxel in stainless steel stirred tank reactors at the world’s
largest GMP plant cell culture facility (Huang and McDonald, 2012). In comparison to cell
suspensions, the complexity of differentiated tissue has constrained their scale-up potential,
and as a result, bioreactors for tissue culture tend to remain bench-scale (Steingroewer et al.,
2013), although there are exceptions. The mass propagation of Stevia rebaudiana shoots in a
500 l bioreactor and Lilium bulblets in 5-20 l non-stirred reactors has been described (Akita et
al., 1994; Lim et al., 1997). Commercial micropropagation processes have typically involved
scaled-out multiplication of plantlets in multiple small or medium-scale vessels (da Silva et
al., 2007; Escalona et al., 1999; Firoozabady and Gutterson, 2003).
1.2.3.4 The temporary immersion culture format
Of all the various plant bioreactor designs, those based on temporary immersion have several
features making them most amenable to semi-automated micropropagation (Aitken-Christie,
1991; Watt, 2012). In temporary immersion (TI) culture, plant biomass is not permanently
submerged in medium, which may adversely affect growth and morphogenesis and induce
hyperhydricity (vitrification), but is periodically immersed (Watt, 2012; Weathers et al.,
34
2010). In TI systems (TIS), nutrient uptake occurs from a thin film of medium retained on the
surface of the plant tissues by capillarity, whose chemical composition is renewed with each
immersion (Debergh, 1983; Teisson and Alvard, 1995). Temporary immersion systems
facilitate adequate oxygen supply (owing to the fact that biomass is not permanently
submerged), reduced shear damage and reduced risk of contamination (Etienne and
Berthouly, 2002; Teisson and Alvard, 1999). Temporary immersion culture has been
acclaimed as “the most natural tissue culture approach”, as tissue morphogenesis under
reduced exposure to liquid medium more closely resembles soil-based plant cultivation
(Arencibia et al., 2008; Watt, 2012; Ziv, 2000; Ziv, 2005). Temporary immersion culture has
been demonstrated to produce high-quality plantlets at high multiplication rates for several
species, including Siraitia grosvenorii (Yan et al., 2010), sugarcane (Lorenzo et al., 1998;
Mordocco et al., 2009), coffee (Coffea Arabica) (Albarran et al., 2005), pineapple (at 300%
and 400% greater multiplication rates than liquid and solid cultures) (Escalona et al., 1999)
and Musa spp. (Alvard et al., 1993; Escalant et al., 1994). Temporary immersion is
efficacious for stimulating shoot proliferation such as for Pinus radiata (Aitken-Christie and
Jones, 1987) and banana (Alvard et al., 1993).
One of the earliest liquid culture systems which was a precursor to modern TI systems
involved growth of Pinus radiata on solid medium with periodic liquid medium
replenishment and allowed monthly harvesting of shoots (Aitken-Christie and Jones, 1987).
The earliest TI bioreactors were modified, adapted Nalgene two-compartment filtration units,
used for Musa propagation (Alvard et al., 1993) and Hevea brasiliensis somatic
embryogenesis (Etienne et al., 1997), or more recently for Curcuma zedoaria and Zingiber
zerumbet plantlet propagation (Stanly et al., 2010). A number of different bioreactor systems
have been adopted for the micropropagation of several species of horticultural, conservation
or pharmaceutical significance (Watt, 2012). Three popular configurations, based on
pneumatic medium transfer, are the Twin Flasks System (BIT®), Recipient for Automated
Temporary Immersion (RITA®) and bioreactor of immersion by bubbles (BIB®) (Escalona
et al., 1999; Mordocco et al., 2009; Soccol et al., 2008; Zhu et al., 2005). The Twin-Flask
system (BIT®) (Escalona et al., 1999) consists of two containers, one for plant biomass and
the other as a reservoir for liquid medium. When a solenoid valve is opened and compressed
air is turned on, the medium is forced into the first flask, immersing the plants. The process is
reversed when another solenoid valve is opened and air pressure forces the medium back into
the original reservoir (Watt, 2012). The RITA® (VITROPIC, France) bioreactor is made of
35
two compartments, an upper one containing biomass and a lower one containing liquid
medium, linked in such a manner that overpressure applied to the lower compartment pushes
the medium into the upper compartment. Immersion of plantlets is achieved through
application of sterile air overpressure. During the immersion period, air flow replaces the
atmosphere inside the vessel as overpressure escapes through an outlet at the top of the
apparatus. When air flow is stopped, the medium returns to the lower compartment under
gravity (Teisson and Alvard, 1999). A more recent TI system, similar to the RITA®, is the
Plantima® (A-Tech Bioscientific Co. Ltd., Taipei, Taiwan) (Yan et al., 2010).The BIB® is
an entirely new TI system based on immersion of biomass in foam, as opposed to liquid
medium (Georgiev et al., 2014). The BIB® has two chambers divided transversely by a
porous plate (Soccol et al., 2008). The system uses a system interlinked by hoses of flexible
rubber for air flow and medium is delivered to the biomass through bubbling (Scheidt et al.,
2011; Scheidt et al., 2009). As well as these three popular configurations, there is a number of
variations of these as well as more ‘unorthodox’ designs (Georgiev et al., 2014; Watt, 2012).
These include the thermo-photo-bioreactor, based on the two-chamber but including a water
bath for temperature control and integrated UV light source, hybrid Ebb-and-Flow with
saturated tubular convective flow, rocker systems, low-cost disposable systems as well as
custom-made temporary immersion systems for individual labs (Georgiev et al., 2014;
Navarro et al., 2011).
Figure 1.1 Technological design and operational
principle of Twin‐Flask system (Georgiev et al., 2014)
(A) period of exposure. The whole volume of liquid medium
is located into the medium storage tank. Air lines of both
containers are closed and the solenoid valves are opened to
atmosphere; (B) dislocation of liquid medium from medium
storage tank to culture chamber. The air line of cultivation
chamber is closed, and the air line of medium storage tank is
opened. The overpressure moves the medium into the
cultivation chamber; (C) period of immersion. The propagules
are immersed into the liquid medium. The medium storage
tank is empty. Air lines for both containers are closed and the
solenoid valves are opened to atmosphere; (D) draining out
the nutrient medium back to the culture medium tank. The air
line of cultivation chamber is opened, whereas the air line of
medium storage tank is closed. The overpressure moves
back the medium into the medium storage tank.
36
Figure 1.2 Technological design
and operational principle of RITA®
system (Georgiev et al., 2014)
(A) period of exposure; (B) Dislocation of
liquid medium. Air pressure is applied to
the bottom compartment through the
central pipe. The liquid medium is moving
to the upper compartment; (C) period of
immersion; (D) draining out the nutrient
medium. The air flow is stopped and the
medium flows back to the bottom
compartment due to gravity.
Figure 1.3 Technological design and
operational principle of bioreactor of
immersion by bubbles system (Georgiev et
al., 2014)
(A) period of exposure; (B) period of
immersion. Air is supplied and foam is formed.
The explants are immersed by culture
medium in a form of bubbles. When aeration
stops, the foam density decreases with time
due to liquid drainage and the explants are
exposed to gaseous environment.
37
1.2.3.5 Downstream processing
Downstream processing (DSP), involving the extraction and purification of recombinant
proteins, is an integral part of any bioprocess, though it was largely ignored by the early
pioneers of the molecular farming boom (Fischer et al., 2012). The exception was Prodigene
Inc., purveyors of plant-derived technical reagents (Menkhaus et al., 2004; Nikolov and
Woodard, 2004). Huge economic savings can be made through optimisation of downstream
processing, since it accounts for approximately 80% of the final product costs (Evangelista et
al., 1998; Kusnadi et al., 1997). Plant cultivation-to-purification processing procedures
should be standardized to ensure the same final product for therapeutic or diagnostic purposes
(Ko and Koprowski, 2005). The downstream processing schemes of most plant-based
bioprocesses can be divided into three general stages: plant material pre-processing, protein
extraction and protein purification (Nikolov and Woodard, 2004). Pre-processing normally
involves grinding or fractionation of plant tissue. The choice of primary recovery operation
differs between leaf and seed-based bioprocesses. Leafy tissue is typically macerated
followed by centrifugation clarification (Nikolov and Woodard, 2004). For seeds, extraction
can be undertaken concurrently with wet grinding or subsequent to dry grinding and
fractionation for volume reduction (Menkhaus et al., 2004). Two important purification
methods are immunoprecipitation and affinity chromatography (Ko and Koprowski, 2005).
These may be supplemented by additional steps for further purification of captured proteins,
such as hydrophobic interaction chromatography (HIC), immobilized metal ion affinity
chromatography (IMAC), ion-exchange and ceramic hydroxyapatite purification (Wilken and
Nikolov, 2012). The integration of clarification and adsorption is possible by using expanded
bed adsorption (EBA) (Bai and Glatz, 2003; Menkhaus and Glatz, 2005; Valdés et al., 2003).
In contrast to conventional ‘fixed bed’ chromatographic modes, EBA involves fluidisation of
chromatography resins, facilitating capture of target proteins from viscous or high-particulate
mixtures (such as macerated biomass) and reducing fouling of resins (Bai and Glatz, 2003;
Menkhaus and Glatz, 2005; Valdés et al., 2003). Protein A or G affinity chromatography
column operations have been ubiquitously used for capture of plant-made antibodies (IgGs),
often followed by an anion exchange polish step (Chen, 2008; Nikolov et al., 2008; Wilken
and Nikolov, 2012).
38
Figure 1.4 Downstream processing routes for seed and leaf crops (Nikolov and Woodard,
2004)
1.2.4 Chloroplast transformation
1.2.4.1 An overview of plant genetic transformation strategies
Stable nuclear transformation, involving integration of a foreign gene construct within the
nuclear genome, thereby conferring stably inheritable traits, has been the most common
method of plant transformation (Obembe et al., 2011). However, there are significant
limitations with nuclear transformation. T-DNA, the section of Agrobacterium Ti plasmid
that is transferred to the host plant genome, can be integrated as multiple copies, as direct or
inverted repeats or other complex patterns, which can lead to transgene silencing (Husaini et
al., 2011). Likewise, transgene silencing is often observed with nuclear transformation by
microprojectile bombardment, caused by integration of multiple copies of the transgene
(Husaini et al., 2011). Nuclear transgene expression is unpredictable and characterised by low
and highly variable expression levels. Therefore nuclear transformation programmes often
have long development times for generation of stable transformants, due to the complex
genetics associated with identifying and stabilising transgenic lines (Hiatt and Pauly, 2006).
In order to circumvent the issues related to nuclear transformation, transient expression
(epichromosomal transformation) may be used (Fischer et al., 2012; Lico et al., 2005).
Epichromosomal transformation does not involve integration of heterologous genes into the
39
plant nuclear genome and therefore does not allow transgene inheritance (Lico et al., 2005).
Epichromosomal transformation may be mediated by a number of vectors: viruses which
infect a number of plant species such as Bromovirus, Hordivirus (BSMV: Barley Stripe
Mosaic Virus), Tobamovirus (TMV: Tobacco Mosaic Virus), Potyvirus, Potexvirus (PVX:
Potato Virus X), Comovirus (CPMV: Cowpea Mosaic Virus), Tombusvirus (TBSV: Tomato
Bushy Stunt Virus) and Alfamovirus (AMV: Alfalfa Mosaic Virus) (Lico et al., 2005; Pogue
et al., 2002) or Agrobacterium tumefacians (“Agroinfiltration”) (Lee and Yang, 2006).
Transient expression is rapid, and process development is not delayed by regeneration times
and the need to establish stable lines by breeding and seed banking (Fischer et al., 2012). As
there is a short time interval between transformation and expression, transient expression is
suited to the rapid, large-scale and cost-effective production of strain-specific vaccines,
especially in the developing world (Merlin et al., 2014). However, there are important
limitations of transient systems, related to the process complexity introduced by the
infiltration process. The large-scale use of GM bacteria or viruses imposes a huge regulatory
and technical burden, related to their containment, fermentation and proper disposal (Fischer
et al., 2012).
Another approach which circumvents the problematic protein expression associated with
stable nuclear transformation, and non-inheritability and technical problems associated with
epichromosomal (transient) expression, is transformation of plastids.
1.2.4.2 The plastid genome as a target for genetic transformation
1.2.4.2.1 Plastids organelles in higher plants
In plants and algae, plastids are semi-autonomous double-membrane bound organelles, which
evolved from free-living cyanobacteria that were acquired by the eukaryotic host through
endosymbiosis (Gould et al., 2008). In plants, plastids play important roles in photosynthesis,
amino acid and lipid synthesis, starch and oil storage, fruit and flower coloration, gravity
sensing, stomatal functioning, and environmental perception (Wise, 2006).
There is a wide diversity of plastid types. All plastids are derived from small undifferentiated
plastids termed proplastids, found in dividing cells in plant meristems. During cell division,
40
proplastids differentiate into the specialised plastid type depending on the type of cell they
reside in (Osteryoung and Pyke, 2014). The different plastid types can be distinguished by
their pigment composition, biochemical contents and structural features. For instance, green
chloroplasts in leaves contain chlorophyll, etioplasts which are formed under low light
conditions lack pigments, white amyloplasts store starch and possess a minimal internal
membrane system, oil-storing elaoiplasts are small and round, and brightly coloured
chromoplasts found in flowers and fruit contain high concentrations of various carotenoids
(Wise, 2006).
Chloroplasts, the plastids found in green leaves and algae, are the best understood plastid type
in terms of biochemistry and morphology. Chloroplasts are important as the site of oxygenic
photosynthesis. Chloroplasts are observable as plano-convex discs (Wise, 2006). Chloroplasts
are delineated by a galactolipid double membrane envelope (Dörmann, 2001), which contain
an internal membranes system known as thylakoids, (Menke, 1962) surrounded by the
stroma, an aqueous matrix. The thylakoids consist of a continuous 3-D membrane
architecture surrounding a single thylakoid lumen. Thylakoid membranes contain cylindrical
stacked membranes known as grana which are connected by unstacked stroma lamellae.
Mature chloroplasts may contain 40 to 60 grana stacks with diameters of 0.3 – 0.6 µm.
Within the thylakoids large protein-chlorophyll complexes are embedded, containing light-
harvesting complexes, photosystem II, photosystem I, cytochrome b6/f and ATPase which
carry out the light reactions of photosynthesis (Dekker and Boekema, 2005). The stroma is
the location of the Calvin-Benson carbon-fixation reactions (Jablonsky et al., 2011).
1.2.4.2.2 Features of the plastid genome
Plastids possess their own genome and ribosomal machinery for protein synthesis, which is
homologous to that of prokaryotes (Harris et al., 1994). The plastid genome (plastome,
plastid DNA or ptDNA) is a double stranded circular DNA molecule of 130 – 160 kb (Tissot
et al., 2008). The plastid genome contains 120 – 130 genes, which mainly orchestrate the
process of photosynthesis and genetic system ‘housekeeping’ functions (Shimada and
Sugiura, 1991). Around 100 genes encode proteins in land plants, and between 30 – 50 genes
encode RNAs (Rivas et al., 2002; Sugiura, 1992). Genes are arranged in operons and are
transcribed polycistronically (Barkan, 1988). An almost universal feature of the plastid
41
genome is a large inverted repeat (IR) sequence, 10 – 25 kilobase pairs (kb) in size, which
separates the remainder of the genome into single copy regions of approximately 80 kb and
20 kb, the large single-copy (LSC) and small single-copy (SSC) regions (Palmer, 1983;
Palmer, 1985). The plastome has significantly reduced in size with evolution, because many
cyanobacterial genes were incorporated into the nuclear genome (Martin et al., 2002). The
ptDNA is localised to membranes in clusters of approximately 10 genomes, known as
nucleoids (Roh and Choi, 2004).
Figure 1.5 Gene map of plastid genome from tobacco (N. tabacum).
Arrows depict direction of transcription. The map was drawn using OGDRAW v1.1
(http://ogdraw.mpimp-golm.mpg.de/index.shtml) using Genbank accession number of N. tabacum
plastome Z00044. Abbreviations: LSC = large single copy; SSC = small single copy; IR = inverted
repeat region.
42
1.2.4.2.3 The plastid genome as a novel target for genetic engineering
The science of plastid genetic engineering is known as ‘transplastomics’ (Chamberlain and
Stewart, 1999). Plastid genetic engineering was first attempted in the microalga
Chlamydomonas reinhardtii (Boynton et al., 1988). After the tobacco plastid genome was
fully sequenced (Shinozaki et al., 1986), chloroplast transformation was successfully
undertaken in tobacco (Nicotiana tabacum) (Svab et al., 1990). Since then, tobacco has been
firmly established as the de facto standard model system for transplastomics (Bock, 2014),
though this has brought both benefits and constraints. Tobacco is a leafy plant with high
biomass yields and high soluble protein, with well-established protocols for chloroplast
modification (Tremblay et al., 2010). However, tobacco is an unpalatable crop which
produces toxic compounds, and is therefore not amenable to the production of ‘edible
vaccines’ (Mishra et al., 2008). Moreover, the scope of the transplastomics field depends on
the diversity of crop species that can be transformed. Chloroplast transformation has been
extended to relatively few other species, including Arabidopsis thaliana (Lutz et al., 2011;
Sikdar et al., 1998), potato (Nguyen et al., 2005; Valkov et al., 2011), oilseed rape (Cheng et
al., 2010), carrot (Kumar et al., 2004), cabbage (Liu et al., 2007), lettuce (Kanamoto et al.,
2006; Ruhlman et al., 2007) and tomato (Ruf et al., 2001) and cauliflower (Nugent et al.,
2006) and rice (Oryza sativa) (Lee et al., 2006). Unfortunately, straightforward, reliable and
reproducible transformation protocols for monocot plants, which include agriculturally
important cereals have been lacking (Bock, 2014; Clarke and Daniell, 2011).
1.2.4.2.4 Methods for transformation of the chloroplast
Stable delivery of transgenes into plastids can be undertaken by tissue bombardment with a
particle gun (biolistics) (Svab et al., 1990), treatment of protoplasts with polyethylene glycol
(PEG) (Golds et al., 1993; Kofer et al., 1998; Spörlein et al., 1991), microinjection or
grafting. Biolistics is the classical and most widely adopted method, as it is less time-
consuming and easier in terms of tissue culture procedures. Plasmids are coated on the
surface of gold or tungsten microparticles (0.4 – 1.0 μm) and shot into leaves using a gene
gun. PEG-mediated transformation is occasionally used, being technically demanding and
laborious (Bock, 2014). A rarely used technique, transformation of plastids by microinjection
has resulted in transient gene expression but stable plastome transformation has not been
43
reported (Knoblauch et al., 1999; Maliga, 2003). A new method of plastid transformation by
horizontal genome transfer has been developed, based on the recent observation that
chloroplast DNA (and presumably chloroplasts) can migrate between cells in grafted plants
(Bock, 2014; Stegemann and Bock, 2009). Plastid transformation by grafting may be
instrumental in extending the technology to new species. However, the applicability of
grafting is restricted by potential incompatibilities between the plastid and nuclear genomes
of distantly related species, given the tight functional and regulatory interactions between the
plastids and nucleus (Greiner and Bock, 2013). The causes for plastid-genome
incompatibility (PGI) may be due to poor complementarity between nuclear and plastidial
proteins that are involved in photosynthetic regulation or plastid protein synthesis (Greiner
and Bock, 2013).
Transgenes are incorporated into the plastome by homologous recombination. Flanking
sequences ensure site-specific insertion of the construct into the inverted repeat region of the
plastid genome (Daniell et al., 1998), eliminating ‘position effects’ observed in nuclear
transformed plants. Moreover, ‘gene silencing’ has not been observed in recombinant plastids
(Daniell et al., 2002). Homologous recombination requires prior knowledge of the sequence
of the inverted repeat region of the plastome. One impediment is the lack of plastome
sequence information for several crop plants (Sabir et al., 2014). In the past 25 years, plastid
genome sequences of just 50 crop plastid genomes have been published, compared to 250
non-crop genomes (Jansen and Ruhlman, 2012; Sabir et al., 2014; Saski et al., 2005). As well
as limited successful transformation of such crop species, this also restricts the availability of
plastome sequences for the design of transformation constructs, further reducing the scope for
a ‘universal vector’ for plastid transformation (Sabir et al., 2014). In the quest for a universal
vector, transformation efficiency may be adversely affected if flanking sequences of a
different species are used. For example, when inverted regions from the petunia plastome
were used as flanking regions for tobacco chloroplast, the transformation efficiency was only
7% (DeGray et al., 2001).
To obtain genetically stable transformed plants, the desired genetic modification must be
present in each ptDNA copy in every cell, a situation known as homoplasmy (also known as
homoplastomy) (Ahmad and Mukhtar, 2013). Failure to achieve homoplasmy results in rapid
somatic segregation and genetic instability. Usually, 2 – 3 rounds of selection and
regeneration allow elimination of residual wild-type genomes still present in primary
44
transformants (Bock and Khan, 2004). Selective enrichment of transformed ptDNA is
undertaken via tissue culture, using an antibiotic selection pressure. The transformation
cassette contains an antibiotic-detoxifying gene which confers a selective advantage to
plastids containing transgenic plastomes. Antibiotic selection pressures such as
spectinomycin, streptomycin or kanamycin are used as these inhibit plastid protein synthesis
(Maliga, 2002). The aadA gene encoding a aminoglycoside 3’’-adenylyl transferase which
confers spectinomycin resistance to transformed chloroplasts is the most commonly used
selectable marker gene included in vector constructs (Svab and Maliga, 1993). Transformants
exhibit green shoots indicating photosynthetically functional chloroplasts and can easily be
distinguished from untransformed shoots which are bleached and display depressed growth
(Maliga, 2004). The tissue culture medium triggers cell division, yielding meristemic cells
with 10 to 14 proplastids, each of which carries only one or two nucleoids. Reduction in
plastid number accelerates plastid sorting during cell division. In the presence of the
antibiotic selection pressure, transgenic plastids divide at a faster rate than plastids containing
only wild-type ptDNA. Non-transformed plastids are lost by dilution during cell division
(Maliga and Bock, 2011). The absence of segregation of antibiotic-resistance is considered a
confirmation of homoplasmy (McCabe et al., 2008). Once homoplasmy is established,
rooting of cultured transformant shoots, followed by ex vitro transfer of regenerated whole
plants is undertaken to produce seeds which can then be banked for future propagation.
1.2.4.2.5 Benefits of plastid transformation
1.2.4.2.5.1 Hyperexpression of transgenic proteins in plastids
One of the most alluring features of plastids for biopharmaceutical production is their
enormous capacity to hyperexpress and accumulate foreign proteins. This is due to
polyploidy with approximately 100 chloroplasts per tobacco leaf cell containing a total of
10,000 copies of ptDNA (Shaver et al., 2006). Successful examples of protein overexpression
include accumulation of insecticidal protein Cry2Aa2 to 46% TSP (total soluble protein) (De
Cosa et al., 2001), tetanus toxin fragment C (TetC) to 25% TSP (total soluble protein) of leaf
tissue (Tregoning et al., 2003) and GFP to 38% TSP (Yabuta et al., 2008). Maximum
transgenic hyperexpression ever achieved was that of antibacterial lysin at over 70% TSP,
which even compromised production of endogenous proteins (Oey et al., 2009). Moreover,
45
chloroplasts can perform post-translational modifications (important for proteins of
eukaryotic origin), including correct folding and disulfide bonding, such as for human
somatotropin (hST) (Staub et al., 2000) and cholera toxin B subunit (CTB) (Daniell et al.,
2001a). Heterologous proteins expressed in plants via nuclear transformation or viral
transfection are susceptible to glycosylation with potentially allergenic non-mammalian
glycans (Sriraman et al., 2004). In contrast, plastid-encoded proteins do not undergo
glycosylation, though a disadvantage is that it precludes the synthesis of glycosylated
antibodies.
1.2.4.2.5.2 Post-transcriptional regulation of plastid protein synthesis and
implications for transformation vectors
Chloroplast protein synthesis is largely regulated at the post-transcriptional level (Stern et al.,
1997; Tillich et al., 2010). To optimise transgene expression, chloroplast transformation
schemes exploit a number of endogenous and heterologous regulatory elements, many of
which influence mRNA processing (Ruhlman et al., 2010), such as 3’- and 5’-untranslated
regions (UTRs). Unlike bacteria, the 3’-UTRs in plastids act as processing and stabilising
elements, but do not terminate transcription (Stern and Gruissem, 1987), so it is possible to
extend existing operons by one or more genes. 5’-untranslated regions (5’-UTRs) are
important for transcript stability and translation efficiency (Singh et al., 2001) and contain a
sequence that forms a stem-loop for binding to ribosomes, analogous to bacterial Shine-
Dalgarno sequences (Eibl et al., 1999; Hirata et al., 2004) . Many vectors contain an intact or
truncated 5’-UTR of a highly expressed plastid gene such as psbA, atpB, rbcL (Kuroda and
Maliga, 2001b; Staub and Maliga, 1994), or gene 10 from the bacteriophage T7 (T7G10)
(Kuroda and Maliga, 2001a). The strong plastid rRNA operon (rrn) promoter (Prrn) is the de
facto standard promoter included in most plastid transformation vectors (Maliga, 2003). The
plastid psbA promoter (PpsbA) and chimeric E. coli trc promoter (Ptrc) have also been used
(Staub and Maliga, 1994; Newall et al., 2003). Where polycistronic transcription is required,
a short intercistronic expression element sequence may be inserted to facilitate intercistronic
mRNA cleavage (Zhou et al., 2007).
46
Figure 1.6 Schematic representation of the chloroplast expression cassette. Map of the chloroplast
expression vector shows possible integration sites (flanking regions), promoters, selectable marker
genes, regulatory elements, and genes of interest (adapted from Verma and Daniell, 2007).
1.2.4.2.5.3 Polycistronic transcription of plastid genes and opportunities for
metabolic pathway engineering
Most plastid genes are organised in operons and are co-transcribed to produce polycistronic
mRNAs (Bogorad, 2000). This allows the possibility for stacking several transgenes in
operons and expressing entire biosynthetic or functional pathways, through a single
transformation event. The scope for metabolic engineering applications is huge (Bock, 2014).
In transformed tobacco chloroplasts expressing Cry2Aa2, the cry2Aa2 operon contains a
small open reading frame (ORF) immediately upstream of the cry2Aa2 gene. This ORF
encodes a putative chaperonin which facilitates the folding of Cry2Aa2 into proteolytically-
stable cuboidal crystals, protecting the foreign protein from protease damage, as well as
aiding downstream purification (De Cosa et al., 2001). The generation of transplastomic
tomatoes with elevated β-carotene (provitamin A) levels represents a milestone in plastid
metabolic pathway engineering for generation of nutritionally enhanced foods. Lycopene, a
caretonoid abundant in ripe red tomato fruits, can be enzymatically converted to β-carotene
by lycopene β-cyclases. A chromoplast transformation scheme which incorporated a β-
47
cyclase transgene from daffodil (Narcissus pseudonarcissus) yielding β-carotene levels
reaching 1 mg g-1 dry weight (Apel and Bock, 2009) was found to be most efficient after
testing cyclase transgenes from various other carotenoid-synthesising bacterial, fungal and
plant species (Wurbs et al., 2007). Proof-of-principle studies have assessed the potential for
transplastomic biosynthesis of novel carotenoids (Wilson and Roberts, 2012). Astaxanthin is
a high-value ketocarotenoid used as a food and feed additive. It is not naturally synthesised in
higher plants but does accumulate in some marine bacteria and algae, though it is chemically
synthesised for commercial use. Hasunuma et al. (2008) succeeded in producing over 0.5%
(dry weight) astaxanthin in tobacco leaves by co-expressing genes encoding β-carotene
ketolase and β-carotene hydroxylase, two enzymes involved in astaxanthin biosynthesis. One
of the most complex metabolic pathways transferred into the plastome, is that for the
synthesis of polyhydroxybutyric acid (PHB), a bacterial biopolyester, introduced into tobacco
chloroplasts (Atkin and Cummins, 1994; Lossl et al., 2005). An operon of three genes phbC-
phbB-phbA, encoding three enzymes derived from Ralstonia eutropha has been introduced
into tobacco chloroplasts. The cytoplasmic mevalonate pathway, consisting of six enzymes,
has been incorporated into tobacco chloroplasts, enabling synthesis of isoprenoids (Kumar et
al., 2012).
1.2.4.2.5.4 Maternal inheritance of the chloroplast genome and implications for
biosafety
Despite the self-evident economic and technical benefits of agricultural cultivation of
transgenic plants, concerns with genetically modified organism (GMO) containment are
hindering its widespread implementation. This issue has been especially poignant in the
recent history of molecular farming, with a number of high-profile ‘leaks’ into the biosphere
(Fox, 2003). Transplastomic plants do provide a greater level of biosafety than nuclear
transformed plants, since plastid genes are largely maternally inherited, limiting the risk of
dissemination of transgenes by pollination (Daniell et al., 2002; Hagemann, 2004). It used to
be assumed that chloroplast transformation offered total transgene containment (Daniell et
al., 2002), although studies have shown that in rare cases, low-level leakage of transgenes in
pollen may occur (Avni and Edelman, 1991; Ruf et al., 2007; Svab and Maliga, 2007).
Another possibility is the transfer of transgenes from the plastome to the nuclear genome
(Huang et al., 2003; Sheppard et al., 2008; Stegemann et al., 2003). Although transplastomic
48
plants do not offer ‘absolute’ biosafety, they may be used in conjunction with in vitro cell
suspension, tissue culture or greenhouse cultivation under fully-contained conditions, as part
of an integrated biosafety strategy (Martine et al., 2009).
1.2.4.2.6 Applications of plastid genetic engineering
Transplastomic plants have huge scope to revolutionise biotechnology, with many innovative
applications currently being envisaged and assessed. The potential applications of this
emerging paradigm hinge upon (but are not limited to) three main areas, engineering crop
plants for improved agronomic traits and nutritionally-enhanced foods, ‘molecular farming’
of biopharmaceuticals, metabolic engineering approaches, and expression of commercial
enzymes and other proteins (Bock, 2007; Bock, 2014).
1.2.4.2.6.1 Plastid engineering for improvement of agronomic traits and Rubisco
activity
Plastid engineering can potentially provide an environmentally benign way to improve the
agronomic traits of field-grown crop plants such as herbicide and insect resistance, since
maternal inheritance precludes dissemination of transgenes through pollination.
Resistance to herbicides is a promising application, yielding extremely herbicide-tolerant
strains of crop plants (Bock, 2007). Successful examples of plastid-encoded herbicide
tolerance include resistances to glyphosate (Ye et al., 2001), sulfonyl-urea herbicides
(Sharma and Shanker Dubey, 2005) and PPT-based herbicides (Lutz et al., 2001). A recent
innovation is the development of a plastid resistance gene against D-amino acids that could
be used as a herbicide (Bock, 2014; Gisby et al., 2012).
Strategies for insect resistance are usually based on the synthesis of Bacillus thuringiensis-
derived Cry insecticidal proteins, and have proved very effective (De Cosa et al., 2001;
Scragg et al., 1988; Steward et al., 1999). Transplastomic plants expressing Cry2Aa2 could
kill insects which were tolerant to insecticidal proteins at concentrations 40,000 times higher
than normal (Zhong et al., 1994).
49
Despite being otherwise nutritionally rich, grain legumes contain limited quantities of the
sulphur-containing amino acids, methionine and cysteine. As well as reducing sulphur
deficiency in humans, methionine and cysteine-enriched legumes as ruminant feed may
increase wool, milk and beef yields in sheep and cows (Higgins et al., 1989; Onodera, 1993;
Pickering and Reis, 1993; Rogers et al., 1979). Attempts to upregulate metabolism of
sulphur-containing amino acids and clone high-sulphur storage proteins have enhanced the
nutritional profile of some species, but improvements have been modest (Sabir et al., 2014).
For example, nuclear expression of sunflower seed albumin in T. subterraneum was 0.3%
TSP (Rafiqul et al., 1996), and 0.2% TSP in Festuca arundinacea Schreb. (Wang et al.,
2001). The demonstrated hyperaccumulation of foreign proteins in chloroplasts may mean
that transplastomic plants may be key in enhancing human and animal diets (Ruhlman and
Daniell, 2007).
Efforts to improve global crop productivity through enhancing photosynthetic efficiency are
underway, focusing on the naturally inefficient carbon-fixing enzyme, Rubisco. Although
little progress has been made, there is huge scope for engineering Rubisco (whose large
subunit is plastid-encoded) through transplastomic approaches (Whitney et al., 2011). A
recent development has been the replacement of tobacco Rubisco large subunit (plastid-
encoded) with the cyanobacterial version, which resulted in more efficient carbon fixation
(Lin et al., 2014).
1.2.4.2.6.2 Molecular farming of protein biopharmaceuticals
Transplastomic plants can provide an ideal platform for high-yield synthesis of
biopharmaceutical proteins such as antibodies, vaccines and anti-microbials (Bock, 2007). An
exciting prospect is the development of whole plant ‘edible vaccines’ that require little or no
downstream purification, no expensive refrigerated storage, and facilitate straightforward
needle-free delivery (Daniell et al., 2005; Mason et al., 2002). Edible vaccines do not require
a high degree of purity, so may be enriched or partially-purified, or simply delivered as a
tissue homogenate without any purification (Walmsley and Arntzen, 2003). Vaccination with
tissue homogenates confers bioencapsulation of the active antigen, which may provide some
protection against degradation or dilution in the digestive system (Gonzalez-Rabade et al.,
50
2011). Alternatively, enrichment strategies may be used, including acid precipitation and ion-
exchange chromatography followed by freeze-drying, which increases the concentration of
the recombinant antigen and removes >95% of potentially toxic polyphenols and alkaloids
(Gonzalez-Rabade et al., 2011). However, an enrichment scheme would remove the
bioencapsulation advantage. For orally administered whole plant vaccines, there is a need to
move away from transplastomic tobacco, which is unpalatable and contains potentially toxic
metabolites such as polyphenols and alkaloids, towards palatable food crops such as lettuce
or tomato (Gonzalez-Rabade et al., 2011). Subunit vaccines against HIV (Gonzalez-Rabade
et al., 2011), tetanus (Michoux et al., 2011; Tregoning et al., 2003), cholera (Daniell et al.,
2001a), anthrax (Kamarajugadda and Daniell, 2006; Koya et al., 2005), plague (Daniell et al.,
2005), Lyme disease (Michoux et al., 2013) and rotavirus (Birch-Machin et al., 2004) have
been successfully expressed in transplastomic plants.
1.2.4.2.6.3 Expression of fusion proteins with affinity tags
Affinity (biospecific) tags are widely used used in lab-scale protein purification for functional
proteomics and structural biology studies, and are becoming increasingly common in
bioprocessing (Waugh, 2005; Wood, 2014). Affinity tags allow selective capture of a fusion
protein, through binding of the tag polypeptide to a ligand, followed by elution and cleavage
of the tag from the target protein using enzymatic or chemical methods (Esposito and
Chatterjee, 2006; Frey and Gorlich, 2014; Waugh, 2005; Wood, 2014). Expression of fusion
tags is especially beneficial in transplastomic plant systems (Ahmad, 2012; Ahmad et al.,
2012a; Daniell et al., 2009; Wilken and Nikolov, 2012). Fusion tags can stabilise and protect
target proteins against proteolytic degradation in plastids and facilitate simplified affinity-
based purifications (Wilken and Nikolov, 2012). Insulin-like growth factor (IGF) fused to the
Z-domain of Staphylococcus aureus, expressed in tobacco chloroplasts, was purified in a
simple procedure using two ammonium sulphate precipitation steps and an affinity column
(Daniell et al., 2009), followed by chemically cleavage with hydroxylamine to release the Z-
domain. Transplatomic fusion protein, cholera toxin B–proinsulin localised as inclusion
bodies in tobacco leaves, and underwent in vitro solubilisation and refolding before
purification of the fusion protein by an IMAC-Ni packed-bed column (Boyhan and Daniell,
2011). Studies undertaken by the Nixon group involved transformation and expression of two
affinity tags glutathione-S-transferase (GST) and a His-tagged derivative of the maltose
51
binding protein (His6-MBP) in tobacco chloroplasts, followed by straightforward affinity-
based purification (Ahmad, 2012; Ahmad et al., 2012a). Elution of GST using a glutathione
resin resulted in 50% recovery when a high DTT concentration of 5 mM in the PEB-A
extraction buffer (Ahmad, 2012; Ahmad et al., 2012a). His6-MBP was purified in a two-step
procedure using immobilised nickel affinity chromatography to bind the His6-tag giving 95%
recovery, followed by an amylose column to bind MBP (Ahmad, 2012; Ahmad et al., 2012a).
1.3 Background to PhD Project
1.3.1 The PhD project in the context of previous studies
The Nixon Group, Imperial College London, has a special interest in biolistic chloroplast
transformation, for the overexpression of a wide variety of proteins. Previous studies have
included the transplastomic expression of the following proteins: GST and His6-MBP affinity
tags (Ahmad, 2012; Ahmad et al., 2012a); membrane proteins plastid terminal oxidase
(PTOX) and NADPH dehydrogenase from Chlamydomonas reinhardtii (Ahmad et al.,
2012b); G-protein coupled receptors (Ahmad, 2012); fragment C of tetanus toxin (TetC)
(Michoux et al., 2011; Tregoning et al., 2004; Tregoning et al., 2003); a variant of green
fluorescent protein (GFP+) (Michoux et al., 2011); a subunit vaccine antigen against tetanus,
and outer surface protein A (OspA) from Borrelia burgdorferi which can be used as a
vaccine antigen against Lyme disease (Michoux et al., 2013). The tobacco species Nicotiana
tabacum has been used as the model organism for all aforementioned studies.
The expression of TetC in chloroplasts is one of special significance as it represents the first
reported successful expression of a subunit vaccine antigen in plant chloroplasts (Tregoning
et al., 2003). This is an attempt to produce a potent vaccine against tetanus that could
potentially be administered mucosally (Tregoning et al., 2004). Initial proof-of-concept
studies demonstrated intrinsic yields of 10% and 25% total soluble protein (TSP) in soil-
grown plants, depending on whether plasmid constructs with AT-rich or GC-rich codons
were used, respectively (Tregoning et al., 2003). The regeneration of shoots from callus
tissue in temporary immersion bioreactors represented the next step in this programme, and
52
TetC yields of 8% total soluble protein were reported (Michoux et al., 2011). This was the
first report of in vitro culture of differentiated shoots used as a host platform for
transplastomic protein synthesis. Further studies, in collaboration with Prof. Heribert
Warzecha (Technische Universität Darmstadt, Germany), demonstrated the expression of
Lyme disease vaccine candidate, OspA (Michoux et al., 2013). Temporary immersion culture
has been demonstrated to result in high-yield synthesis of transplastomic proteins in a
contained and controlled manner. The studies outlined in this PhD thesis are in continuity
with these preliminary experiments. The studies outlined in this thesis continue in the
investigation of transplastomic overexpression of TetC, under a wider range of culture
treatments, as well as the expression of a number of other proteins of biotherapeutic
significance.
1.3.2 Aims of Study
The successful establishment of molecular farming as a route to inexpensive
biopharmaceuticals will require the effective integration of a plant genetic engineering with
plant propagation and conventional bioprocessing approaches (such as those associated with
established microbial and mammalian cell host platforms). In particular, this PhD dissertation
focusses on the convergence of transplastomics and in vitro micropropagation for the high-
yield biosynthesis of biopharmaceuticals. The studies described in this PhD dissertation are
based on the in vitro Nicotiana tabacum shoot regeneration from callus in temporary
immersion culture as a platform for the biosynthesis of biopharmaceutical proteins with
special emphasis on transplastomic (plastid-encoded) vaccine antigens. Chapter 3 investigates
various in vitro culture conditions and the influence on biomass growth and transplastomic
TetC protein expression, and relates the synthesis of transplastomic proteins to the maturation
of chloroplasts and increase in chloroplast number during the in vitro morphogenesis process.
Chapter 4 investigates the nuclear expression and assembly of fully-functional monoclonal
antibodies via temporary immersion shoot regeneration, and discusses the implications of in
vitro differentiated shoots for industrial production of complex proteins such as antibodies.
Chapter 5 focusses on a major bottleneck in transplastomic protein expression, the proteolytic
degradation of transplastomic proteins, within the context of in vitro temporary immersion
shoot regeneration, through investigation of two proteins particularly susceptible to
proteolysis, VP6 and p24. Chapter 6 will outline experiments entailing novel developments in
53
in vitro shoot production for molecular farming, scale-up of culture systems and germplasm
preservation, and the impact of biomass growth and TetC expression. The scale-up of
temporary immersion shoot regeneration from 0.5 l to 60 l in a mechanical bioreactor will be
described, which represents one of the first attempts of scaling-up in vitro organogenesis for
biopharmaceutical production. Finally, the feasibility of alginate encapsulation of callus
germplasm for medium-term preservation prior to temporary immersion regeneration was
investigated.
54
Chapter 2. Materials and Methods
2.1 Stock Solutions
2.1.1 Standard solutions and buffers
All buffers, solutions and media were prepared in reverse osmosis (RO) filtered water
(Neptune model L993162, Purite, UK). All chemicals were procured from Sigma-Aldrich
Chemicals (USA), Melford Laboratories (UK), Thermo Fisher Scientific (USA), or Merck
Chemicals (Germany), unless otherwise stated.
2.1.2 Antibodies
Primary and secondary antibodies used in immunoblotting and ELISA studies are listed in
Table 2.1. All antibodies are diluted in Tris-buffered saline (TBS) or phosphate-buffered
saline (PBS) to the required dilution. All secondary antibodies are horseradish-peroxidase
(HRP) conjugated.
55
Table 2.1 List of antibodies used
Primary
antibody
Epitope
Primary
antibody
working
dilution
Source
Secondary
antibody
Secondary
antibody
working
dilution
α-TetC
Fragment C of
tetanus antigen
(TetC)
1:3,000
Prof. N.
Fairweather,
Imperial College
London
anti-rabbit
IgG
1:10,000
α-p24
HIV-1 p24 (HIV
antigen)
1:500
D7320, Aalto
Bioreagents,
Dublin, Ireland
(donated by
Prof. J. Gray,
Cambridge
University)
sheep anti-
goat
monoclonal
antibody
(A9452,
Sigma)
1:1000
α-VP6 (rabbit
polyclonal anti-
rotavirus VP6
antiserum)
VP6 bovine
rotavirus antigen
1:3000
Prof. J. Gray,
Cambridge
University
anti-rabbit
IgG
1;10,000
Guy’s 13
monoclonal
antibody in
transgenic
tobacco lysates
(i.e. analyte is the
primary
antibody)
For indirect
ELISA,
Streptococcal
antigen SA I/II
(conformational
epitope)
N/A for
Western
blot; serial
10-fold
dilution
series from
120 µg/ml
or neat.
Tobacco lysates
from temporary
immersion
culture; lines
donated from
Prof. J. Ma,
SGUL.
Anti-mouse
IgG
1:1,000
56
2.2 Cultivation of transgenic Nicotiana tabacum
2.2.1 Nicotiana tabacum growth conditions
Nicotiana tabacum cv. Petit Havana was used in these studies, except in the monoclonal
antibody expression studies where N. tabacum cv. Xanthii was used. All in vitro
micropropagated plants including seedlings, calli, suspension cultures and regenerated
plantlets were grown in an indoor air-conditioned plant cultivation facility at 25ºC, under a 16
hour photoperiod, at an approximate light intensity of 45 – 120 µmol photons m-2 s-1. Soil-
grown seedlings used as positive or negative controls were grown in a research greenhouse
facility at 25 ºC / 20 ºC (day / night) under 16 hour photoperiod, irradiance of 120 µmol m-2 s-
1 and 40% humidity.
Media at all stages of in vitro micropropation were based on MS basal medium (Duchefa
Biochemie, Netherlands) supplemented with 3% (w/v) sucrose (30 g l-1) and set at pH 5.8
(although certain TI cultures deviated from this recipe). Solid cultures, such as seedling
germination and callus induction, were performed on MS medium supplemented with 8 g l-1
agar (Melford Laboratories Ltd, Suffolk, England) as a gelling agent. To exclude microbial
contamination, 500 mg l-1 spectinomycin and 1 ml l-1 Plant Preservative Mixture™ broad
spectrum biocide (PPM) (supplied by Plant Cell Technology, Washington DC, USA) were
added. PPM is a mixture of two isothiazolones, methylisothiazolone and
chloromethylisothiazolone, which have been found to be effective at eradicating surface and
endophytic bacteria and fungi in plant tissue cultures, while having minimal impact on
explant growth and morphogenesis of several species (Compton and Koch, 2001; George and
Tripepi, 2001; Miyazaki et al., 2010; Niedz, 1998; Niedz and Bausher, 2002). Sucrose and all
plant growth regulators were provided by Sigma (St. Louis, MO, USA). All media, culture
vessels and other materials were sterilised by autoclaving at 120 ºC (103 kPa) for 20 minutes.
57
2.2.2 In vitro micropropagation of transgenic N. tabacum
2.2.2.1 In vitro germination of sterile seedlings
Seeds from stable transformant lines were obtained from the Nixon group or from
collaborators. Seeds were germinated in vitro in sterile Magenta vessels on semi-solid MS
medium containing 8 g l-1 agar (Melford Laboratories Ltd., Suffolk, England) gelling agent.
Prior to sowing, seeds were surface sterilised in mild bleach (50% v/v) with 0.1% (v/v)
Tween-20 for 15 minutes, and washed in 3 times for 5 minutes in autoclaved dH2O.
Seedlings were grown for 3 – 4 weeks to provide donor material for generation of callus
germplasm.
2.2.2.2 Callus induction and proliferation, and suspension cultures
For callus initiation, proliferation and shaken suspension, ‘callus induction’ medium (CIM)
was used (4.4 g l-1 MS basal medium, 3% (w/v) sucrose, 1 mg l-1 1-napthaleneacetic acid
(NAA), 0.1 mg l-1 kinetin, pH 5.8). Sections of vascularised leaves from sown plantlets were
cut with a sterile scalpel and plated on culture plates, containing semi-solid CIM (with 8 g l-1
agar), abaxial side down. After 2 – 4 weeks, undifferentiated primary callus, induced mainly
at the cut edges of leaf sections, was plated to new solid CIM for further growth for 2 – 3
weeks. In fresh medium, isolated from the original functional leaf tissue, callus tissue rapidly
proliferates. This may be repeated 2 – 3 times if necessary.
Further liquid callus suspension cultures were undertaken to generate fine uniform callus
aggregates as suitable inocula for temporary immersion culture. Erlenmeyer flasks were
loaded with 200 ml of CIM and small pieces of friable callus from the second or third callus
subculture. Erlenmeyer flasks were orbital shaken at 140 rpm for a period of 2 – 3 weeks to
generate inocula for regeneration. Suspension cultures may be subcultured to fresh CIM 2 – 3
times. Replenishment of media and dilution of waste products extend the duration of the
‘exponential phase’ and allow generation of large quantities of fine aggregates.
58
2.2.2.3 Temporary immersion regeneration of shoots from callus
Plantlet regeneration from undifferentiated callus was undertaken in temporary immersion
(TI) cultures in RITA® two-compartment bioreactors (Vitropic, France), with a biomass
chamber volume of 0.5 l. Air for immersion of cultures is delivered using a vacuum pump
and manifold via silicone tubing. Periodic pneumatic immersion is performed with
application of air over-pressure via an air-pump and solenoid valve medium delivery from a
lower compartment containing medium to an upper biomass chamber. Air bubbled into the
plant chamber during immersion also provides gentle agitation and renews the headspace
atmosphere (Etienne and Berthouly, 2002), providing high oxygen concentrations (Roels et
al., 2006). An immersion duration and frequency of 4 mins every 8 hours was used.
Using a sterile scalpel, 0.5g of fine callus aggregates from suspension culture and 300 ml of
‘plantlet regeneration’ medium were loaded into each RITA® bioreactor. The composition of
‘plantlet regeneration’ medium is 4.4 gl-1 MS basal medium, 3% sucrose and 0.1 µM
thidiazuron (TDZ) at pH 5.8. The duration of temporary immersion culture was 40 days,
during which the callus undergoes organogenesis and vegetative biomass accumulation.
2.2.2.4 Temporary immersion organogenesis from encapsulated calli (modified
procedure)
A variation of the above temporary immersion regeneration procedure was employed using
callus inoculum encapsulated in an alginate matrix. For generation of the calcium alginate
matrix, 3% (w/v) low-viscosity sodium alginate (Sigma, St. Louis, MO) was used as the
gelling matrix and 100 mM CaCl2 (Sigma, St. Louis, MO) as the complexing agent (both
autoclaved solutions) (Hung and Trueman, 2012; Naik and Chand, 2006; Rai et al., 2008;
Singh et al., 2010; Singh et al., 2009). Callus suspensions were filtered using the Corning®
filter system. Using a sterile scalpel, 0.5g of calli were incubated for at least 10 minutes in
sterile falcon tubes containing 25 ml of plantlet regeneration medium supplemented with 3%
sodium alginate (per bioreactor). The solution was carefully decanted. The calli were then
transferred to sterile falcon tubes containing 25 ml 100 mM CaCl2, and incubated for 30
minutes to allow the complexation reaction to occur. The alginate matrix is hardened through
ion exchange between Ca2+ and Na+. The CaCl2 solution was then discarded and the
59
encapsulated calli were washed twice in autoclaved water. The calli encapsulated in the
alginate matrix was then dried for 3 hours by air flow in a laminar flow hood. 0.5 g of
encapsulated callus inoculum was loaded into each RITA bioreactor, and allowed to undergo
organogenesis for 40 days, as per the standard procedure.
2.2.2.5 Regeneration of shoots in a large-scale hydraulic bioreactor
2.2.2.5.1 Construction of large-scale bioreactor
We invented, constructed and operated a prototype large-scale ‘box-in-a-bag’ mechanical
temporary immersion bioreactor for the regeneration of N. tabacum shoots. This consisted of
a biomass-containing chamber within a sealed, sterile plastic bag containing liquid plantlet
regeneration medium. Nutrient delivery to the biomass worked according to the principle of
periodic contacting of biomass with liquid medium through deformation of the bag, which
was achieved through vertical displacement of the box-bag assembly by a hydraulic car jack.
The biomass chamber consisted of a 60 l polypropylene box. Large holes were drilled in the
base and a stainless steel 0.5 mm wire mesh placed on the inner surface of the base to allow
flooding and drainage of medium. The chamber was placed in a 200 l sterile BIOEAZE®
Polyethylene (PE) Bag (Sigma, Aldrich, St. Louis, MO, USA), which was heat-sealed to
close any openings and maintain sterility. Sterile air filters were placed at the inoculation
ports of the BIOEAZE® bag. The box-bag assembly was sterilised by gamma irradiation
(Synergy Health, Swindon, UK). The displacement mechanism was provided by an
Automatic Jack ™ mechanical car jack (product no. 86025, Universal Power Group, Texas,
USA), connected to a mains operated universal digital timer (Tempatron, Maldon, UK). The
mechanical jack was modified to lift to a maximum of 12 cm. The box-bag assembly was
placed upon the car jack piston to facilitate the up / down vertical displacement, and housed
in a larger 145 l box for support and stability.
2.2.2.5.2 Operation of large-scale bioreactor
16 l of plantlet regeneration medium was loaded into the BIOEAZE® Polyethylene (PE) Bag,
followed by inoculation of 60 g callus suspension. Medium and inoculum addition was
60
undertaken via an autoclaved funnel and inoculation port of the bag placed in a laminar flow
hood (this was a compromise as it is physically impossible to place the entire box-bag
assembly in the hood). Shoot regeneration culture was undertaken for 50 – 80 days. The
biomass media immersion mechanism occurred as follows. In between immersions, the
mechanical jack and biomass chamber is raised in the default ‘up’ position. The liquid
medium settles under gravity in the ‘bulge’ beneath the biomass chamber. During
immersions, the mechanical jack piston descends to the ‘down’ position, so that biomass
chamber and bag are level. The deformation of the bag and the disappearance of the ‘budge’
cause the medium to be displaced so that it contacts the biomass and nutrient delivery can
take place. When the immersion cycle has ended, the jack piston and biomass chamber are
once more raised to their default position and the liquid medium drains away from the
chamber, settling at the bottom of the bag. Similar to the RITA temporary immersion system,
in this system, the callus inoculum undergoes organogenesis and vegetative biomass
accumulation.
Figure 2.1 Operation of large-scale mechanical bioreactor
(A) When the mechanical jack piston is in the default ‘up’ position, the biomass chamber and bag
assembly are raised and the medium settles in the bulges beneath the biomass chamber.
(B) The piston is lowered for nutrient delivery. The displacement of liquid medium and the space
constraint causes the biomass to be immersed.
2.2.2.6 Harvest of regenerated shoots, and fresh and dry weight determination
At the end of the allotted culture period, plantlet biomass was harvested and weighed to
determine fresh weight. Small representative samples of shoot tissue were excised and frozen
at -80°C for further analysis. Additionally, large healthy leaves were excised for chlorophyll
fluorescence analysis. After fresh weight determination, plantlets were dried in an oven at
A B
61
80ºC for 24 hours, for total moisture removal from tissues. Dried biomass was then allowed
to cool to room temperature and weighed. The ratio of dry-to-wet weight was taken, and the
dry weight is calculated.
2.3 Protein Analysis
2.3.1 Total Soluble Protein Extraction
Total soluble protein (TSP) extractions were undertaken from frozen regenerated plant
biomass (healthy leaves, vitrified leaves, undifferentiated shoot primordia and representative
samples of total biomass) harvested from temporary immersion culture. Frozen plant extracts
were ground with a pestle and mortar in liquid nitrogen. The resulting fine powder was mixed
with protein extraction buffer (PEB-A) (50 mM HEPES-KOH (pH 7.5), 2 mM DTT, 1 mM
EDTA, 10 mM potassium acetate, 5 mM magnesium acetate, and 1 tablet of cOmplete Mini
protease inhibitors EDTA-free cocktail (Roche Applied Sciences, Germany) per 5 ml buffer)
in a ratio of approximately 100 mg biomass to 100 µl. Samples were vortex mixed for 1
minute. Samples were centrifuged at 18,000 g for 30 minutes. The supernatants were
collected and the pellets were discarded. The supernatants were centrifuged again to remove
any residual pellets. Lysate extracts containing total soluble protein (TSP) were subjected to
SDS-PAGE as described in 2.3.3.
2.3.2 Determinination of protein concentration
The concentration of proteins in the lysates was determined using the Bradford Assay (Sigma
Aldrich, St. Louis, MO, USA), according to the manufacturer’s instructions (Bradford, 1976).
A dilution series of known concentrations of bovine serum albumin (BSA) were used as a
standard. The absorbance at 595 nm was plotted against the concentration to compute the
extinction coefficient against which the protein concentrations of the samples were
calculated.
62
2.3.3 SDS-PAGE
Soluble proteins were separated by sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) using the Bio-Rad mini-gel electrophoresis system (Bio Rad
Laboratories, USA). 15% (w/v) gels were made. Resolving gel recipe (6 mini-gels): 9.38 ml
40% acrylamide; 5 ml 3 M Tris-HCl buffer (pH8.9); 300 µl 10% SDS; 6 M urea; dH2O added
to 25 ml; 0.01% (w/v) N,N,N,N-tetramethylenediamine (TEMED); 0.1% (w/v) ammonium
persulphate (APS). 5% stacking gel recipe (6 mini-gels): 1.25ml 40% acrylamide; 1.2 ml 1 M
Tris-HCl (pH 6.8), 100 µl 10% SDS; H2O added to 10 ml; 0.01% (w/v) TEMED; 0.1% (w/v)
APS. Prior to loading onto gels, TSP samples were solubilised in 4 × solubilisation buffer
(250 mM Tris-HCl (pH 6.9), 8% (w/v) SDS, 40% (w/v) glycerol, 0.1% (w/v) bromophenol
blue and 10% (w/v) β-mercaptoethanol (added freshly for each use)) for 5 mins at 100 °C. 8–
12 µg of protein were loaded into each lane. Gels were run at room temperature at 130 V for
2–3 hours in SDS-PAGE running buffer (25 mM Tris-HCl (pH 8.3), 190 mM glycine, 0.1%
(w/v) SDS). Precision Plus Protein™ Dual Color Standards (Bio Rad Laboratories, USA)
were used to determine molecular weights of migrated protein bands. Mini-gels were run as
duplicates, with one mini-gel stained and the other for immunoblot transfer.
2.3.4 Staining of polyacrylamide gels
Gels were stained for 3 – 5 hours with Coomassie blue (0.2% (w/v) Coomassie Brilliant Blue
‘R-250’, 40% (v/v) ethanol and 10% (v/v) acetic acid) or SYPRO® Orange Protein Gel Stain
(Life Technologies) (5,000 x dilution of concentrate in 10% (w/v) acetic acid). Prior to
visualisation, Coomassie-stained gels were de-stained overnight or SYPRO® Orange-stained
were de-stained for 5 minutes, in both cases with 10% (v/v) acetic acid. Visualisation of total
protein staining was undertaken using a standard gel imager for Coomassie or LAS-3000
CCD digital imaging system (FujiFilm, USA) for SYPRO® Orange, according to the
manufacturer’s instructions.
2.3.5 Immunoblotting and Enhanced Chemiluminescence (ECL) Detection
At all stages after protein electrotransfer, all buffers used, including blocking, incubation and
washing are based on TBS-Tween (2.42 g l-1 Tris-base, 80 g l-1 NaCl, pH 7.6, 0.1% (v/v)
63
Tween-20) unless otherwise stated. Following electrophoresis, ‘wet’ transfer of proteins to a
0.2 µm nitrocellulose membrane was conducted using the Bio-Rad Mini Trans-Blot®
Electrophoretic Transfer Cell (Bio-Rad Laboratories, USA) at 60 V for 1 hour, according to
the manufacturers’ instructions, using transfer buffer (0.84 g l-1 NaHCO3, 0.30 g l-1 Na2CO3,
20% (v/v) methanol) (Towbin et al., 1979). The membranes were blocked in 5% (w/v) dried
skimmed milk (Marvel brand) for 1 hour. Alternatively, proteins were transferred to a
polyvinylidene fluoride (PVDF) membrane using the iBlot® Dry Blotting System
(Invitrogen, USA). The membranes were then incubated in primary antibody overnight at 4
ºC or for 1 hour at room temperature, washed 3 × 20 minutes, and subsequently incubated
with secondary antibody conjugated with horseradish peroxidase (HRP) for 1 hour at room
temperature. The membranes were washed 3 × 10 minutes in TBS-Tween, followed by a 10
minute final wash in TBS without Tween.
Enhanced Chemiluminescence (ECL) was undertaken using the Enhanced
Chemiluminescence (ECL) Detection kit (Amersham Pharmacia, UK), according to the
manufacturer’s instructions. Chemiluminescent detection was conducted using a LAS-3000
CCD digital imaging system (FujiFilm, USA), according to manufacturer’s instructions.
Alternatively, chemiluminescent detection may be undertaken on X-ray film (Kodak, USA)
developed in a Curix60 table top processor (AGFA Healthcare, Belgium), according to the
manufacturers’ instructions.
For semi-quantitative analysis of target proteins, densitometry was conducted on exposures
(digital or film) that were not overexposures, using ImageJ software (National Institutes of
Health, USA). Normalisation to stained total protein was used as a loading control (Aldridge
et al., 2008).
2.3.6 Indirect ELISA to assess functional activity of plant-expressed monoclonal
antibodies
Costar® 3603 96-well polystyrene plates (Corning) were used as the solid phase in all
instances. Phosphate buffer saline (PBS) (7.5 mM Na2HPO4, 2.5 mM NaH2PO4, 150 mM
NaCl, pH 7.4) was used as a universal buffer at all stages, including coating, blocking,
64
antibody incubations and washing, unless otherwise stated. TSP lysates containing expressed
antibodies were prepared according to the procedure described in 2.3.1 and total protein
concentrations were determined, as described in 2.3.2. Serial dilutions of lysates were made
in PBS. The antigen, diluted 1/1000 to approximately 5 µg ml-1, was immobilised (‘coated’)
to the bottom of the wells by passive adsorption, for 2 hours at room temperature, or 4ºC
overnight. After washing twice with distilled water, the wells were blocked with 5% (w/v)
dried skimmed milk (Marvel brand) for 3 hours at room temperature or overnight at 4ºC,
followed by 2 × washes with distilled water with 0.1% (v/v) Tween-20. Wells were then
incubated in plant TSP lysates as serial 10-fold or 2-fold dilutions (‘log10’ or ‘log2’ dilutions
respectively) for 2 hours at room temperature, to facilitate binding of the immobilised antigen
to antibodies expressed in the lysates. After washing 5 times with distilled water and 0.1%
Tween-20, incubation of horseradish peroxidase (HRP) conjugated secondary antibody
(‘conjugate’) with 5% milk was undertaken for 2 hours to allow binding to the primary
antibody. Detection was undertaken by adding equal volumes of 3,3′,5,5′-
Tetramethylbenzidine (TMB) solution (Merck-Millipore, USA) and stop solution (1 N
H2SO4), and reading absorbances at 450 nm, according to the manufacturers’ instructions.
2.4 Analysis of in vitro regenerated plant biomass
2.4.1 Viability assay of in vitro regenerated shoots
This viability assay is based on the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) to
insoluble red triphenylformazan (TF) by mitochondrial dehydrogenase activity, as an
indicator of plant cell viability (Clemensson-Lindell and Persson, 1995). 400 mg of fresh
biomass were incubated in 7.5 ml of 0.1 M sodium phosphate buffer (pH 7.0) with 0.6%
(w/v) TTC and 0.05% Tween-20, for 20 hours at 30°C in darkness (Ruf and Brunner, 2003).
After incubation, the formazan was extracted by decanting the buffer, grinding biomass in
liquid nitrogen, transferring the powder to eppendorf tubes, addition of 1.7ml 96% ethanol,
and centrifugation at 10,000g for 2 minutes. The supernatants containing TF were collected
and the absorbance at 520 nm was determined.
65
2.4.2 Chlorophyll Fluorometry
Chlorophyll fluorescence measurements were undertaken using a pulse-modulated
fluorometer (DUAL PAM 2000, Walz Germany) with DUAL-PAM-100 measuring system
and DUAL-PAM v1.11 software. Recordings of photosystem II (PSII) parameters such as Fm
(maximum fluorescence), Y(II) (effective PSII quantum yield), Fv/Fm (maximum PSII
quantum yield) were taken (Maxwell and Johnson, 2000).
Medium or large healthy TIB-regenerated leaves were used for measurements. Leaves were
dark-adapted for at least 15 minutes beforehand. Fluorometry was undertaken at room
temperature. The minimal fluorescence (F0) and maximum fluorescence (Fm) were taken
using a saturation pulse of 6000 µmol photons m-2 s-1 for 0.6 seconds in dark-adapted leaves.
66
Chapter 3. Parameters affecting the dynamics of
biomass growth and transplastomic protein accumulation
in temporary immersion culture
3.1 Introduction
3.1.1 In vitro differentiated plant tissues for molecular farming
With the advent of plant-made biopharmaceuticals, there is a need to establish robust
bioprocessing and regulatory strategies (Spök et al., 2008b). Within the bioproduction
process, the ‘upstream’ steps involving cultivation and harvesting require the most innovation
and development, in order to exploit the distinctive features of the plant host system (Colgan
et al., 2010). In contrast, for the later downstream processing (DSP) steps involving crude
extraction and further purification, much of the established approaches from existing
microbial and mammalian cell-based platforms can be used as they will not differ greatly
from plant-based bioprocesses (Fischer et al., 2012). There is increasing interest in the in
vitro regeneration and differentiation of shoots as the heart of this suite of upstream
bioprocessing technologies for large-scale biosynthesis of biopharmaceutical proteins (Doran,
2000; Steingroewer et al., 2013). Fortunately, decades of expertise from the commercial
micropropagation industries can be applied to new molecular farming applications. There are
several challenges that the in vitro plant regeneration paradigm must face, if it is to compete
with established recombinant host systems. Existing bacterial, fungal and mammalian cell
biotechnologies are built on decades of bioprocess innovation and infrastructure, which have
resulted in fairly in-depth biological and process characterisation of fermentation culture
systems (Shuler and Kargi, 2002). Moreover, conventional bacterial and yeast hosts are
unicellular and undergo little significant developmental change during bioprocessing,
whereas plant morphogenic growth involves a greater degree of complexity which
profoundly impacts product yield. This is especially true with cell and tissue culture in which
culture parameters can be evaluated in a systematic way and ‘tuned’ in order to optimise
heterologous protein yield (Doran, 2000). The studies outlined in this chapter describe the
67
influence of various temporary immersion culture parameters on the growth and
morphogenesis of Nicotiana tabacum callus tissue and TetC biosynthesis.
3.1.2 Nt-pJST12 as a rational model host system for investigating the impact of the
culture environment on recombinant protein expression
Clostridium tetani, a bacterium widespread in nature, produces tetanus toxin (also known as
tetanospasmin, TeNT or TeTx), an extremely potent neurotoxin, which is responsible for the
clinical syndrome of tetanus. C. tetani spores germinate and synthesise TeNT under
conditions of low oxygen tension, slight acidity and nutrient availability such as wounds and
skin ruptures (Popoff, 1995). Tetanus usually occurs after an acute injury, and is
characterised by prolonged contraction of skeletal muscle fibres and over-activity of the
autonomic nervous system, which causes spasms and vasoconstriction, leading to a rise in
blood pressure (Tregoning et al., 2004). Tetanus toxin may constitute more than 5% of the
total mass of the organism. It is a single polypeptide of approximately 150 kDa and 1315
amino acid residues, which forms a two-chain activated molecule composed of a heavy chain
(Hc) and light chain (Lc) linked by a disulphide bond (Calvo et al., 2012). Parenteral
immunisation has been very effective at preventing tetanus, by causing production of anti-
tetanus toxin antibodies, thus blocking its action. Conventionally, vaccine preparations
against tetanus are produced by formaldehyde detoxification of tetanus toxoid, although this
is a time-consuming and intensive process (Anderson et al., 1996) and causes a reduction in
antigenicity (Metz et al., 2013). Tetanus vaccine antigens have been routinely administered as
a component of the trivalent diphtheria–tetanus–pertussis (DTP) vaccine since the 1940s
(Bernstein et al., 1995; Brodzik et al., 2009; Orenstein et al., 1990). Fragment C, also known
as TetC, is a non-toxic 47 kDa cleavage product of tetanus toxin that can be used as a subunit
vaccine against tetanus (Makoff et al., 1989; Tregoning et al., 2003). TetC has been
successfully expressed in chloroplasts, in the stable N. tabacum cv. Petit Havana line Nt-
pJST12, generated by the Nixon group, Imperial College London (Tregoning et al., 2003).
This transplastomic line was used in all the studies described in this chapter.
68
3.1.3 Aims of studies
Preliminary studies undertaken by the Nixon group into the feasibility of in vitro shoot
morphogenesis in RITA® temporary immersion bioreactors (TIBs) for high-level and
contained expression of TetC have proved effective (Michoux et al., 2013). Moreover the
yield of TetC can be modulated by changing various culture conditions. The aim of these
studies, using TetC expression in Nt-pJST12 as a model system, is to:
gain understanding of the in vitro morphogenesis process in TIBs, and the
implications on transplastomic protein yields;
investigate the influence of various culture conditions on biomass growth and
transplastomic protein yields.
Nt-pJST12 shoot biomass was regenerated from callus tissue under a range of different
conditions in RITA® temporary immersion bioreactors, through changing the composition of
the standard culture media or the culture protocol.
3.2 In vitro shoot regeneration via organogenesis of N. tabacum callus
and the influence on transplastomic protein expression
3.2.1 Callus organogenesis as a developmental pathway for in vitro plantlet
regeneration
As a biotechnological host, plants are unique, in terms of growth and development.
Organogenesis is a defining feature of plants, owing to their multicellularity, while being
absent in alternative bacterial and yeast expression systems. Although organogenesis is a
feature of animals, the developmental strategies of plants are entirely different. Most land
plants (embryophytes) undergo indeterminate growth as a mode of irreversible volume
increase, involving the continuous formation of new tissues and organs as a result of
perpetual meristemic activity throughout their lifetime (Kutschera and Niklas, 2013). In
contrast, animals undergo determinate growth, in which body size and body size and organ
number are predetermined, and organogenesis only occurs during embryogenesis (Woodward
69
et al., 2006). In plant tissue culture, the indeterminate mode of growth is closely related to
totipotency, the capacity to regenerate organs and even whole plants from differentiated cells
(George et al., 2007). From a biotechnological perspective, indeterminate growth of plants in
organogenic tissue culture from callus tissue is an advantageous feature, as it facilitates
continual formation and proliferation of shoot tissue for recombinant protein expression.
3.2.2 In vitro morphogenesis dynamics during temporary immersion culture
3.2.2.1 Design of experiment
In order to determine growth kinetic parameters during temporary immersion organogenesis,
several temporary immersion cultures in RITA® bioreactors were undertaken in parallel,
using the same callus suspension inoculum in order to minimise batch-to-batch variation.
Temporary immersion cultures were allowed to grow for an allotted time interval, and then
the biomass was harvested for fresh and dry weight determination and total soluble protein
(TSP) extraction.
3.2.2.2 Results and Discussion
3.2.2.2.1 Dynamics of biomass growth and morphogenesis
Callus tissue is a disorganised, apparently undifferentiated cell mass, which in nature is
generated as a stress response to wounding (Stobbe et al., 2002). The totipotency of callus
tissue is widely exploited in tissue culture, and callus formation is induced through
exogenous application of auxin and cytokinin (Ikeuchi et al., 2013). In vitro morphogenesis
involves the transition of callus to fully-differentiated shoot tissue. This is a complex
phenomenon, involving morphological, physiological and metabolic changes as a
consequence of reprogramming of gene expression and induction of the totipotent state of
somatic cells (Ovečka et al., 1997).
70
In vitro morphogenesis involves localisation of rapidly dividing cells, and aggregation of
friable calli, accompanied with an increase in metabolism (Fournier et al., 1991; George et
al., 2007). This localisation into clusters was observed to result in formation of meristemoids.
These are calluses in which there is a layer of proliferating shoot meristems overlaying an
inner core of vacuolated cells acting as a mechanical and nutritional support (George et al.,
2007). This observation is consistent with previous reports of a meristemoid stage in organ-
forming cultures of N. tabacum (Ovečka et al., 1997; Ross et al., 1973; Yoshikawa and
Furuya, 1983). The formation of meristemoids in temporary immersion culture involves the
transition of callus aggregates to compact clusters, and subsequently the formation of
primordia leading to shoot buds (Ovečka et al., 1997; Yoshikawa and Furuya, 1983).
Primordia represent an intermediate stage between undifferentiated calli and fully
differentiated shoots, having the appearance of nodular outgrowths that will develop into
shoot buds. In temporary immersion culture, the meristemoid developmental sequence, with
zones of preferential cell division and subsequent shoot primordia formation is not entirely
age dependent, but occurs continuously over several days, starting early in some calluses but
later in others. Localisation of cell division on the surface of callus clusters leading to
meristem formation was observed between days 6 and 17, with subsequent formation and
proliferation of shoot primordia occurring between days 12 and 25. This apparently continual
developmental phenomenon without clear-cut milestones is consistent with other reports
(Ross et al., 1973). In vitro morphogenesis is instigated by 0.1 µM thidiazuron (TDZ), a
cytokinin. TDZ is known to be effective at promoting shoot regeneration (Ivanova and van
Staden, 2008; Thomas and Katterman, 1986) and somatic embryogenesis (Gill and Saxena,
1993).
The kinetics of increase in biomass accumulation during a temporary immersion culture can
be modelled using a logistic growth model, similar to bacterial growth models (Figure 3.2).
There is a ‘lag’ phase between days 0 – 20, followed by an exponential increase in biomass
from approximately day 25 to 35. The increase in biomass growth slows down from
approximately day 40 to 100. In practice, TIB cultures are normally harvested at day 40. The
biomass growth curves are reflective of the morphological and physiological changes that
accompany the transition of callus aggregates to differentiated shoots that were observed
during morphogenesis.
71
Figure 3.1 Callus-meristemoid transition and shoot bud formation during morphogenesis. (A)
Callus aggregates on day 0 (inoculum from callus suspension culture) (B) Callus aggregates on day 6.
(C) Primordia formation at meristems in callus clusters on day 17. (D) Shoot bud formation in
meristemoids on day 25. (E) Leaf and shoot formation on day 30.
(F) Fully-differentiated shoot clusters on day 40.
72
Figure 3.2 Logistic increase of fresh and dry biomass accumulation during in vitro
organogenesis in RITA® TIBs.
3.2.2.2.2 Differential expression of TetC during in vitro organogenesis
SDS-PAGE and immunoblot analysis was undertaken to demonstrate the differential
expression of TetC at different time intervals during the morphogenesis process in TIB
culture (Figure 3.3). A comparative analysis of TetC ‘absolute’ volumetric yield (milligrams
per litre of bioreactor) (estimated using densitometry of immunoblots) and fresh biomass
increase is shown in Figure 3.4. There is a clear increase in expression between days 0 and
25, closely following the transition of calli to shoots. The intrinsic yield of TetC (as a
proportion of total soluble protein) is 15-fold higher in shoot clusters than in undifferentiated
calli used as inoculum. A small decline in TetC expression is observed in older tissues, from
73
90 to 100 days. This is possibly due to senescence-related proteolysis in the chloroplasts.
TetC is exhibited as a doublet of two bands of size 43 kDa and 47kDa, consistent with
previous work undertaken by the Nixon group (Michoux et al., 2011; Tregoning et al., 2003).
The doublet may be attributed to partial degradation of TetC in the chloroplast or during total
soluble protein (TSP) extraction (Michoux et al., 2011; Tregoning et al., 2003).
The increase in intrinsic TetC yield during morphogenesis reflects the maturation of plastids,
involving the transition of proplastids to mature chloroplasts, accompanied by development
of photosynthetic apparatus and increased plastidial protein synthesis (Ladygin et al., 2008).
The number of plastids and nucleoids increase during in vitro cell and tissue culture. There is
a striking correlation between transplastomic protein expression and the increase in
chloroplast number and developmental status. This may be inferred by the increasing
abundance of chloroplast-encoded Rubisco large subunit, which as one of the most abundant
proteins in plants, is represented by a distinct band at approximately 50 kDa on a gel (Ma et
al., 2009). It has been previously observed that plastid copy number increases rapidly in
exponential phase N. tabacum cell cultures (Takeda et al., 1999), with the highest frequency
of chloroplast division occurring in the early exponential phase. Moreover, the intrinsic
features of the transplastomic transformation vector can greatly influence expression. Prrn,
the plastid 16S rRNA promoter included in most plastid transformation vectors is similar to
the 16S rRNA promoter in rice (Michoux et al., 2011) which has seven-fold lower activity in
rice embryogenic calli compared to leaves (Silhavy and Maliga, 1998). It was found that
plastid-encoded mRNA levels of embryogenic rice calli were much lower than in leaves, for
rbcL (153-fold lower), atpB (37-fold lower), 16SrDNA (7-fold lower) (Silhavy and Maliga,
1998).
74
Figure 3.3 SDS-PAGE and immunoblot showing differential expression of TetC. TetC
expression during (A) callus-to-shoot morphogenesis and (B) stationary phase. 12% acrylamide;
LWM marker (A); Precision Plus marker (B); 8µg protein loading per well; Coomassie staining.
A B
Figure 3.4 Increase in TetC volumetric yield and fresh biomass. Error bars
denote standard errors.
75
3.3 Impact of Hyperhydricity on Expression of TetC
3.3.1 The hyperhydricity phenomenon in in vitro micropropagation
Hyperhydricity (sometimes known as ‘vitrification’) is a common and problematic
phenomenon in micropropagative tissue culture, much to the chagrin of commercial
micropropagators. Ever since Debergh et al. (1981) first reported and defined the
phenomenon of ‘vitrification’, many comprehensive studies have been undertaken to attempt
to understand and reduce this phenomenon. Although a number of factors have been
implicated in the induction of hyperhydricity, it is not entirely predictable (Olmos et al.,
1997). Hyperhydricity encompasses a range of morphological disorders, characterised by a
thick, translucent, brittle stems and leaves (Kevers et al., 2004; Olmos and Hellın, 1998; van
den Dries et al., 2013). In temporary immersion culture, vitrified shoots are easily
distinguished by translucency of shoots and leaves, thick waterlogged leaves with little
deposition of epicuticular waxes (Figure 3.5). It is recognised that the hyperhydricity
phenomenon in in vitro culture is due, in part, to high humidity, the presence of liquid media,
low air exchange and sealed culture vessels (Kevers et al., 2004; Olmos and Hellın, 1998; van
den Dries et al., 2013). In the temporary immersion culture system, regular infiltration of
biomass with liquid medium contributes to hyperhydricity.
76
Figure 3.5 Visual comparison between non-vitrified and vitrified shoots
A, B, C: Non-vitrified biomass
D, E, F: Vitrified biomass
3.3.2 TetC accumulation in vitrified and non-vitrified leaves
Regenerated biomass harvested from a number of TIB cultures was separated into vitrified
and non-vitrified (healthy) samples and TSPs were extracted for SDS-PAGE and immunoblot
analysis. Hyperhydricity was found to have little notable influence on the expression of TetC,
at different stages of morphogenesis (Figure 3.6) or at different sucrose and light irradiances
(Figure 3.7). However, the immunoblots indicate that the intensity of the lower 44 kDa band
of the TetC doublet is lower in vitrified shoots compared to healthy shoots. As the expected
A B C
D E F
77
size of TetC is 47 kDa, this suggests that hyperhydricity may inadvertently lead to increased
TetC stability.
Figure 3.6 Investigation of hyperhydricity on TetC accumulation at different time intervals
of temporary immersion culture, by SDS-PAGE and immunoblot. (A) 28 days; (B) 33 days; (C)
40 days. 8-10 µg protein analysed by SDS-PAGE on 12% (w/v) polyacrylamide gel followed by
Coomassie or Sypro Orange staining.
A B C
78
Figure 3.7 Effect of hyperhydricity on TetC accumulation at various sucrose
concentrations and irradiances. 11 µg protein loading; 12% acrylamide; Sypro Orange staining;
Precision Plus marker.
3.3.3 Discussion on the influence of hyperhydricity on transplastomic protein
expression
These studies indicate that hyperhydricity conditions have little significant impact on
transplastomic protein expression (Figures 3.6 and 3.7). The reasons for this are unknown,
although this may be associated with the expression and localisation of the heterologous
proteins in the plastids. Hyperhydric waterlogging of plant tissues is a largely apoplastic
phenomenon (van den Dries et al., 2013) and although there is increased expression of stress-
associated genes, such as hypoxia-responsive genes (van den Dries et al., 2013) and
antioxidant enzymes (Balen et al., 2009; Dewir et al., 2006), these are mainly nuclear-
79
encoded. The transcriptional regulatory circuits which cause upregulated expression of
certain proteins under stress conditions mainly act on nuclear genes (Aarts and Fiers, 2003;
Ciarmiello et al., 2011), and not the plastid genome. Therefore incorporation of the transgenic
construct into the plastid genome may offer protection against the stress-related changes in
protein synthesis. The observation that hyperhydricity may contribute to increased stability of
transplastomically expressed proteins is unexpected and intriguing. The reasons behind this
observation are currently unknown, though worthy of future investigation.
3.4 The Impact of Tissue Culture Media on Morphogenesis and TetC
yield
3.4.1 The impact of ammonium : nitrate ratio on TetC yield
3.4.1.1 The nitrogen requirements of in vitro plant growth
Nitrogen (N) is an essential element for plant life. After carbon, N is required in largest
quantity by plants, being a constituent of proteins, DNA, RNA, chlorophylls, co-enzymes,
phytohormones and secondary metabolites (Hawkesford et al., 2012). Fertilisers and culture
media usually include inorganic nitrogen, in the form of nitrate (NO3-) or ammonium (NH4
+),
though organic sources such as glutamine or urea may be used. Standard Murashige & Skoog
(1 ×) media contains 60 mM inorganic nitrogen, a higher amount than other in vitro culture
media and more than 2/3 of the total mineral content (Murashige and Skoog, 1962), in a ratio
of 40 mM NO3- : 20 mM NH4
+. Despite the ubiquity of MS media in plant tissue culture, very
few studies have attempted to establish a physiological basis for this particular nitrogen pool
(George et al., 2007). However, the absolute and relative amounts of nitrogen have been
found to significantly influence growth and morphogenesis (George et al., 2007), in in vitro
tissue culture (Chaleff, 1982; Evans, 1993; Ivanova and Van Staden, 2009), cell suspensions
(Gorret et al., 2004; Holland et al., 2010; Vasilev et al., 2013) and seedling cultivation (Bar-
Yosef et al., 2009; Tan et al., 2000).
80
The aim of this set of studies is to investigate the influence of the inorganic nitrogen pool
composition on biomass growth, organogenesis and TetC expression in temporary immersion
culture. The first study involved assessing the change in media pH over the course of a 40-
day TI culture duration, and inferring the nitrogen assimilation kinetics from this. The second
study entailed investigation of biomass accumulation and transplastomic protein expression at
various NO3-: NH4
+ ratios, while keeping the total inorganic nitrogen content at 60 mM.
3.4.1.2 Design of experiment
The ratio of NO3-: NH4
+ was modulated, while keeping the total concentration of inorganic
nitrogen constant at 60 mM, as in standard MS media (Murashige and Skoog, 1962). MS
basal media including vitamins was made de novo, to the same composition as the
commercial version (M0222, Duchefa Biochemie) (http://www.duchefa-
biochemie.com/product/details/number/M0222), though without inorganic nitrogen. Nitrogen
was added later according to the required ratio of NO3-: NH4
+. TIB shoot regeneration
cultures were undertaken at seven ratios of NO3-: NH4
+ – 0:60, 10:50, 20:40, 30:30, 40:20,
50:10, and 60:0 (mM : mM). The ionic balance of the media was maintained using K+ and Cl-
as balancing ions when individual minerals were removed. Shoot regeneration cultures were
undertaken as duplicates for each ratio tested, in RITA® TIBs. After a 40-day culture period,
biomass was harvested and TSPs were extracted for SDS-PAGE and immunoblot analysis.
3.4.1.3 Results
3.4.1.3.1 Influence of nitrogen source ratio on growth and TetC expression
3.4.1.3.1.1 Influence of nitrogen source ratio on biomass accumulation and
morphogenesis
The biomass growth of N. tabacum was highly affected by the NO3-: NH4
+ ratio at an initial
total nitrogen content of 60 mM (Figure 3.9). There is a positive relationship between NO3-
content and fresh and dry biomass accumulation, and extent of morphogenesis (Figure 3.8).
No morphogenesis occurs at 0:60 and 10:50; no differentiated leaf tissue was observed for
81
these treatments. At all treatments between 20:40 and 60:0, fully-differentiated leaf and shoot
tissue is observed, with no visually notable differences between them. When NH4+ was used
as the sole nitrogen source, there was virtually no biomass accumulation, 16.8 g l-1 fresh
weight and 0.42 g l-1 dry weight. At 10:50, the biomass accumulation was 212 g l-1 fresh
weight and 8 g l-1 dry weight, but biomass consisted of undifferentiated callus clusters.
Interestingly, the callus biomass observed at 0:60 and 10:50 exhibited a deep orange colour,
indicating a lack of chlorophyll and presumably little photosynthetic capacity. The curve
plateaus at 20:40, with maximum fresh biomass accumulation of 291 g l-1 observed at this
ratio (Figure 3.9). Between 10:50 and 60:0, there was a marginal increase in dry weight, from
9 g l-1 to 11.6 g l-1.
It is obvious that in vitro regenerated tobacco has a preference for nitrate, compared to
ammonium, as a N source. Quantitatively, this preference may be determined on the basis of
total mean dry biomass generated in ammonium alone, as compared to nitrate alone. For the
sake of symmetry around ‘zero’ and for simple comparison between species or propagation
regimes, the preference ratio is log2-transformed as:
𝑙𝑜𝑔2𝑃𝑅 = 𝑙𝑜𝑔2 (𝐷𝑊𝑎𝑚𝑚𝑜𝑛𝑖𝑢𝑚
𝐷𝑊𝑛𝑖𝑡𝑟𝑎𝑡𝑒)
with log2PR denoting the log2 preference ratio (Bartelheimer and Poschlod, 2014). In this
study, the log2PR for in vitro regenerated tobacco was found to be -4.77, indicating a strong
preference for nitrate.
82
Figure 3.8 Effect of NO3-: NH4
+ ratio on developmental status of N. tabacum regenerated
shoots after 40-day temporary immersion culture. (A) 0:60; (B) 10:50; (C) 20:40; (D) 30:30; (E)
40:20; (F) 50:10; (G) 60:0; (H) proliferation of callus aggregates lacking chlorophyll under 10:60
treatment; (I) fully-differentiated leaves observed under 60:0 treatment.
83
Figure 3.9 Influence of NO3-: NH4
+ ratio on fresh and dry biomass accumulation. Error bars
represent standard errors.
3.4.1.3.1.2 Influence of nitrogen source ratio on yield of TetC
Immunoblot analysis was undertaken to assess the impact of NO3-: NH4
+ ratio on TetC
expression after a 40-day culture period (Figure 3.10). Hyperexpression of TetC is observed
in shoots having undergone morphogenesis, at NO3-: NH4
+ ratios between 20:40 and 60:0. In
comparison, very low expression is observed in undifferentiated callus at NO3-: NH4
+ ratios
0:60 and 10:50 (high ammonium treatments). Densitometric analysis was undertaken to
quantify TetC expression and estimate volumetric yields (Figure 3.11). The nitrate-related
shoot regeneration resulted in TetC intrinsic yields of 7–9 ng µg-1, compared to 2 ng µg-1 in
undifferentiated biomass, an approximate 4-fold increase. In absolute terms, the estimated
volumetric TetC yield is 2–2.5 mg l-1 in regenerated tissues, compared to approximately 100
µg l-1 in undifferentiated callus when NH4+ is the sole nitrogen source, a 20 – 25 fold
increase.
84
Figure 3.10 SDS-PAGE and immunoblot analysis of lysates to assess TetC expression
under various NO3-: NH4
+ ratios. 12% acrylamide gel; 7 µg protein loading; Sypro Orange staining;
Precision Plus ladder.
Figure 3.11 Densitometric quantification of TetC intrinsic yields and volumetric yields
under various NO3-: NH4
+ ratios, from immunoblot data in Figure 3.10. Error bars represent
standard errors.
85
3.4.1.3.2 pH shift of media during temporary immersion culture
The preference of N. tabacum biomass for nitrate is further validated by the observed shift in
pH during temporary immersion regeneration culture, shown in Figure 3.12. 1 ml samples of
media were withdrawn from duplicate TIB cultures every 2-3 days and the mean pH was
determined. It was observed that during temporary immersion regeneration, the pH of the
media steadily rises from approximately pH 5.5 (post-autoclave pH) to 7.0. Gradual media
alkalinisation during liquid culture is a well-documented phenomenon in plant suspension
culture (George et al., 2007; McDonald and Jackman, 1989; Srinivasan et al., 1995). The
nitrogen assimilation dynamics can be inferred, as an increase in pH is related to uptake of
nitrates. Uptake of nitrate takes place effectively in an acid pH, but is accompanied by
extrusion of anions, leading to the medium gradually becoming more alkaline. In contrast,
uptake of ammonium results in the cells excreting protons into the medium, making it more
acidic (George et al., 2007). This study indicates that in vitro culture for N. tabacum callus
organogenesis has a preference for nitrate as a nitrogen source, highlighted by the gradual pH
rise of the media. It is widely reported that for suspension cultures there is an initial sharp
uptake of NH4+ characterised by a sharp fall in pH to approx. 4.2-4.6 (George et al., 2007),
followed by a gradual alkalinisation during which NO3- absorption is stimulated. Although
there is a small decrease in pH to 5.4, this is not as pronounced as in other studies (Gorret et
al., 2004; McDonald and Jackman, 1989; Srinivasan et al., 1995), indicating a very weak
preference for NH4+.
Figure 3.12 Shift in medium pH over temporary immersion culture period. Error bars denote
standard errors.
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
0 2 4 7 9 11 14 16 18 21 23 25 28 30 32 35 37
pH
Time (days)
86
3.4.1.4 Discussion on the influence of nitrogen pool on in vitro regeneration and
transplastomic protein expression
These studies have demonstrated the preference for NO3- over NH4
+ for in vitro N. tabacum
shoot differentiation, organogenesis and transplastomic protein expression. It was found that
NH4+ as a sole nitrogen source is ineffective for growth, whereas NO3
- alone was sufficient to
induce morphogenesis. Although a NO3-:NH4
+ ratio of 10:50 resulted in high biomass
accumulation, no differentiation and functionalization of leaves occurred. High biomass
growth and shoot regeneration were observed for every NO3-:NH4
+ ratio tested between 20:40
and 60:0.
The superiority of nitrate over ammonium for promoting growth and protein expression is
consistent with previous several previous studies in plant tissue culture. Successful shoot
formation and proliferation has been reported when NO3- was the only nitrogen source
(Cousson and Van, 1993; Ivanova and Van Staden, 2009; Nagakubo et al., 1993; Ramage and
Williams, 2002; Tsai and Saunders, 1999; Woodward et al., 2006). Our study demonstrates
high shoot regeneration when a combination of NO3- and NH4
+ are used, indicating a
synergistic effect between the two ions (Gamborg, 1970; Ivanova and Van Staden, 2009;
Ramage and Williams, 2002; Shirdel et al., 2011). The inclusion of NH4+ mitigates the
alkalinisation of the media, and thus may contribute towards enhanced NO3- assimilation
(Shirdel et al., 2011). The highest regeneration of Aloe polyphylla shoots occurred with
NH4+:NO3
- (mM) of 20:40, 30:30 and 40:20 (Ivanova and Van Staden, 2009) and greatest
shoot formation of tobacco leaf discs was reported when media containing 40mM NO3-
:20mM NH4+ was used (Ramage and Williams, 2002). Nitrogen assimilation in plants occurs
through progressive reduction of NO3- to NO2
- (nitrite) by nitrate reductase and then to NH4+
by nitrite reductase, followed by ammonium assimiliation into amino acids (Masclaux-
Daubresse et al., 2010; Xu et al., 2012a). Despite this, ammonium as the sole nitrogen source
has been found to inhibit growth and shoot morphogenesis in a number of species (Castro-
Concha et al., 2006; Chaleff, 1982; Cousson and Van, 1993; Murthy et al., 1998; Ramage and
Williams, 2002; Richter et al., 2007; Walch-Liu et al., 2000). This is the result of reduced cell
division and elongation (Walch-Liu et al., 2000). Reduced growth potential caused by high
NH4+ levels is attributed to a number of possible factors including acidification of media and
87
corresponding growth inhibition, the toxic effects of intracellular free ammonia (NH3) which
is related to disturbances in pH homeostasis, uncoupling of photophosphorylation and a
reduction of photosynthesis (Ivanova and Van Staden, 2009; Walch-Liu et al., 2000). It has
also been suggested that at low pH, NH4+ inhibits uptake of essential inorganic elements or
causes leakage of nitrogenous metabolites (Murthy et al., 1998). However, assimilation of
NH4+ as the sole nitrogen source is possible in buffered media or at a pH close to neutrality
(to mitigate the influence of acidification), or in the presence of an organic acid citrate,
malate or pyruvate (George et al., 2007).
These studies demonstrate the associations between inorganic nitrogen source, chloroplast
maturation, developmental status of biomass and expression of transplastomic proteins. The
complex interactions between these factors may be explained when considering that nitrogen
assimilation in plants is developmentally regulated, and the chloroplasts play a major role in
this (Xu et al., 2012a). Reduction of nitrite to ammonium is catalysed by nitrite reductase
localised in chloroplasts (Meyer and Stitt, 2001). Assimilation of ammonium to amino acids
by the so-called GS/GOGAT cycle also occurs in the chloroplast. Ammonium is fixed to a
glutamate molecule by glutamine synthetase (GS) to form glutamine (Masclaux-Daubresse et
al., 2010). This glutamine reacts subsequently with 2-oxoglutarate to form two glutamate
molecules, catalysed by glutamine 2-oxoglutarate amino transferase (GOGAT) (Masclaux-
Daubresse et al., 2010). In chloroplasts, GS and GOGAT are both reduced by the electron
carrier protein ferrodoxin (Fd) or NADPH, which must first be reduced by photosystem I
(Hanke et al., 2004). Moreover, GS is dependent on hydrolysis of ATP (Eisenberg et al.,
2000). Organic assimilation of nitrogen into amino acids requires the availability of carbon
skeletons and especially keto-acids (Masclaux-Daubresse et al., 2010). Hence, the link
between photosynthesis and plastidial nitrogen assimilation, in the context of transplastomic
protein expression, becomes apparent. The supply of ATP, Fd, NADPH and carbon skeletons
for nitrogen assimilation requires actively photosynthesising chloroplasts. It is known that
nitrate reductase (a cytosolic enzyme) and nitrite reductase are induced in the presence of
nitrate (Faye et al., 1986; George et al., 2007; Grimes and Hodges, 1990); this is corroborated
by these studies. The reduced capability of the chloroplasts to undertake nitrogen reduction
and amino acid synthesis explains the decreased TetC expression when NH4+ was the sole
nitrogen source and when 50 mM NH4+ : 10 mM NO3
-.
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The demonstrated requirement of nitrate for the expression of recombinant protein expression
is consistent with previous reports. Nitrate enrichment of the culture medium resulted in
increased accumulation of accumulation of 2G12 monoclonal antibody in N. tabacum BY-2
cell suspension cultures (Holland et al., 2010). Through a combination of fractional factorial
designs (FFDs) and response surface methodology (RSM), it was found that KNO3 and
NH4NO3 were among the nutrients that has the most significant influence on human antibody
M12 accumulation in N. tabacum BY-2 cell suspension cultures (Vasilev et al., 2013). The
estimated volumetric yield of TetC observed when NO3- and NH4
+ are used in combination is
similar to that when NO3- alone is used, with the optimum yield observed at 20:40 NO3
-
:NH4+, suggesting a synergistic effect of using both ions on protein metabolism. From the
perspective of plants as protein factories, NH4+ is the more ideal nitrogen source. NO3
-
uptake requires expenditure of considerable ATP (Atkin and Cummins, 1994). In
comparison, NH4+ uptake can be driven by the electrochemical gradient generated during
normal metabolism, although energy is required for re-establishment of the original gradient
(Raven et al., 1992). NH4+ directly goes towards amino acid synthesis (and therefore protein
synthesis) via glutamate, without first being reduced (George et al., 2007). Unfortunately, in
this study low assimilation of NH4+ was observed. This may be partially attributed to initial
low pH of 5.8 and the further possible acidification during culture, limiting uptake of NH4+.
3.4.2 Effect of initial media pH on biomass growth and TetC expression
3.4.2.1 Design of experiment
As a standard practice, plant tissue culture media is titrated to slightly acid conditions, pH
5.4–5.8, prior to autoclaving (George et al., 2007). In our protocol, the medium pH is set at
5.8. Many in vitro plant cells and tissues will tolerate pH in the range of 4.0-7.2 (Butenko et
al., 1984). The medium pH is an important parameter which determines a range of metabolic,
biotransport and morphogenic activities in cultured tissues (George et al., 2007). As
demonstrated in 3.4.1.3.2, the pH is expected to drift over the course of the culture period as
well as after autoclaving, especially in unbuffered medium. The impact of initial medium pH
on biomass accumulation and TetC expression was investigated, by undertaking 40-day
temporary immersion regeneration cultures in media titrated to a range of pH levels, 3.8, 4.8,
89
5.8 and 6.8 (pre-autoclave pH). Cultures were undertaken as triplicates, and TSPs from
regenerated biomass were used to undertake SDS-PAGE and immunoblot analysis.
3.4.2.2 Results and Discussion
3.4.2.2.1 The influence of initial medium pH on biomass growth and TetC expression
High shoot morphogenesis was observed in all treatments tested (Figure 3.13). Initial media
pH was found to have no significant effect on either fresh or dry biomass accumulation.
There was also a small impact on TetC intrinsic yield, with more acidic conditions favouring
TetC expression (Figure 3.14). A small increase in TetC expression is observed with pH 3.8
and 4.8, in comparison to 5.8 and 6.8.
Media pH is an important parameter as it influences the uptake of nutrients and
phytohormones and regulation a wide range of biochemical reactions in plant tissues,
especially those catalysed by enzymes (Owen et al., 1991). However, in this study, pH was
found to have no discernable influence on growth or recombinant protein expression. This
study is in concordance with others in which initial pH has little influence on biomass growth
in suspension and regeneration cultures (Bhatia and Ashwath, 2005; Butenko et al., 1984;
Kaul and Staba, 1968; Martin and Rose, 1976). These results suggest that In vitro cultured N.
tabacum callus and shoots are tolerant to a wide range of acidic pH.
90
Figure 3.13 Influence
of media pH on fresh
and dry biomass
accumulation. Error bars
represent standard errors.
Figure 3.14 SDS PAGE
and immunoblot analysis
of of media pH effects on
TetC expression. 12%
acrylamide gel; 14µg protein
loading; Sypro Orange
staining.
91
3.4.3 The Impact of Varying Sucrose and Irradiance for Photomixotrophic
Propagation Regimes
3.4.3.1 The importance of exogenous saccharides and irradiance in in vitro plant
tissue culture
Light and sugars both play an important role in plant grown and development. As well as
being the main source of energy via photosynthesis, light plays a signalling and regulatory
role in several developmental processes (Franklin et al., 2005). High light may be a source of
stress and damage the photosynthetic apparatus through photoinhibition (Vass et al., 2007).
Sugars also play a major role in metabolism, as respiratory substrates and precursors for
synthesis of metabolically important compounds such as amino acids, fatty acids and
structural compounds such as cellulose. In vitro tissue culture usually involves addition of the
dissacharide sucrose to the medium and growth in sterile airtight vessels (Pospóšilová et al.,
1999), in order to promote an increase in multiplication rate. However, features of this culture
environment include low air exchange rates, high humidity (>95%), low photosynthetic
photon flux (PPF) (50-100 µmol photons m-2 s-1), low photoperiod CO2 concentrations,
ethylene accumulation and stagnant air movements (Zobayed et al., 2000). These conditions
can cause several physiological and metabolic abnormalities. Photoautotrophic culture
methods have been advocated to overcome these problems and enable plantlets to develop
full photosynthetic capabilities to produce endogenous carbohydrates for growth.
Photoautotrophic growth conditions are regarded to promote growth and physiological
development, a higher multiplication rate than conventional heterotrophic culture regimes
and successful ex vitro acclimatization (Kozai et al., 1991). Photoautotrophy is induced
through using sugar-free medium and can be promoted through forced ventilation or
enhanced diffusive ventilation (Kozai and Kubota, 2001), CO2-enriched air (Kozai, 1991;
Solárová and Pospíšilová, 1997) and high light intensities. Photoautotrophy is difficult to
achieve in practice, so a directed photomixotrophic culture strategy may be used, without
eliminating sugar from the medium completely (Gonzalez-Olmedo et al., 2005). Most
micropropagative protocols involve photomixotrophic growth of plant tissues, which
assimilate carbon via both exogenous sugars and photosynthesis.
92
3.4.3.2 Design of experiment
In typical in vitro cell, tissue and organ cultures, 3% (w/v) or 30 g l-1 sucrose is typically
used, the de facto standard level since Murashige and Skoog first formulated their MS media
(Murashige and Skoog, 1962). This is the case with our TI culture method for heterologous
protein expression in N. tabacum (Michoux et al., 2013; Michoux et al., 2011). It appears that
this 3% sucrose level is based mainly on tradition, without reference to the intrinsic
photosynthetic capabilities of in vitro regenerated plantlets. In typical micropropagation
facilities including our own, irradiance levels are typically low, approximately 45 µmol m-2 s-
1. Hence, for the purposes of this study, 3% sucrose and irradiance of 45 µmol m-2 s-1 will be
considered ‘standard’ conditions, and other sets of sucrose and light intensities will be
considered ‘deviations’ from standard conditions.
The aim of this study is to investigate the impact of sucrose concentration and light level on
vegetative biomass growth in temporary immersion culture and yield of TetC. Temporary
immersion organogenesis cultures were undertaken under varying sucrose concentrations, 0
(0%), 7.5 (0.75%), 15 (1.5%), 30 (3%) or 60 g/l (6% w/v), denoted S0, S7.5, S15, S30 and S60
treatments and three irradiance levels under three difference irradiance levels, 45, 75 and 120
µmol m-2 s-1. PAM fluorometry was undertaken on regenerated leaves by temporary
immersion culture to confirm their photosynthetic activity.
3.4.3.3 Results and Discussion
3.4.3.3.1 Effect of sucrose and irradiance on biomass accumulation and TetC
expression
When cultured in sucrose-free media, tobacco callus displayed virtually no growth and
underwent necrosis, demonstrating that exogenous carbohydrates are essential for shoot
multiplication (Figure 3.15). This may be due to insufficient light intensity to establish truly
photoautotrophic cultures. These results in are agreement with those of Schnapp and Preece
(1986) who showed that reducing the sucrose concentration to 0 produced fewer and shorter
axillary shoots in in vitro grown carnation plants (Schnapp and Preece, 1986). The supply of
exogenous sucrose positively influenced in vitro growth, both fresh and dry biomass
93
accumulation (Figure 3.16). The data show that there is an apparently linear relationship
between sucrose concentration and dry biomass accumulation, for all three irradiances
investigated. The positive association between exogenous sucrose and vegetative growth
traits is consistent with findings by previous authors (Galzy and Compan, 1992; Klughammer
and Schreiber, 1994; Kovtun and Daie, 1995; Kubota et al., 2002; Schnapp and Preece, 1986;
Tichá et al., 1998). Notably, the fresh biomass accumulation for S15 treatments was almost
identical to S30, despite having half the sugar supply. The regenerated shoots for both S15 and
S30 treatments showed visually healthy phenotypes, with a high degree of differentiation and
expansion. A reduction in fresh weight accumulation is observed for S60 under all light levels
tested, indicating that high sucrose levels exert an inhibitory effect on growth. Several
authors have reported that excessive sugar-feeding can be detrimental and impede in vitro
biomass accumulation, although the optimum level of sucrose varies between species and
culture protocol (Kovtun and Daie, 1995; Park et al., 2004b; Zhang et al., 1996; Zych et al.,
2005).
Figure 3.15 Visual demonstration of the effect of sucrose concentration on shoot
morphogenesis after 40-day temporary immersion culture at 45 µmol m-2 s-1. (A) 0 g/l (0%); (B)
7.5 (0.75%); (C) 15 g/l (1.5%); (D) 30 g/l (3%) (E) 60 gl-1 (6% w/v).
94
Figure 3.16 Effect of sucrose concentration and irradiance on fresh and dry biomass
accumulation. Error bars denote standard errors.
3.4.3.3.2 Effect of sucrose and irradiance on TetC expression
Densitometric analysis of immunoblots (Figures 3.17, 3.18 and 3.19) show that under
conditions of sucrose deprivation (0.75% and 1.5% sucrose), transplastomic protein
expression is greatly increased. From a standard 3% sucrose concentration, a 2-fold reduction
to 1.5% results in approximately 50% increase in TetC yield, whereas a 4-fold reduction to
0.75% gave an approximate 4-fold increase in TetC expression. The impact of sucrose
reduction on transplastomic protein yield increase was so great that at 45 µmol m-2 s-1
irradiance, a 3-fold increase in absolute TetC yield from 2 mg l-1 to 6 mg l-1 is possible by
reducing sucrose concentration from 3 to 0.75% w/v, even after accounting for a 32%
reduction in fresh biomass. Conversely, high sucrose concentrations seem to inhibit
heterologous protein expression, though this may be associated with reduction in growth
95
potential. The doubling of sucrose concentration from 3% to 6% resulted in approximately
86% and 89% reduction in intrinsic and absolute TetC yields. Irradiance levels were not
found to profoundly influence TetC expression levels. The light levels investigated in this
study were very low (characteristic of typical micropropagative facilities), and may not
greatly influence the development of photosynthetic capabilities.
Figure 3.17 SDS-PAGE and immunoblot showing the effect of sucrose and light on Tetc
expression. 10% acrylamide gel; 7 µg protein loading; Coomassie staining.
96
Figure 3.18 Influence of sucrose and light levels on TetC intrinsic yield (ng TetC per µg total
soluble protein (TSP)), determined densitometrically. (A) sucrose concentration as independent
variable; (B) irradiance as independent variable. Error bars denote standard errors.
Figure 3.19 Influence of sucrose and light levels on estimated absolute TetC yield (µg TetC
per litre of bioreactor)
0
1000
2000
3000
4000
5000
6000
0 1 2 3 4 5 6
Estimated absolute
TetC yield (µg/l)
Sucrose concentration (% w/v)
A B
97
The suppression of transplastomic protein expression with increasing sucrose supply may be
due to a number of factors. Firstly, increased sucrose supply is associated with a reduction in
photosynthetic capacity, through a number of mechanisms. Importantly, there is
downregulation of plastidial gene transcription associated with photosynthesis (Sheen, 1990).
The repression of transplastomic gene expression by exogenously supplied sugars is clearly
observed, though the exact mechanism is not fully known. In plants, the end-products of
photosynthesis, sucrose and monosaccharide cleavage products glucose and fructose are the
main molecules in carbohydrate metabolism, translocation and polysaccharide formation
(Roitsch and González, 2004). Triose phosphates, products of the photosynthetic Calvin-
Benson cycle are substrates for sucrose biosynthesis in the cytosol. Sucrose is then
translocated in phloem to sinks or stored in the vacuole (Wind et al., 2010). The Calvin-
Benson cycle intermediate fructose 6-phosphate is a precursor for plastidial starch
biosynthesis (Zeeman et al., 2010). Given their important metabolic roles, sucrose and its
derivatives are important in sensing and signalling, especially with regulation of
photosynthesis (Häusler et al., 2014). In soil-grown plants, accumulated saccharides are
implicated in repressing photosynthesis (Neales and Incoll, 1968), which is generally
accepted to be a feedback mechanism to ensure balanced carbon flow between the source and
sink (Ainsworth and Bush, 2011; Jang and Sheen, 1994; Paul and Pellny, 2003). In tissue
culture, this effect is even more pronounced with the artificially high supply of exogenous
sucrose. High exogenous sucrose levels have been shown to reduce the photosynthetic rate of
in vitro plantlets (Arigita et al., 2002; de la Viña et al., 1999).
3.4.3.3.3 Pulse amplitude modulation (PAM) fluorometry to assess photosynthetic
activity of in vitro regenerated shoots
‘Pulse amplitude modulation’ (PAM) fluorometry for determination of chlorophyll a (Chl a)
fluorescence is a powerful tool to elucidate information on photosystem II activity and the
potential for photosynthetic electron flow (Schreiber et al., 1986). Chlorophyll fluorescence
measurements confirmed that in vitro regenerated plantlets are photosynthetically active,
despite the presence of exogenous sucrose. Chlorophyll fluorescence studies demonstrated
the photochemical functionality of photosystem II. Fv/Fm is a quantitative measure of the
intrinsic (maximum) photochemical efficiency of photosystem II (Maxwell and Johnson,
2000). Maximum photochemical efficiency was measured for S30 and S15 treatments at 45
98
and 120 µmol m-2 s-1 (Table 3.1). The Fv/Fm values for all treatments measured varied greatly
between 0.4-0.7, though there is no clear relationship between sucrose concentration and
irradiance levels. This apparent independence of PSII photochemical efficiency and sugar and
irradiance in in vitro micropropagated plantlets is consistent with previous findings
(Kadleček et al., 2003). The great variation in Fv/Fm is thought to be due to differences in the
in vitro culture environment of the temporary immersion bioreactors (despite our best efforts
to maintain uniformity). The estimated value of Fv/Fm for higher C3 plants of several species,
subject to optimal growing conditions, is remarkably constant at 0.832±0.004 (Guerra et al.,
2001). The reduced PSII photochemical efficiency suggests physiological stress under
temporary immersion culture conditions.
Average Fv/Fm Standard
Deviation
30g/l sucrose; 120 µmol m-2 s-1 0.575 0.091 (n=9)
30g/l sucrose; 45 µmol m-2 s-1 0.613 0.090 (n=6)
15g/l sucrose; 120 µmol m-2 s-1 0.449 0.200 (n=6)
15g/l sucrose; 45 µmol m-2 s-1 0.635 0.112 (n=9)
Table 3.1 Variable fluorescence / maximal fluorescence (Fv/Fm) measurements for leaves
regenerated by temporary immersion culture of Nt-pJST12 under different photomixotrophic
treatments.
3.4.3.4 Implications of these findings on transplastomic molecular farming
Sugars are considered to be a major modulator of plant cell division and growth (Riou-
Khamlichi et al., 2000), because their availability to proliferating cells in meristems is
indicative of overall photosynthetic activity and hence prevailing growth conditions (Koch,
1996). It is not surprising that sucrose wields such great influence on the vegetative growth
and transplastomic protein expression in in vitro shoot regeneration. However, the finding
that sucrose causes repression of transplastomic protein expression was unexpected.
Expression in the plastids provides an explanation for this phenomenon. Plastidial protein
expression is dependent on the developmental status of chloroplasts (Barkan, 2011; Klein and
Mullet, 1987; Møller et al., 2014; Silhavy and Maliga, 1998). The maturation of chloroplasts
99
is influenced by conditions favouring photosynthesis, and conditions of high sucrose supply
and low irradiances common in plant tissue culture, can impede plastid development and
photosynthetic capacity. These studies suggest, perhaps counter-intuitively, that for high
transplastomic protein biosynthesis, exogenous sucrose levels at a quarter of the standard
level (0.75% as opposed to 3%) are required. Light level was found to have little influence on
TetC expression, although far higher irradiances are probably required to stimulate
photosynthetic activity.
3.4.4 The influence of altered MS media strength on biomass accumulation and yield
3.4.4.1 Design of experiment
Murashige & Skoog (MS) media is the de facto standard basal media for micropropagation
(Murashige and Skoog, 1962). Although initially developed for the optimal growth of
tobacco callus, this formulation has successfully been used to satisfy the micro- and macro-
nutrient requirements for a range of cell and tissue culture methods and several species
(George et al., 2007). 4.4 g l-1 MS media (i.e. 1 × or 100% MS strength) corresponds to the
standard concentration used in most protocols. Deviations from this standard, 25%, 50%,
100% and 200% MS media strength were investigated for the impact on temporary
immersion biomass regeneration and TetC expression. For each treatment, cultures were
undertaken as duplicates for 40-day durations, and TSPs from harvested biomass were used
for SDS-PAGE and immunoblot analysis.
3.4.4.2 Results and Discussion
3.4.4.2.1 Influence of altered MS media strength on biomass accumulation and
TetC expression
Biomass accumulation is positively correlated with %MS media strength, and the fresh
biomass accumulation curve (Figure 3.20) demonstrates a classic rectangular hyperbolic
relationship, with marginal yield increases at higher strengths. Shoots grown on ½ and ¼ MS
basal medium strength were green-yellow in colour with high differentiation but little
100
expansion (Figures 3.21 and 3.22). The biomass fresh weights for both ½ and ¼ strength
media were much reduced compared to the full-strength medium, at 59% and 26% of that of
the full-strength medium, indicating a rapid and fairly linear decline in biomass generation
with medium dilution. Visually, the health of plantlets grown on half and quarter- strength
medium can be considered as poor, relative to the control. The discolouration indicates
chlorosis, insufficiency to produce chlorophyll, possibly due to nitrogen, iron or magnesium
deficiency (Kumar Tewari et al., 2006; Petrović, 2013; Tagliavini et al., 2000; Will, 1966).
However, there is no significant difference in dry weight accumulation, between full- and
half-strength MS concentrations, despite nearly double fresh weight difference. In contrast to
these findings, some authors have reported that half-strength MS medium has resulted in
reduced necrotic or discoloured tissue formation (Abbasin et al., 2010; North et al., 2011),
possibly due to reduced osmotic damage from lowered salt concentrations. There is only a
marginal increase in biomass accumulation of 5% upon doubling the strength from 100% to
200%, though this is probably due to the space limitations of the TIB.
Immunoblot analysis reveals that the relationship between TetC intrinsic yield and media
strength is fairly linear (Figure 3.23). Using densitometry, it has been determined that dilution
of media from 100% (standard) to 50% and 25% results in % reductions in TetC expression
of 63% and 84% respectively. Conversely, doubling the MS strength from 100% to 200%
results in a 61% increase in TetC yield.
This investigation shows that a simple way of enhancing transplastomic protein yields is to
double the MS concentration from the standard 4.4 g/l. There is also the need to establish an
‘optimum’ MS strength which results in maximum TetC yield. From this study, it is clear that
the ‘optimum’ medium concentration is greater than 100% MS. Further studies would need to
be undertaken to establish this optimum.
101
Figure 3.20 Effect of MS media strength on fresh and dry biomass accumulation. Error bars
denote population standard errors.
Figure 3.21 TIB cultures at 40 days (prior to harvest) at 100%, 50%, 25% MS medium strength,
from left to right.
102
Figure 3.22 Visual comparison of 40-day temporary immersion cultures at 100% and 200% MS
medium strength, from left to right.
Figure 3.23 SDS-PAGE and immunoblot showing the effect of MS media strength on Tetc
expression. 10% acrylamide gel; 14 µg protein loading; Coomassie staining. WT culture used as
negative control.
103
3.5 Effect of Temporary Immersion Culture Hydrodynamics on
Viability, Growth and Transplastomic Protein Expression in early-stage
Callus Morphogenesis
3.5.1 The importance of hydrodynamics in plant cell and tissue cultures
Mechanical forces are known to have profound impacts on cell structure, physiology and
metabolism (Gloe et al., 2002), so from a biotechnological perspective, it is important to gain
insight into these phenomena. Culture of microbes, plant suspensions or mammalian cells
requires effective nutrient and oxygen transfer, and homogeneous cell suspensions. This is
achieved through mixing, via impeller agitation and sparging of air in stirred tank bioreactors
and sparging alone in bubble column and airlift reactors (Dufourmantel et al., 2007).
Hydrodynamic shear stresses are required for adequate mixing, associated with velocity
gradients within the agitated or sparged liquid (Merchuk, 1991). However, numerous studies
regarding microbial, mammalian and plant cell cultures have demonstrated that
hydrodynamic shear can adversely affect growth, metabolism and product formation. For
plant aggregate suspensions, hydrodynamic shear forces can cause changes in morphology,
release of intracellular compounds, alterations of morphology and productivity, and loss of
cellular viability (Meijer et al., 1993).
Consideration of rheology is important for understanding the fluid mechanical and biological
aspects of hydrodynamic shear stress. Shear stress τ is a force per unit area acting on a
surface in the tangential direction (dimensions, N m-2 or Pa), that arises from velocity
fluctuations in the fluid. The spatial velocity gradient is known as the shear rate and is
expressed as
𝛾 =𝑑𝑢𝑥
𝑑𝑦
Where 𝑢𝑥 is the velocity in the x direction and y is perpendicular to x (dimensions, s-1)
(Dufourmantel et al., 2007). The shear stress is directly proportional to the shear rate; this
relationship, Newton’s law of viscosity, may be expressed as
𝜏 = −𝜇𝑑𝑢𝑥
𝑑𝑦 or 𝜏 = −𝜇𝛾
104
in which the proportionality constant, µ is the viscosity of the fluid (dimensions, N m-2 s or Pa
s). This only applies to Newtonian fluids, in which µ is considered constant. An extension of
this, the two-parameter Ostwald-de Waele model (power law) (Coulson et al., 1999) explains
the rheological behaviour of non-Newtonian fluids
𝜏 = 𝑘𝛾𝑛
In which k is the consistency coefficient and n is the power-law index. For Newtonian fluids,
𝑛 = 1 and k is equivalent to the viscosity µ, and this correlation simplifies to Newton’s law
of viscosity.
A major limitation in the large-scale culture of plant cells, either as suspension cultures or in
the initial phases of plantlet regeneration is their fragile nature coupled with the shear stresses
of the hydrodynamic flow field of the culture vessel. The shear sensitivity of plant cells has
been extensively investigated, and the concept of ‘viability’ is often invoked in such studies.
‘Viability’ is generally considered the propensity of cells to grow and divide (Dunlop et al.,
1994), though it is possible for cells to remain viable without discernable biomass increase
(Liu et al., 2008). Alternative viability assays based on membrane integrity, metabolic
activity or ATP content (Crouch et al., 1993) are useful in determining the health of cells
after imposition of stress conditions, though these parameters do not determine whether cells
can undergo growth and morphogenesis. Arguably, characterisations of cellular viability
should include the potential for growth, division and (where appropriate in micropropagation
applications) morphogenesis, as these are more important, from a biotechnological viewpoint
(Kieran et al., 2000). Many studies have attempted to quantify the viability of plant
suspensions (Kota et al., 1999; Liu et al., 2008). Although insightful, flow regimes are far too
complex to draw a simple relationship between the fluid dynamics and cell viability (Sowana
et al., 2001).
3.5.2 Investigation of the effects of fluid hydrodynamics on callus morphogenesis,
viability and heterologous protein turnover during pneumatic immersion
3.5.2.1 Aims of experiment
In temporary immersion culture, periodic suspension of liquid media is the mode of nutrient
transfer to plant biomass. This occurs through pneumatic power input provided by the
105
isothermal expansion of sparged air (Sánchez Pérez et al., 2006). Observations of previous
cultures undertaken by the Nixon group suggest that during early temporary immersion
culture, excessive air flow damages cell aggregates and inhibits growth and morphogenesis
(data not shown). Previous to the study described below, there has not been any quantitative,
systematic investigation into these complex hydrodynamic phenomena and the biological
responses in terms of morphogenesis and product formation.
This study aims to correlate N. tabacum biomass growth, morphogenesis, viability and
heterologous protein turnover with pneumatic immersion hydrodynamics. Mechanisms
accounting for shear damage of callus aggregates and organogenic clusters are postulated.
Furthermore, the results of this study will provide the context of potential strategies for the
scale-up of pneumatic immersion culture for biopharmaceutical synthesis.
3.5.3 Characterisation of key parameters
3.5.3.1 Characterisation of the rheological and hydrodynamic properties of the
pneumatic submersion of plant biomass
As gas inflow is the only source of fluid motion during periodic media suspension in
temporary immersion bioreactors (Sánchez Pérez et al., 2006), gas flow rate is indicative of
the prevailing shear conditions. In order to gain insight into the hydrodynamic flow field of
the biomass chamber during pneumatically driven suspension of media and the biological
responses of biomass, it is first necessary to determine a number of rheological and
hydrodynamic parameters.
3.5.3.2 Characterisation of the rheological properties of tissue culture media
The hydrodynamics of multiphase reactors such as temporary immersion bioreactors depend
on the rheological properties of the media and its density (Sánchez Pérez et al., 2006). The
tissue culture media, composed of 1 × MS media (4.4 g l-1) and 3% (w/v) (30 g l-1) sucrose, is
a Newtonian fluid; hence Newton’s law of viscosity applies (Kato et al., 1978; Rodrıguez-
Monroy and Galindo, 1999; Tanaka, 1982; Trejo-Tapia et al., 2001). For aqueous solutions
106
and suspensions, viscosity is a function of solute concentration (Jones and Talley, 1933). The
viscosity (µ) of the media was estimated using Mooney’s commonly used semi-empirical
equation relating viscosity to solute concentration (Mooney, 1951):
μ = μsexp (2.5φ
1 −φφ∗
)
In this, µs is the viscosity of the solvent (water), and φ is the volume concentration of
spherical solute particles. This theory describes the effect of infinite viscosity increase when
approaching a critical volume concentration of spherical particles, φ∗ = 0.74, corresponding
to the close packing of uniform spheres (de Bruijn, 1942). In this case, µs (viscosity of water)
is 0.000891 Pa s at 25ºC. The pre-dissolution volume of the non-aqueous components of the
media, 1 × MS basal salt mixture (4.4 g/l) and 30 g l-1 sucrose was determined to be 35 ml,
giving a volume concentration φ of 0.035 (35 ml l-1). Inputting these values into the Mooney
expression, the viscosity of media is estimated to be 0.000976 Pa s. The density (ρ) of the
tissue culture media is 1.034 g l-1.
3.5.3.3 Parameters for characterising the hydrodynamic flow field
In bubble column type bioreactors, the pneumatic air supply is the only source of power,
through isothermal expansion of gas. Two important parameters, the average shear rate and
volumetric power input can be determined from the superficial gas velocity (Ug) (m s-1)
(Sánchez Pérez et al., 2006). The superficial gas velocity is simply the volumetric flow rate
(m3 s-1) divided by the cross-sectional area (m2).
3.5.3.4 Estimation of average shear rate
In bubble-column reactors, the average shear rate of a Newtonian fluid depends on the
superficial gas velocity Ug, as follows this mechanistically-derived relationship (Sánchez
Pérez et al., 2006):
γ = [gρUg
μ]
1 2⁄
107
3.5.3.5 Estimation of specific power input
The specific pneumatic power input, also known as the rate of energy dissipation (Pg
V) (power
per unit volume) (W m-3) which applies when the isothermal expansion of gas is the
predominant source of power (Chisti and Moo-Young, 1989; Sánchez Pérez et al., 2006), is
given by:
Pg
V= ρgUg
This can also be expressed in terms of as energy dissipation per unit mass (ϵ) (W kg-1) by
dividing the above equation by the fluid density.
3.5.3.6 Cumulative Energy Dissipation
It may be more appropriate to assess cumulative biological responses against cumulative
energy dissipation (instead of specific power dissipation) (J kg-1) (Dunlop et al., 1994;
Sowana et al., 2001). This is simply a product of the specific power dissipation (ϵ) (W kg-1)
and the exposure time (s). In this study the total energy dissipation over the initial 20 days of
TI culture is easily calculated, given the immersion duration / frequency of 4 mins / 8 hours.
The total exposure time over the initial 20 days is 14400 s (4 h).
3.5.4 Design of experiment
Temporary immersion cultures were undertaken at various air flow rates during pneumatic
immersion. Differential air flow rates of 38, 45, 165, 376 and 440 ml / min, correspond to
average shear rates of 28.5, 31.0, 59.2, 89.3 and 96.7 s-1 respectively, isothermal gas
expansion power rates of 0.77, 0.90, 3.31, 7.53 and 8.82 mW kg-1 respectively, and total
energy dissipation over an initial 20-day duration of 11.1, 13.0, 47.6, 108.5 and 127.0 J
respectively. Cultures were undertaken as duplicates and biomass was harvested after 3, 20 or
40-day durations. The estimated shear rates and power dissipation rates are the initial rates at
the start of the culture period, because callus aggregates are most susceptible to shear damage
just after inoculation, and before formation of functional organs. Since each TIB is inoculated
with only 0.5 g callus, the impact of inocula on system properties is assumed to be negligible
and the system is considered a two-phase system. After the allotted culture durations,
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biomass was harvested and weighed. Samples were taken for analysis of mitochondrial
activity (as a measure of biomass viability) and TetC expression. Mitochondrial activity
assays were based on mitochondrial activity based on the reduction of 2,3,5-
triphenyltetrazolium chloride (TTC) to insoluble red triphenylformazan (TF) (Towill and
Mazur, 1975). SDS-PAGE and immunoblot analysis was undertaken to assess TetC
expression.
3.5.5 Results
3.5.5.1 Effect of shear rate and power dissipation on biomass accumulation
Accumulation of fresh and dry biomass declined with increasing flow rate for both 20 and
40-day old cultures, corresponding to increasing shear rate and energy dissipation rates
(Figures 3.24). An increase of air flow rate from 38 and 376 ml min-1 (corresponding to a
28.5 to 89.3 s-1 change in shear rate, or 0.77 to 7.53 mW kg-1 change in energy dissipation),
resulted in 50% and 14% reductions in fresh weight at 20 and 40 days respectively. However,
the most significant decrease in growth occurred at 440 ml min-1 (equivalent to a shear rate of
96.7 s-1 or energy dissipation rate of 8.82 mW kg-1), giving fresh weights of only 4.2 g and
25.9 g per bioreactor at 20 and 40 days, respectively, corresponding to 82% and 80%
decreases relative to that at 38 ml/min. No significant reductions in biomass growth (either
fresh or dry weights) were observed in 3-day cultures.
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Figure 3.24 Plots showing the influence of average shear rate and energy dissipation rate
on fresh and dry biomass accumulation, after 3, 20 and 40-day cultures. Error bars denote
standard errors.
3.5.5.2 Effect of shear rate and gassed power input on mitochondrial activity
Mitochondrial dehydrogenase activity, an indicator of overall cellular viability and metabolic
activity was expressed in terms of absorbance (520 nm) of reduced triphenylformazan
(Castro-Concha et al., 2006; Ruf and Brunner, 2003; Towill and Mazur, 1975). Plots showing
the effect of hydrodynamic parameters on mitochondrial dehydrogenase activity after 3, 20
and 40-day cultures are in Figure 3.25. Mitochondrial activity was largely unaffected by air
flow rate (and derived parameters, shear rate and energy dissipation rate) at 3 and 20 days
between 38 and 376 ml min-1, though a shallow increase in mitochondrial activity with
increasing hydrodynamics was observed for the 40 days culture. Significant impairment of
mitochondrial function is observed at a 440 ml min-1, corresponding to an average shear rate
of 96.7 s-1 and energy dissipation rate of 8.82 mW kg-1. At 20 and 40 days, the mitochondrial
activity is 60% and 67% of that at 376 ml/min. This is reflective of the reduction in biomass
growth at this flow rate. It is better to express cell viability as a function of total cumulative
energy dissipation (Sowana et al., 2001), which is a product of the energy dissipation rate and
exposure time. A plot of mitochondrial activity against total energy dissipation over the first
20 days of temporary immersion culture is presented in Figure 3.24. The drop in
mitochondrial function is observed between 108.5 and 127 J kg-1.
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3.5.5.3 Effect of shear rate and gassed power input on TetC expression
SDS-PAGE and immunoblot analysis was undertaken on TSPs extracted from biomass
grown at various air flow rates at 3, 20 and 40-day intervals (Figure 3.26). For 3 and 20 days,
air flow rate was found to have no discernable effect on TetC expression. However, for the
40-day cultures, TetC yields decrease with increasing air flow rate. Densitometric analysis
was undertaken on this immunoblot to visualise the trend in TetC yield reduction with
parameters derived from air flow rate, shear rate and energy dissipation rate after 40-day
culture. Apparent exponential decay relationships are observed for both intrinsic and
volumetric TetC yields with respect to both parameters (Figure 3.27).
Figure 3.25 Plots showing the
influence of average shear rate,
energy dissipation rate and total
energy dissipation (after 20 days
culture only) on mitochondrial
respiratory activity after 0, 3, 20 and
40-day cultures. Error bars denote
standard errors.
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A B
Figure 3.26 SDS-PAGE and
immunoblots showing the effect
of air flow rate on TetC
expression. (A) 3-day TI culture;
(B). 20-day TI culture; (C) 40-day TI
culture. 10% acrylamide gel; 7 µg
protein loading; Coomassie staining.
A B
C
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Figure 3.27 Decline in intrinsic and volumetric TetC yields with hydrodynamics after 40-day
culture. Intrinsic yield values (ng/µg) were estimated densitometrically, from immunoblot data.
Volumetric yields (g l-1) were determined as the product of intrinsic TetC yield (ng µg-1), total soluble
protein extracted (µg/g fresh biomass) and fresh biomass per unit volume (g/l). Intrinsic and
volumetric yields were expressed as proportions of the maximum yield (obtained at 38 ml/min air flow
rate), on a relative scale between 0 and 1 (A) Average shear rate as independent variable; (B) Energy
dissipation rate as independent variable.
3.5.6 Discussion of the influence of hydrodynamic shear on temporary immersion
regeneration of N. tabacum shoots and TetC expression
Most previous studies investigating shear effects in in vitro plant culture have focussed on
suspension cultures. This is the first known attempt to systematically quantify the impact of
hydrodynamics on in vitro organogenesis, especially in the context of transplastomic protein
synthesis. Most protocols involving pneumatically-driven temporary immersion culture for
differentiated plant growth recommend a low gas flow rate for pneumatic immersion
(Steingroewer et al., 2013), although this is usually anecdotal and lacks any quantitative
basis. This study represents an attempt to empirically correlate the hydrodynamic flow field
associated with periodic pneumatic infiltration with cumulative biological responses in terms
of differentiated biomass growth, metabolic activity and transplastomic protein expression,
revealing important implications for industrial bioprocessing and scale-up.
In pneumatic two-phase systems such as TIBs, the hydrodynamics are determined by the air
flow rate (Sánchez Pérez et al., 2006). Although the fluid dynamics are too complex to fully
characterise, two parameters mechanistically-derived from the aeration rate, the average shear
rate and isothermal energy dissipation may be correlated against biological responses.
Importantly, the shear environment had a major impact in terms of reduction in biomass
growth. This may be attributed to decreased cell division under stress conditions or cell lysis.
It is speculated that differentiation of plant tissues may protect cells against lysis; therefore
for the 40-day old culture, reduced cell division may be the primary mode of damage. An
alternative explanation is that lysis of undifferentiated callus clusters during the early phases
of culture would reduce the amount of viable inocula for further biomass growth. Between 38
and 376 ml min-1 mitochondrial activity was largely unaffected by increasing shear for 3-day
and 20-day cultures, although a steady increase was noted for 40-day cultures. This increased
respiratory activity may be due to increased protein synthesis as a plant stress response (Aarts
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and Fiers, 2003). At a critical flow rate of 440 ml min-1, corresponding to an average shear
rate of 96.7 s-1 and energy dissipation rate of 8.82 mW kg-1, significant cell damage is
observed for both 20 and 40-day cultures, indicated by reduction in biomass accumulation
and mitochondrial activity. Several authors have suggested the need to establish critical
values for energy dissipation to avoid, and thus reduce excessive shear damage of cells
(Dunlop et al., 1994; Kieran et al., 2000; MacLoughlin et al., 1998; Sowana et al., 2001).
These results also suggest a critical total energy dissipation of 127 J kg-1. Total energy
dissipation is a more reliable critical parameter than energy dissipation rate, as biological
responses against shear damage tend to be cumulative over time (Sowana et al., 2001). Total
energy dissipation over 20 days was chosen because the initial 20 days of culture
approximate to the ‘lag’ phase of organogenesis, when there is very little biomass increase
and virtually no morphogenesis, hence the culture can be approximated to a two-phase
system. During this phase, the callus inoculum slowly proliferates just prior to the rapid
increase in meristemic shoot formation. Indeed, this early phase is important in determining
the later physiological and metabolic status of the biomass. TetC expression was found to be
especially sensitive to shear damage, and exhibits an apparent exponential decrease in yield
with increasing shear conditions. Unlike growth and mitochondrial activity, which exhibited
a significant decline with a high air flow rate, the reduction in TetC intrinsic yield was across
the entire range of conditions tested.
Although no known comprehensive studies of hydrodynamic effects in callus morphogenesis
have been undertaken, the results of this study indicate a number of fundamental approaches
that can be employed for the design and scale-up of pneumatic immersion cultures. These
results indicate that low aeration rates promote growth and transplastomic protein expression.
These results suggest that to avoid critical cell damage, it is advisable not to exceed an
average shear rate of 89.3 s-1, or total energy dissipation of 108.5 J kg-1, with bioprocess
scale-up. However, a steady decline in transgenic protein expression was observed, even at
moderate aeration rates, which demonstrates that low air flow conditions are necessary to
maintain high yields of transplastomic protein expression.
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3.6 Discussion on in vitro morphogenesis of callus in TIBs and
transplastomic protein expression, and the various parameters
affecting these
Conventional transgenic plant host systems, based on nuclear or transient expression, have
unfortunately been hampered by low yields which have rendered these systems uneconomical
compared to existing bacterial, fungal and mammalian cell host systems. In order to compete
with established bioproduction technologies, it is important that transgenic plant expression
systems provide heterologous protein yields comparable to these incumbents. Transplastomic
plants have been proven to be technically feasible protein ‘factories’, able to provide
recombinant protein hyperexpression in chloroplasts, several orders of magnitude greater
than nuclear transformants (Bock, 2014). These studies have shown that for in vitro
transplastomic plant systems, recombinant protein expression can be modulated, by tuning
plastid development and morphogenic status of biomass through culture conditions.
These studies have presented in vitro morphogenesis of shoots from calli (shoot regeneration)
in RITA® temporary immersion bioreactors (TIBs) as a viable method for the biosynthesis of
transplastomic proteins. The intrinsic yield of TetC in TIB regenerated shoots is
approximately 15-fold greater than in callus suspensions. This is of significance, as plant
suspension cultures are an alternative promising host technology. In addition, TIB
regeneration provides 500-fold biomass increase after 40-day culture. The increase in
transplastomic protein yields is associated with the increase in chloroplast number and
maturation of chloroplasts’ photosynthetic capabilities during morphogenesis. As a future
work, this association may be quantitatively determined through correlating transplastomic
protein expression against a highly expressed plastid-encoded protein such as Rubisco large
subunit (encoded by rbcL), D1 subunit of photosystem II (encoded by PsbA), ATP synthase
beta subunit (encoded by atpB), P700 reaction centre of photosystem I (encoded by psaA) or
chloroplast RNA polymerase alpha subunit (encoded by rpoA). These proteins would be ideal
candidates as their expression is directly related to the chloroplast’s ability to undertake
photosynthesis, or in the case of RNA polymerase, related to the chloroplast’s functional
development (Erickson, 1998; Kim et al., 1993; Tiller and Bock, 2014; Weihe et al., 2012).
115
In both in vitro and soil-based plant cultivation, the intuitive approach has been to optimise
growth conditions for visually-observable plant phenotypic health and vigour. The traditional
agricultural and horticultural industries have focussed their efforts on the production of high
quality plant biomass, because the biomass itself is a useful product. This approach has
extended to micropropagative tissue culture (which inherently imposes stresses on plants), in
which plantlet vigour is often measured in terms of ex vitro survival rates. However, as these
studies have shown, there is little association between visual plantlet health and heterologous
protein turnover. This is an important theme in the emerging molecular farming field,
consistent with findings of other authors (Colgan et al., 2010). This principle is especially
evident in the observations that hyperhydricity has little effect on transplastomic protein
expression, and that increased shoot development at high sucrose concentrations did not lead
to improved transplastomic protein expression. Although plant health per se is not wholly
important for recombinant protein expression, there is a strong correlation between
chloroplast development, photosynthetic capability, and plantlet developmental status and
transplastomic protein synthesis. This is demonstrated with the sucrose inhibition of TetC
expression, and the increased TetC expression observed in regenerated shoots when both
NO3- and NH4
+ are used as a nitrogen source. Moreover, these studies suggest that the
imposition of excessive abiotic stresses, such as high shear during pneumatic immersion of
biomass will impinge upon product synthesis.
When investigating the role of certain bioprocess parameters on transplastomic protein
expression, it is clear that overall yield is dependent on two factors, the intrinsic yield of
heterologous protein and overall biomass yield (Sabalza et al., 2014; Twyman et al., 2013).
The intrinsic yield is the heterologous protein yield as a proportion to total soluble protein
(TSP) (often expressed as % TSP), or as a proportion to fresh or dry biomass. For the
purposes of these studies, intrinsic yield is understood to mean yield as a proportion of total
soluble protein, for simple comparison between treatments. The relationship between
‘absolute yield’, intrinsic yield and fresh biomass yield can be demonstrated with this simple
equation.
Absolute heterologous protein yield (g
l) = Intrinsic yield (
ng
ng) × TSP from fresh biomass (
g
l)
Any approach to increase heterologous protein must focus on maximising either or both of
these variables (Figure 3.28). Rational approaches to increasing transgenic protein outputs
may be based on recombinant DNA technology (such as codon optimisation, the utilisation of
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strong promoters and strategic use of plastid gene regulatory elements such as 5’-UTRs) or
optimisation of growth conditions. These studies have focussed on the latter approach.
Figure 3.28 Absolute recombinant protein yield depends on both intrinsic yield and biomass
growth. (A) This equation underpinning protein yield is presented graphically. Intrinsic yield is often
expressed as %TSP. (B) The absolute yield can be thought of as a function of culture conditions.
A B
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Chapter 4. Expression and assembly of Guy’s 13
monoclonal antibody via temporary immersion shoot
regeneration
4.1 Introduction : Monoclonal antibody production in transgenic plants
4.1.1 Monoclonal antibodies as biopharmaceuticals
The discovery of monoclonal antibodies (mAbs) in hybridoma cell lines (Kohler and
Milstein, 1975) and their subsequent exploitation have revolutionised medicine and
biochemistry (Reichert et al., 2005). Monoclonal antibodies and antibody fragments are
amongst the most important biotherapeutic agents used in the treatment of a number of
diseases. Immunized sera and intravenous immunoglobulin (IVIG) have been conventionally
used to provide passive immunity to patients with immunoglobulin deficiency and infectious
diseases (Casadevall and Scharff, 1995; Goswami et al., 2013), though these have been
largely replaced by mAb therapeutics. There is resurgence in the development of antibody-
vaccines against infectious diseases, which has been driven by a number of worrying
paradigms: the increasing resistance of pathogens to multiple antibiotics, the emergence of
new pathogens, and the growing population of immunocompromised individuals (Berry,
2005; Berry and Gaudet, 2011; Casadevall, 1998). For example, patients at high-risk from
severe respiratory syncytial virus (RSV) infection, one of the leading causes of hospital
admissions for paediatric respiratory illness, are given a mAb, palivizumab, which binds to
the RSV F protein and thereby directs the immune-mediated clearance of the virus from the
body (Leader et al., 2008). A major application of mAb therapeutics is the treatment of
inflammatory diseases, by targeting tumour necrosis factor (TNF), a cytokine that stimulates
increased activity of the immune system. These include Infliximab (Remicade;
Centocor/Merck) and adalimumab (Humira; Trudexa/Abbott), which are routinely used to
treat rheumatoid arthritis, as well as Crohn's disease and plaque psoriasis. MAbs have
become one of the largest classes of new therapies in oncology applications (Pillay et al.,
2011). Over 40 mAbs and mAb fragments have been approved by the Food and Drug
Administration (FDA) as therapeutics and diagnostics over the past 25 years (Brorson and
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Jia, 2014). One of the major strengths of antibody therapeutics is their relatively high
approval success rates, currently 23% for canonical antibodies, and 35% for antibody drug
conjugates (ADCs) (Reichert, 2014), compared to 7% for small molecule “new molecular
entities” (NMEs) (Hay et al., 2014). In principle, mAbs represent ‘ideal’ pharmaceutical
agents. They bind to specific targets, have slow clearance rates and elicit reduced side effects
than many small molecule drugs (Brorson and Jia, 2014). However, mAbs generally exhibit
low potencies, and are therefore required in high doses for chronic diseases (Jain and Kumar,
2008). This means that mAbs are among the most expensive therapeutics. For example, two
licenced antibodies, palivizumab and infliximab, are used at doses of 10 – 15 mg/kg body
weight (Ma et al., 2005a). For some mAb therapeutics, intensive large-scale
biomanufacturing processes producing 100 – 1000 kg/year would be required to cope with
market demands (Ma et al., 2005a).
Antibodies, also known as immunoglobulins (Ig), comprise of five main classes, IgG, IgA,
IgM, IgD and IgE, of which IgG is the most abundant in humans (Schroeder and Cavacini,
2010). The general structure of an immunoglobulin G (IgG) molecule is shown in Figure 4.1
(Nelson et al., 2008). An IgG molecule consists of the constant Fc (crystallizable fragment)
and an antigen binding domain comprising the Fv (variable fragment) and the Fab fragment
(antibody fragment) (Figure 4.1) (Nelson et al., 2008). Antibodies are heteromultimeric
proteins comprised of two heavy chains and two light chains. Therefore production of
antibodies requires not only gene expression but also oligomerization of subunits for
functional antibodies. To avoid challenges associated with antibody assembly and reduce the
size and complexity of the molecule, antibodies have been engineered by connecting two of
the variable, antigen-binding domains of the light and heavy chain with a small linker peptide
to generate a continuous polypeptide chain (Bird and Walker, 1991). However, this single-
chain Fv will often have a lower affinity for the antigen than the parent antibody, which may
be a significant bottleneck in applications requiring high affinity binding (Bird and Walker,
1991; Tavladoraki et al., 1993). Although the mechanisms of N-linked glycosylation differ in
plants and mammals (Schoberer and Strasser, 2011), high mannose type N-glycans in plants
have structures identical to those in other eukaryotes.
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Figure 4.1 Structure of immunoglobulin G (IgG) (Nelson et al., 2008)
4.1.2 Plant systems for antibody production
Currently, mAbs have been expressed mainly in mammalian cells culture, particularly
myeloma cell lines and Chinese hamster ovary cells (Dinnis and James, 2005). However,
mammalian cell culture is slow, expensive and productivities are low (Dietmair et al., 2012).
Alternative expression systems, such as yeast and bacteria, do not have the specific
machinery for post-translational modifications of active (or partially-active) mAbs. In 1989,
pioneering work undertaken by Hiatt and colleagues revealed the prospect of expressing
functional antibodies in plants (Hiatt et al., 1989). Since then, sustained research efforts on
this theme have demonstrated the feasibility of using transgenic plant host systems for
producing therapeutic antibodies. Plant systems offer several advantages including low
upstream cost inputs, an absence of human or animal pathogen contaminants, and the ability
to employ post-translational modifications such as glycosylation and disulphide bond
formation (Brodzik et al., 2006; Ko et al., 2003; Obembe et al., 2011; Richter et al., 2000). To
express complete antibodies in plants, the original approach was to clone light and heavy
chains into separate plants and subsequent crossing. This approach is time-consuming. Co-
transformation of heavy and light chains is a faster alternative, but may suffer from a large
proportion of low antibody expressors (Engelen et al., 1994; Neve et al., 1993; Nicholson et
al., 2005). Nicholson et al. (2005) transformed rice to express the components of a secretory
antibody, with the transgenes on different plasmids delivered simultaneously. Approximately
20% of transformants carried the four transgenes encoding all the components required for
expression of a complete secretory antibody, namely the light chain (LC), heavy chain (HC),
joining chain (JC) and secretory component (SC).
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4.1.3 Guy’s 13 monoclonal antibody as a topical immunotherapy agent for prevention
of dental caries
Dental caries are caused by colonisation of the tooth surface by cariogenic bacteria of the
mutans streptococci group, such as Streptococcus mutans and S. sobrinus (Featherstone,
2000; Loesche, 1986). Adhesion of Streptococcus mutans to the tooth surface is mediated by
a cell surface glycoprotein, SA I/II (Munro et al., 1993). Guy’s 13 is a mouse monoclonal
antibody (IgG1) which recognises streptococcal antigen, SA I/II. Guy’s 13 is a promising
immunotherapy agent for prevention of dental caries, which could have the potential to
revolutionise dental health. Guy’s 13, when used as a passive mucosal vaccine, could affect
the adhesion function of SA I/II, inhibiting S. mutans colonisation of the buccal cavity. It is
suggested that Guy’s 13 may block adhesion epitopes by exerting their effect, either locally
(through steric hindrance) or at a distance (through induced conformational changes in the
SA I/II molecule) (van Dolleweerd et al., 2004). In 1994, pioneering research by Prof. Julian
Ma and colleagues led to the expression and assembly of Guy’s 13 monoclonal in Nicotiana
tabacum plants (Ma et al., 1994). A major disadvantage of biosynthesis of Guy’s 13 in soil-
grown plants is secretion of the antibody into the soil, which is a regulatory and
environmental bottleneck, calling for the development of in vitro or contained growth
systems. Continuous innovation by Prof. Ma’s lab (St. George’s University of London) have
led to expression of Guy’s 13 in hydroponic rhizosecretion, hairy root cultures, teratomas,
cell suspensions and hydroponic seedlings (Drake et al., 2003; Drake et al., 2009; Sharp and
Doran, 2001b). An IgA/G chimeric secretory variant of Guy’s 13 is in commercial
development by Planet Biotechnology Inc. (USA) under the trade name CaroRx™ (Ma et al.,
2005b). The studies outlined below demonstrate the expression of Guy’s 13 in in vitro shoots
regenerated from callus tissue, in RITA® temporary immersion cultures.
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4.2 Expression and assembly of Guy’s 13 mAb by temporary immersion
regeneration of N. tabacum cv. Xanthii
4.2.1 Design of experiment
Temporary immersion shoot regeneration cultures of Nicotiana tabacum cv. Xanthii callus
suspensions expressing Guy’s 13 mAb were undertaken. Seeds from N. tabacum lines which
were demonstrated to stably express the monomeric Guy’s 13 antibody (Ma et al., 1994) were
kindly donated by Prof. Julian Ma and Dr. Pascal Drake of St. George’s University of
London for this purpose. The gene constructs and transformation procedures used for
generating the antibody-producing plant have been described in Ma et al. (1994). Briefly,
tobacco leaf disks were infected with Agrobacterium tumefaciens containing Guy’s 13 heavy-
or light-chain cDNA including the native (mouse) immunoglobulin leader sequence, then
regenerated plants expressing heavy chains were crossed with those expressing light chains to
generate progeny plants which produce the full antibody (Ma et al., 1994).
After a 40-day culture period in RITA® temporary immersion bioreactors, harvest of
biomass, and total soluble protein extractions were undertaken, followed by SDS-PAGE and
Western immunoblotting under non-reducing conditions to confirm expression and assembly
of Guy’s 13, followed by functional ELISA to investigate antigen binding activity of the
expressed antibody. For both immunoblotting and ELISA protocols, immunodetection of the
expressed antibody was undertaken using a HRP-conjugated anti-mouse IgG (no ‘primary’
antibody is necessary as the expressed Guy’s 13 is a murine antibody).
4.2.2 Results
4.2.2.1 Non-reducing Western Immunoblotting to confirm expression and
assembly of Guy’s 13 IgG1 in transgenic tobacco
Non-reducing SDS-PAGE and immunoblot analysis (Figure 4.2) demonstrates the expression
and oligomerisation of the heavy chain (H) (~57 kDa), light chain (L) (~25 kDa), and the
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full-length IgG1, in both tissue culture-grown (temporary immersion culture) and soil-grown
biomass. Guy’s 13 mAb expression and assembly is observed in both healthy and vitrified
shoots. An additional band observed at 80 kDa, might correspond to the HL assembly
intermediate. The accumulation of the full-length antibody is significantly greater than
accumulation of unassembled heavy and light chains for all samples, confirming that
transgenic plants possess the apparatus for folding, assembly, disulphide bond formation and
stabilisation of complex multimers such as antibodies. This is in agreement with several
previous reports of full antibody production in plant systems. However, this is the first report,
to our knowledge, of immunoglobulin production in leaves generated by in vitro
organogenesis from callus tissue. The presence of bands corresponding to H, L and HL
fragments may be attributed to assembly intermediates (Khoudi et al., 1999; Neve et al.,
1993; Wongsamuth and Doran, 1997) or proteolysis of the expressed antibody possibly
during sample homogenisation (Ma et al., 1994; Sharp and Doran, 2001a). For tobacco-
expressed Guy’s 13 mAb, additional bands have been observed in previous studies, notably at
40, 45, 120 and 135 kDa (Sharp and Doran, 2001a; Wongsamuth and Doran, 1997). The
absence of these bands in this study suggests that the fragments observed are not attributable
to in planta proteolysis. In mice, the order of immunoglobulin multimer assembly varies
according to the heavy chain isotype. IgM and IgG2b assemble first as HL and then H2L2,
whereas for IgG1 and IgG2a, H2 dimers are formed first, then H2L and finally H2L2 (Baumal
et al., 1971; Percy et al., 1976). The subunit assembly pathway of Guy’s 13 IgG1 is unknown
in plants (Wongsamuth and Doran, 1997), but these results suggest assembly of HL (putative
80 kDa fragment), analogous to murine IgM and IgG2b, though this is open to conjecture.
Densitometric analysis suggests that biosynthesis of the full antibody in TIB-regenerated
shoots is comparable to that in soil-grown plants. Despite the inclusion of TDZ in the media,
which promotes shoot formation and supresses rooting, a small degree of adventitious rooting
was observed (<1% of total fresh weight). Yield of the full antibody is approximately 2.72-
fold higher in adventitious roots than in healthy (non-vitrified) shoots, determined
densitometrically. Despite this, given the extremely low proportion of biomass comprised of
roots, the current system would not be feasible for the large-scale root production.
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Figure 4.2 SDS-PAGE and Western Immunoblot of lysates of in vitro and soil-cultivated
biomass under non-reducing conditions. Assembly of full-length IgG is confirmed, as well as
fragment intermediates. 7.5 µg TSP loaded per well. Coomassie-stained 15% acrylamide gel with
LMW ladder. No β-mercaptoethanol or DTT was added, to maintain non-reducing conditions.
4.2.2.2 Antigen binding assay for functional studies of expressed Guy’s 13
monoclonal antibody
Functional ELISAs were undertaken to investigate the antigen-binding specificities of
expressed Guy’s 13 mAb, on lysate TSPs from healthy non-vitrified leaves, vitrified leaves
both regenerated via temporary immersion culture, and leaves from soil-cultivated plants. A
positive control, high purity Guy’s 13 mAb derived from the supernatant of hybridoma
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culture (kindly donated by Dr. P. Drake, SGUL) was used and temporary immersion-grown
biomass which does not express Guy’s 13 was used as a negative control. Samples were run
as duplicates, and the data from four different microplate assays conducted in parallel were
combined. Figure 4.3 shows the binding titration curves of the lysates, based on average
response absorbance. The titration curves of the temporary immersion-grown tissue (non-
vitrified and vitrified) are similar to that of soil-grown plants, indicating comparable antigen-
binding activity of in vitro and soil-grown biomass.
Figure 4.3 Lysate titration curve showing the binding of Guy’s 13 mAb to the purified SWCF
fragment of SA I/II. (A) Antigen binding as a function of protein concentration, with 4-PL logistic
curves fitted. (B) Antigen binding as a function of a relative dilution from 120 µg/ml starting
concentration. The plotted data produced sigmoid curves for transformant lines grown as in vitro TIB
cultures and as soil grown plants. The positive control, Guy’s 13 derived from mouse hybridoma
culture supernatant gave a steep sigmoidal curve. Error bars denote the population standard
deviation.
A
B
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4-parameter logistic (4-PL) (sigmoidal) models were fitted to the antibody titration curves
using a Levenberg Marquardt iteration algorithm and weighting, with OriginPro 9.1. The
equation describing the 4-PL model is as follows:
𝑦 = 𝐷 +𝐴 − 𝐷
(1 + (𝑥𝐶)
𝐵
)
in which y is the response (absorbance at 450 nm), x is the protein concentration, D is the
response at infinite antibody concentration, A is the response at zero antibody concentration,
C is the inflection point or the concentration at which the response is half-way between A and
D, and B is the Hill slope factor (Findlay and Dillard, 2007). C is the EC50 value, the serum
(or lysate) titre corresponding to 50% antigen binding to the antibody. The lysate EC50 values
are shown in Table 4.1. The lysate EC50 of vitrified shoots is 35.7% of that of non-vitrified
shoots, demonstrating a 2.8-fold enhanced avidity of vitrified shoot-expressed Guy’s 13. The
avidity of Guy’s 13 antibody expressed in soil-grown plants is comparable to that in vitrified
shoots.
Non-vitrified shoots
Vitrified shoots
Soil-grown plant
Positive control
EC50 titre of
lysate (µg/ml)
117.71 (±16.36)
42.00 (±1.09)
49.68 (±0.41)
1.29 (±0.05)
EC50 dilution
(from 120
µg/ml initial
concentration)
1.02
2.86
2.42
92.76
Table 4.1 EC50 titres and EC50 dilutions of plant lysates with standard errors, derived from
4-parameter logistic curve fitting to titration curves
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4.2.3 Discussion
4.2.3.1 Plants possess the relevant machinery for expression of functional
antibodies
A number of studies have observed that plants have the capability to synthesise and assemble
functional immunoglobulins, despite the absence of homologous proteins in plants,
suggesting that the mechanisms of protein assembly in plants must be similar to mammals
(Hiatt et al., 1989). This is the first known study of the expression and assembly of functional
immunoglobulins in in vitro regenerated leaves from callus tissue. This study demonstrates
that suitable post-translational machinery required for synthesis of complex proteins can be
developed by in vitro organogenesis from callus in TIBs. Oligomerisation of antibodies is
important for increasing the functional affinity to antigens (Batra et al., 2002), giving
improved pharmacokinetics. In mammals, immunoglobulins are synthesised in B-cells and
plasma cells. The heavy and light chains are synthesised independently. This is followed by
translocation to the lumen of the rough endoplasmic reticulum (RER), directed by N-terminal
signal sequences. In the RER, formation of disulphide bonds and folding and assembly of the
full immunoglobulin occurs, guided by a number of chaperones and foldases. The assembled
antibodies are transported to the Golgi and then post-Golgi vesicles, for export by exocytosis.
Throughout this process, carbohydrates are continuously added to H-chains, from the nascent
chain being attached to the ribosome to the point of secretion. It is thought that a similar
pathway occurs in plants, although this is only partially understood (Ma and Hein, 1995). In
plants, it is known that a significant amount of expressed antibodies accumulate in the
apoplastic space (Hein et al., 1991). Antibody engineering approaches may be employed for
enhanced apoplastic accumulation, through inclusion of a secretory component such as for
IgA (Ma et al., 1994). It has been demonstrated that targeting recombinant proteins to the
apoplast can reduce proteolytic degradation, due to the lower abundance of proteases
compared to that in the cytosol (Benchabane et al., 2008).
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4.2.3.2 The impact of hyperhydricity on functional Guy’s 13 mAb titre in
temporary immersion regeneration
The observation that hyperhydricity positively influences functional mAb titre is an
unexpected finding. The reasons for this are unknown, although a number of explanations can
be put forward, including an increase in chaperone activity or provision of a more favourable
environment for antibody accumulation in the apoplast.
It is known that upregulated synthesis of BiP (binding protein), a 78 kDa ER-resident
molecular chaperone and member of the Hsp70 family occurs under various stress conditions,
including water stress (Alvim et al., 2001). Importantly, it has been shown that
hyperhydricity significantly induced synthesis of BiP in a number of species, including
pepper (Fontes et al., 1999) and eggplant (Picoli et al., 2001). It is possible that the same
phenomenon is occurring in the RITA® temporary immersion culture system. Elevated
activity of BiP (and perhaps other chaperones) may be enhancing correct assembly of Guy’s
13 H2L2 multimers, and thus improving folding of the epitope-binding sites. This would
account for the increased avidity of expressed antibodies.
In temporary immersion culture, infiltration with liquid media and high humidity associated
with a sealed culture vessel exacerbate the hyperhydricity syndrome. Hyperhydric (vitrified)
plants are unable to maintain a correct water balance and accumulate water (Rojas-Martinez
et al., 2010). In particular, excess water is localised mainly in the apoplast (Fukao and Bailey-
Serres, 2004; Gribble et al., 1998). Previous studies have confirmed that Guy’s 13 antibody
accumulates in the apoplast and is even rhizosecreted from hairy roots (Drake et al., 2009),
despite the absence of secretory domains (Ma et al., 1994). It is possible that the unnaturally
large influx of water in the apoplast may provide a stabilised environment for accumulated
antibodies through dilution effects. This may be because of reduced proteolysis by dilution of
intercellular proteases. It was found that the majority of proteolytic degradation of
monoclonal antibodies occurs in the apoplastic space (Hehle et al., 2011). Alternatively,
reduced aggregation of assembled antibody through dilution may contribute to increased
functional activity.
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Although hyperhydric conditions are known to induce changes in protein synthesis, related to
photosynthesis, cellulose and lignin synthesis and stress responses (Rojas-Martinez et al.,
2010), non-reducing Western immunoblot analysis suggests similar abundances of the full
antibody in vitrified and non-vitrified tissue. The increased titre of the expressed antibody
(and its fragments) in hyperhydric shoots, as demonstrated by ELISA, is probably due to
greater avidity of the synthesised antibody.
4.2.3.3 Demonstration of mAb production in in vitro shoot regeneration via
temporary immersion culture
The majority of studies involving expression of mAbs in plants have involved soil cultivation
of whole plants (Artsaenko et al., 1998; Busse et al., 2002; Ko et al., 2009; Stoger et al.,
2000), although there have been some innovations in the development of root rhizosecretion
systems (Drake et al., 2003; Drake et al., 2009) and cell suspensions (Holland et al., 2010;
Vasilev et al., 2013). This is the first known report of mAb production by in vitro
regeneration of leaves from callus. These studies demonstrate that in vitro regeneration can
yield titres of functional antibody comparable to that from soil-grown plants. This suggests
that TIB biomass growth can be a feasible alternative to conventional agricultural growth
methods. The observation that hyperhydricity results in higher titres presents the possibility
of promoting hyperhydric conditions in tissue culture to modulate the functional antibody
titre. If Guy’s 13 mAb was to be commercialised, a production throughput of over 1,000 kg a
year would be needed, just for administration to the child population of Europe alone (Ma et
al., 2005a). Scaled-out production in TIBs may be the only reasonable way of achieving this
target. Vitrified biomass from just one RITA® TIB can produce enough antibody for an
estimated 60 patient doses at the EC50 dosage. This is based on the assumptions that
approximately 1 mg total soluble protein is extractable from 1 g fresh biomass (based on
previous observations), dilution of lysates to the EC50 dosage, and there are no downstream
losses of the Guy’s 13 mAb.
As the ‘plantibody’ paradigm progresses, plant suspension cultures have also been implicated
as being industrially feasible for mAb expression. Despite being more expensive and giving
lower mAb yields than production in soil grown plants, plant cell culture allows shorter
production cycles as well as higher batch-to-batch consistency (Boivin et al., 2010; Magy et
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al., 2014). These studies demonstrate the feasibility of in vitro shoot regeneration as a viable
alternative to plant suspension culture, offering the same process consistency under
controlled environmental conditions as in vitro suspension culture (bioreactors and
micropropagative multiplication), while yielding titres comparable to that of soil-grown
plants. Scaled-out temporary immersion culture systems may be a feasible way of large-scale
biosynthesis of Guy’s 13 necessary to provide enough doses for a global oral health
campaign.
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Chapter 5 Expression of transplastomic proteolytically
unstable proteins via temporary immersion shoot
regeneration
5.1 Introduction
5.1.1 In planta proteolysis of recombinant proteins
Proteolytic degradation of recombinant proteins is one of the most significant technical
challenges in expression of biopharmaceuticals in plant systems (Benchabane et al., 2008).
The accumulation of foreign proteins in planta can span several orders of magnitude, from
less than 0.02% total soluble protein (TSP) for human serum protein C, interferon β,
erythropoietin and epidermal growth factor (Daniell et al., 2001b) to 47% TSP for plastid-
encoded Bt Cry2Aa2 (De Cosa et al., 2001) and over 70% for a plastid-encoded phage lytic
protein (Oey et al., 2009). The great variability in recombinant protein accumulation is due,
in part, to proteolysis by native proteases. Hundreds of plant genes encode proteins involved
in proteolytic processes (Rawlings et al., 2008), with over 800 protease genes in Arabidopsis
(van der Hoorn, 2008). The number of susceptible cleavage sites accessible to endogenous
proteases for peptide bond cleavage is important in determining a foreign protein’s tendency
to undergo complete hydrolysis or ‘partial trimming’ (Benchabane et al., 2008). The specific
tissue or organ in which recombinant protein expression or accumulation occurs has a strong
influence on yield and integrity (Potenza et al., 2004). Although leaves are the target organ of
choice for recombinant proteins (especially transplastomic proteins), the lower metabolic
rates of seeds and tubers seem to confer greater foreign protein stability due to a lower
abundance of proteases (Artsaenko et al., 1998; Stoger et al., 2005). Additionally, protein
sequestration in specific organelles or cellular compartments determines the stability and
yield of foreign proteins in planta (Benchabane et al., 2008).
Transplastomic host systems offer several benefits over nuclear transformants. One major
advantage is the hyperexpression of foreign proteins via the plastid genome and apparently
greater stability of sequestered protein. It is observed that when nuclear-encoded proteins are
targeted to the chloroplast they accumulate to high levels. For example, targeting a fungal
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xylanase to the chloroplasts with the ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco) activase transit peptide resulted in high accumulation in Arabidopsis (Hyunjong et
al., 2006). In a similar manner, transferring human growth hormone to the plastids with a
Rubisco small subunit chloroplast transit peptide resulted in improved turnover in N.
benthamiana (Gils et al., 2005). However, there are a number of endogenous proteases in the
chloroplast (Adam et al., 2006), which can potentially degrade plastid-encoded foreign
proteins. A number of studies have reported the age-related proteolysis of transplatomic
proteins in mature or senescing leaves (Birch-Machin et al., 2004; De Cosa et al., 2001; Zhou
et al., 2008). However, in relative terms, degradation of foreign proteins may not be a
significant problem, since high net accumulation will usually still occur given the high rate of
plastidial protein synthesis.
5.1.2 Transplastomic expression of vaccine subunits susceptible to proteolytic
degradation
In the following studies, the impact of temporary immersion shoot regeneration on
transplastomic protein accumulation was investigated for two proteins known to be
susceptible to age-related proteolytic degradation in the chloroplast, VP6 bovine rotavirus
capsid protein and HIV-1 p24 antigen. Both proteins are of special significance as subunit
vaccines against rotavirus and HIV respectively. N. tabacum cv. Petit Havana lines
expressing VP6 and p24 were obtained from Professor John Gray, University of Cambridge.
In previous studies, accumulation of VP6 was found to be reduced in maturing leaves of N.
tabacum cv. Petit Havana despite constant mRNA transcript levels, possibly as a result of
proteolytic degradation in the chloroplasts. Similarly, it was observed that p24 accumulation
in N. tabacum cv. Petit Havana and N. tabacum cv. Maryland Mammoth were susceptible to
declining accumulation in mature leaves, probably due to degradation or translational
limitations (McCabe et al., 2008; Zhou et al., 2008). These previous investigations
confirming the age-related degradation of transplastomic proteins were undertaken by Prof.
Gray’s group. In the following described studies, biomass of lines expressing VP6 and p24
were grown via in vitro organogenesis in 40-day temporary immersion culture, and intrinsic
yields were compared to soil-grown plants.
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5.1.2.1 VP6 as a potential subunit vaccine against rotavirus infection
Rotavirus infection is the most common cause of severe diarrhoea in infants and young
children and is responsible for the deaths of about 600,000 children every year (Dennehy,
2008). Over 80% of all rotavirus deaths occur in developing countries in south Asia and sub-
Saharan Africa (Parashar et al., 2006). The development of a vaccine is therefore a high
priority (Shann and Steinhoff, 1999). Vaccines based on live, attenuated rotavirus strains
have been commercially available since 2005-2006, including Rotarix (GlaxoSmithKline)
and RotaTeq (Merck) (Ward and McNeal, 2010). In 1999, the Rotashield vaccine was
withdrawn from the market after one year of universal use because of associations with
intussusception, even though the probability of this is very low and several lives could have
been saved (Rennels, 2000). Though current vaccines are found to be fairly safe and
efficacious (Justino et al., 2012), the ‘Rotashield’ incident does demonstrate the existence of
safety concerns with live rotavirus vaccines. While there is no urgent requirement to
withdraw current live vaccines, given their life-saving potential and minimal safety risks,
there is a need to develop alternative candidates, with high immunogenicity and fewer safety
issues (Ward and McNeal, 2010), such as subunit vaccines. One such subunit candidate is the
rotavirus inner coat protein VP6. VP6 is highly abundant with 760 molecules in the inner
capsid layer, making up over half the total protein mass in the rotavirus particle and is a
highly conserved protein (Inka Borchers et al., 2012). VP6 has been found to be highly
immunogenic (Choi et al., 2000) and the majority of antibodies generated on rotavirus
infection are against VP6 (Svensson et al., 1987). Oral administration of VP6 is found to
induce serum IgG and mucosal IgA immunoglobulins in mice and calves thus protect against
rotavirus infection (Dong et al., 2005; Gonzalez et al., 2010; Zhou et al., 2010). VP6 is thus
an ideal subunit vaccine against rotavirus infection.
5.1.2.2 p24 as a subunit vaccines against HIV
The acquired immunodeficiency syndrome (AIDS) resulting from human immunodeficiency
virus (HIV) infection is arguably the greatest medical and scientific challenges to face
humankind over the past three decades (Meyers et al., 2008). Over 30 years since the
discovery of HIV and AIDS, no effective vaccine has been developed. HIV has a high
mutation rate and there are various subtypes prevalent in different geographical regions,
133
complicating the potential for a universal vaccine (Kalish et al., 1995; Spira et al., 2003). A
multi-component vaccine comprising several proteins may be necessary to elicit immunity.
Current efforts in vaccine development have focussed on subunit vaccines which target
epitopes within conserved regions of the virus. The HIV-1 Gag precursor protein Pr55 Gag as
well as its derivatives, proteins p17 (matrix protein) and p24 (capsid protein) resulting from
cleavage of Pr55Gag by the viral protease, are potentially good vaccine candidates (Meyers et
al., 2008). Moreover Pr55Gag and possibly p17-p24 fusion proteins can form non-infectious
highly immunogenic virus-like particles (VLPs) morphologically similar to immature HIV
particles (Morikawa et al., 2000), which are potent stimulators of cellular and humoral
responses (Doan et al., 2005). The 24 kDa capsid protein p24 is an ideal candidate as there is
80% conservation of identical residues across HIV-1 clades because of structure / function
constraints (Hanke and McMichael, 2000). A number of serological studies have
demonstrated that the risk of AIDS increases with falling serum titres of anti-p24 antibodies
(Cheingsong-Popov et al., 1991; De Wolf et al., 1987; Dyer et al., 2002; Novitsky et al.,
2003), indicating that high anti-p24 antibody titres are required for a disease-free state
(Meyers et al., 2008).
5.2 Expression of transplastomic proteins susceptible to degradation via
temporary immersion shoot regeneration
5.2.1 Accumulation of plastid-expressed rotavirus VP6 via temporary immersion
shoot regeneration and comparison to soil-grown seedlings
5.2.1.1 Design of experiment
A stable transplastomic transformant N. tabacum (cv. Petit Havana) line, Nt- Prrn-VP6 line
7A, which expresses VP6 was used in this study. This line was generated by Professor John
Gray at Cambridge University as described in Birch-Machin et al. (2004). Seeds donated by
Prof. John Gray were used as donor material for the induction of callus germplasm. Plantlet
regeneration from callus via 40-day temporary immersion culture in RITA® bioreactors was
undertaken to investigate the expression of VP6. In parallel, seedlings were grown on soil.
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Biomass was harvested and total soluble protein was extracted for SDS-PAGE and
immunoblot analysis.
5.2.1.2 Results
5.2.1.2.1 VP6 stability in soil-grown tobacco leaves
To assess the accumulation of VP6 in leaves of different ages, total soluble protein extracts
were prepared from a single 8 week-old plant grown on soil under greenhouse conditions,
and subjected to SDS-PAGE and Western immunoblotting (Figure 5.1). Leaves were
numbered from the bottom (leaf 1) upwards towards the youngest leaf (leaf 12) closest to the
shoot apex. Leaves 1-3 had presumably undergone senescence-related proteolysis, as TSP
levels were too low for comparative analysis, therefore TSPs of leaves 4-12 were analysed.
The chronological ‘distance’ between each leaf represents a plastochron, the period of time
between the initiation of two successive leaf primordia (Hill and Lord, 1990). Therefore, leaf
number is a good indication of morphologic development. On an immunoblot, VP6 was
detectable as a band at approximately 40 kDa. The amount of VP6 detected is fairly constant
in the youngest leaves, leaves 8-12. VP6 accumulation is significantly lower in the oldest
leaves (leaves 4-7), evidenced by reduction in immunoblot band intensity, suggesting
extensive proteolytic breakdown of synthesised protein in the chloroplast. Despite relative
stability in the youngest leaves (8-12), the observation of doublet bands at approximately 40
kDa suggests a partial cleavage. The age-related degradation of VP6 observed in this study is
in agreement with previous immunoblot studies undertaken by Birch-Machin et al. (2004).
Previous Northern blot analysis has revealed similar VP6 mRNA levels at each leaf
development stage, indicating that reduction in VP6 accumulation is probably because of
age-related protein degradation and not decreased transcription rates (Birch-Machin et al.,
2004).
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Figure 5.1 SDS-PAGE and immunoblot showing VP6 accumulation in the leaves of an 8-week
old soil-grown plant. 12% acrylamide gel; Precision Plus marker; Coomassie staining. Leaf number
corresponds to number of plastochrons, with leaf 1 (1st plastochron) being the oldest and leaf 12 (12th
plastochron) being the youngest. The immunoblot demonstrates a gradient in VP6 accumulation,
suggesting age-related proteolytic degradation of VP6 in the chloroplasts.
5.2.1.2.2 Expression of VP6 in in vitro temporary immersion-regenerated biomass
SDS-PAGE and Western immunoblot analysis of TSPs was undertaken to assess the impact
of temporary immersion callus-to-shoot regeneration on VP6 accumulation. Samples from
the youngest leaves of a soil-grown seedling (leaf 12) and sterile in vitro seedling were
included for comparison (Figure 5.2 (A)). Expression of VP6 was detectable in TIB-grown
shoots, albeit at a much lower level than that in both soil-grown and in vitro plants. This may
be because of proteolytic degradation by plastidial proteases. The low expression of VP6 in
TI-regenerated leaves suggests that, despite different developmental pathways, age-dependent
proteolysis of recombinant proteins still occurs. This may occur during the later stages of 40-
day temporary immersion plantlet regeneration.
SDS-PAGE and immunoblot analysis demonstrate the increased expression of VP6 in
regenerated leaves compared to callus suspension (inoculum) (Figure 5.2 (B)). VP6
expression in undifferentiated calli is negligible, and below the limit of detection for Western
136
immunoblot. This dependence of transplastomic protein expression on plant physiological
development is expected, and reflects the upregulated plastidial gene expression associated
with chloroplast maturation.
Figure 5.2 SDS-PAGE and Western immunoblot for demonstration of the expression of VP6 in
TIB-grown biomass. (A) Comparison of VP6 accumulation in in vitro grown biomass and
seedlings. Accumulation of VP6 was compared between shoots grown in RITA® temporary
immersion bioreactors (TIBs), the youngest leaf of an in vitro seedling and the youngest leaf (leaf 12)
of an 8-week old soil-grown seedling. (B) Comparison of VP6 expression in callus and in vitro
regenerated shoots. Approx. 11 µg protein loading. 12% acrylamide; Coomassie staining; Precision
Plus marker.
5.2.2 Accumulation of HIV-1 p24 antigen via temporary immersion shoot
regeneration and comparison to soil-grown seedlings
5.2.2.1 Design of experiment
A stable transplastomic transformant N. tabacum (cv. Petit Havana) line, Nt-pZSJH1p24 line
which expresses p24 was used in this study. This line was generated by Professor John Gray
at Cambridge University (Zhou et al., 2008). Seeds were kindly donated by Prof. John Gray
A
B
137
were used as donor material for the induction of callus germplasm. Plantlet regeneration from
callus via 40-day temporary immersion culture in RITA® bioreactors was undertaken to
investigate the expression of p24. In parallel, seedlings were grown on soil. Biomass was
harvested and total soluble protein was extracted for SDS-PAGE and immunoblot analysis.
5.2.2.2 Results
5.2.2.2.1 p24 stability in soil-grown tobacco leaves
The impact of leaf development on p24 expression in soil-grown Nt-pZSJH1p24
transformants was investigated. SDS-PAGE and Western immunoblot analysis was
undertaken on total soluble protein extracts, prepared from a single 8 week-old seedling
grown under greenhouse conditions (Figure 5.3). Leaves were numbered from the bottom
(leaf 1) upwards towards the youngest leaf (leaf 12) closest to the shoot apex. TSP levels in
the oldest leaves, leaves 1 to 3, were too low to undertake any meaningful assessment, so
TSPs from leaves 4-12 were analysed. On an immunoblot, bands at 24 kDa represent the
expression of the p24 capsid protein. Accumulation of p24 was found to be dependent on leaf
age, similar to accumulation of VP6 in Nt- Prrn-VP6, as described previously. There is a
steady, proportional decrease in p24 accumulation with leaf age, with barely detectable levels
in leaves 4 and 5. These observations corroborate with previous studies demonstrating the
age-related degradation of p24 in both Maryland Mammoth and Petit Havana varieties of N.
tabacum (McCabe et al., 2008; Zhou et al., 2008).
138
Figure 5.3 SDS-PAGE and immunoblot showing p24 accumulation in the leaves of an 8-
week old soil-grown plant. 15% acrylamide gel; Precision Plus marker; Coomassie staining. 11µg
protein loading. Leaf number corresponds to number of plastochrons, with leaf 1 (1st plastochron)
being the oldest and leaf 12 (12th plastochron) being the youngest. The immunoblot demonstrates a
gradient in VP6 accumulation, suggesting age-related proteolytic degradation of VP6 in the
chloroplasts.
5.2.2.2.2 Expression of p24 in temporary immersion-regenerated shoots
SDS-PAGE and Western immunoblot analysis was undertaken on TSPs to investigate the
impact of in vitro TI callus-to-shoot organogenesis on p24 expression (Figure 5.4 (A)). For
comparison, leaves 12 and 6 from a soil-grown plant were included, corresponding to the
most recent plastochron cycle (youngest leaf) and the plastochron mid-way through plant
development, respectively. Expression of p24 in TIB-regenerated shoots is comparable to that
in leaf 6, suggesting that vegetative biomass growth provides some mitigation against
proteolytic decay of the recombinant protein. Expression in the in vitro grown plantlet is
analogous to that in the youngest leaves.
139
The effect of developmental status of TIB-grown biomass on p24 accumulation was also
investigated (Figure 5.4 (B)), comparing p24 intrinsic yields in callus suspension (TIB
inoculum), shoot primordia and regenerated shoots after a 40-day culture period.
Accumulation of p24 is detected in leaves and shoot primordia buds (i.e. intermediate
between callus and fully differentiated leaves). In comparison, no detectable expression was
observed in callus suspension. Accumulation in leaves was greater than that in primordia.
This analysis demonstrates the dependence of transplastomic protein expression on the
degree of morphogenesis of plant biomass. This reflects the upregulated plastidial gene
expression associated with chloroplast development.
Figure 5.4 SDS-PAGE and Western immunoblot for demonstration of the expression of p24 in
TIB-grown biomass. (A) Comparison of p24 accumulation in in vitro grown biomass and
seedlings. Accumulation of p24 was compared between shoots grown in RITA® temporary
immersion bioreactors (TIBs), the youngest leaf of an in vitro seedling and leaves 6 and 12 of an 8-
week old soil-grown seedling. (B) The influence of in vitro callus morphogenesis on p24
expression in TIB. Approx. 11 µg protein loading. 15% acrylamide; Coomassie staining; Precision
Plus marker.
140
5.2.3 Discussion on the expression of proteolytically unstable transplastomic proteins
via TI regeneration
These studies involving VP6 and p24 synthesis in transplastomic tobacco confirm previous
reports of proteolytic instability of the foreign protein in soil-grown plants. In vitro
micropropagative shoot regeneration is an alternative cultivation technology, and so
experiments were performed to assess the impact of this different developmental pathway on
transplastomic protein expression. For p24 expression, in vitro organogenesis seems to
provide some protection against proteolytic degradation, though this effect is less apparent
for VP6. VP6 appears to be more susceptible to proteolytic degradation than p24, as
evidenced by lower expression relative to the soil-grown equivalent. Despite recombinant
protein expression in both transplastomic strains being dependent on the same plastid gene
expression regulatory mechanisms and similar plastid protease environments, VP6 and p24
exhibit different accumulation levels. It must be remembered that the intrinsic physico-
chemical properties of recombinant proteins also influence their susceptibility to degradation,
e.g. number of endopeptidase sites, steric access of proteases, tendency to aggregate. For p24
expression via in vitro biomass growth, protease degradation has little real significance, as
the elevated expression associated with morphogenesis and chloroplast development far
exceeds the rate of degradation.
The differences in proteolytic landscapes between in vitro regeneration and soil cultivation
may be explained by invoking the different morphological developmental pathways. Seedling
germination and growth involves bud initiation culminating in leaf formation at the shoot
apical meristem. This occurs in a sequential manner, giving a linear stem with nodes giving
rise to petioles and leaves, and the internodes between each leaf representing the plastochrons
between each successive bud initiation. In contrast, in vitro morphogenesis in TIB culture
involves bud initiations from multiple meristemic nodes in callus clusters. Moreover there is
absence of apical dominance, TDZ-induced suppression of root formation and lack of
tropisms (due to continual displacement of biomass). This gives rise to complex phyllotaxic
arrangements, since several leaves appear concurrently within a short space of time. The in
vitro rapid proliferation of shoots and leaves does not promote age-related protease activity.
In contrast, the oldest leaves in soil-cultivated plants are prone to senescence, involving high
protease activity. From a regulatory perspective, the gradient in transplastomic protein
expression with successive plastochrons in soil-grown plants is unfavourable, as this reduces
141
batch-to-batch consistency and makes it difficult to accurately determine overall recombinant
protein yields. In comparison, rapid shoot proliferation in temporary immersion culture
should be more reproducible. Accumulation of both VP6 and p24 are greater in soil-grown
plants than in vitro regenerated shoots. The reduced protein yield in in vitro regenerated
shoots may be a result of the imposition of aberrant environmental conditions (compared to
greenhouse or field conditions) (Gaspar et al., 2002).
It must be remembered that differential intracellular protein accumulation represents the
difference between protein synthesis and protein degradation (Doran, 2006). There has been
much progress in the development of molecular biology strategies for modulating
heterologous protein expression. However, high transcription does not guarantee high levels
of heterologous protein accumulation for a number of transgenic plant systems (Doran,
2006). The literature contains several examples of transgenic plant systems in which there is
little correlation between mRNA transcript level and protein yield for both nuclear
transformants (Ohtani et al., 1991; Outchkourov et al., 2003; Richter et al., 2000) and
chloroplast transformants (Birch-Machin et al., 2004), suggesting that proteins are effectively
expressed but subsequently degraded.
Green leaves have become the de facto host organ of choice for molecular farming
applications, in spite of the high metabolic rate and the proteolytic activity associated with
endogenous protein and turnover. This is because of their rapid growth rate, the application of
conventional agricultural methods, and the availability of numerous regulatory sequences
adapted to transgene expression in the leaf cell environment (Benchabane et al., 2008). As
demonstrated in Chapter 3, leaves are especially suitable for transplastomic protein
expression, due to transgene hyperexpression associated with upregulation of plastid gene
expression during photosynthetic development. For transplastomic proteins, although
proteolysis is a major issue, its impact is largely mitigated by the inherently high protein
synthesis rates.
To summarise the results of the studies outlined in this chapter, expression of subunit vaccine
candidates VP6 and p24, against rotavirus and HIV respectively, is possible via temporary
immersion regeneration. Although the intrinsic yields for both proteins are less than that for
soil cultivation, it must be remembered that high biomass yields are possible with TIB
culture, 250 – 300 g/l, with near 100% volume occupancy of the culture vessel. Moreover,
142
since in vitro shoot proliferation occurs in a short time, there is a reduced gradient of
expression with increasing age of biomass, giving reduced batch-to-batch variability. With
optimisation of culture conditions or cellular engineering strategies such as protease-
knockout, it may be possible to undertake large scale manufacture of subunit vaccines for
vaccination programmes in endemic regions. VP6 and p24 are of special importance, as
subunit vaccines against two highly dangerous viruses, especially prevalent in the developing
world. Viral capsid proteins, like VP6 and p24 are ideal candidates for subunit vaccines
because of their high immunogenicity and conservation as structural proteins between virus
subtypes (Novitsky et al., 2001). In a number of viral infections, immune responses directed
against viral coat proteins have been found to be protective, like hepatitis B and influenza
(Gottlieb and Ben-Yedidia; Iwarson et al., 1985; Russell and Liew, 1980).
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Chapter 6. Developing new tools for in vitro molecular
farming
6.1 Development of large-scale mechanical bioreactor
6.1.1 The need for scale-up of in vitro organogenesis for molecular farming purposes
In principle, field-cultivated plants as biopharmaceutical protein biofactories should have
almost infinite scalability, in stark contrast to conventional microbial and mammalian cell-
based systems (Fischer et al., 2013; Rybicki, 2009). However, the world’s productive
agricultural land is not unlimited, and is set to be under considerable pressure from a rising
population, intensification of food production as well as demand for biofuels (Lymbery,
2014). Although field production to provide cheap vaccines to developing countries is
morally laudable, it is possible that at a large-scale, this will divert agricultural capacity away
from much-needed food crops, and may cause food prices to rise, not to mention the
possibility of transgene release. In this context, the strength of in vitro plant growth platforms
is the decoupling of biomass growth from agricultural resources. Nevertheless, the challenge
of scalability still remains. For the supply of high-value vaccines and biotherapeutics from in
vitro micropropagative approaches to be feasible, high-throughput bioprocessing will be
needed. In Chapter 3, we saw that with in vitro shoot regeneration in RITA® temporary
immersion bioreactors, typical yields of 2 mg l-1 of TetC antigen were possible, which is
enough to provide enough vaccine doses, either intranasally or orally for 250 or 20
individuals, respectively. An estimated 3,350 RITA® cultures alone would be required to
supply intranasal tetanus vaccines against all the babies born in the UK in 2013 (not factoring
yield losses in downstream processing). There is a need for a high-yield biomass growth
platform capable of large-scale biotherapeutics production. This study describes the
demonstration of a custom-made 60 l mechanical temporary immersion bioreactor for large-
scale transplastomic protein synthesis.
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6.1.2 Design of experiment
A prototype 60 l mechanical (hydraulic) temporary immersion bioreactor was custom-built
(Figure 6.1). Duplicate temporary immersion shoot regeneration cultures of Nt-pJST12
biomass were undertaken according to the methods described in section 2.2.2.5, for 50 and 80
days respectively. After the allotted time periods, the biomass was harvested, weighed and
total soluble proteins (TSPs) were extracted for SDS-PAGE and immunoblot analysis of TetC
expression. Biomass growth and TetC yields were compared to that of standard RITA®
TIBs. We invented, designed and constructed the large temporary immersion bioreactor for
this study, as described in section 2.2.2.5.1.
Figure 6.1 Large 60 l mechanical bioreactor in operation
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Figure 6.2 Schematic showing operation of large bioreactor.
(A) When the mechanical jack piston is in the default ‘up’ position, the biomass chamber and bag
assembly are raised and the medium settles in the bulges beneath the biomass chamber.
(B) The piston is lowered for nutrient delivery. The displacement of liquid medium and the space
constraint causes the biomass to be immersed.
6.1.3 Results
6.1.3.1 Biomass Accumulation and Organogenesis
After 50 or 80 days culture in the 60 l mechanical bioreactor, the biomass was harvested,
visually analysed and weighed. Regenerated biomass underwent complete morphogenesis,
similar to regenerated biomass grown in 0.5 l RITA® bioreactors (Figure 6.3). Shoots
displayed a higher degree of leaf formation and expansion than those grown in the RITA®.
This is probably related to reduced space limitations in the small-scale vessel.
A B
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Fresh and dry biomass accumulation were 1183.4 g and 56.1 g, and 2324.2 g and 98.1 g, after
50 and 80 days respectively. Between 50 and 80 days, biomass accumulated by 96% and 75%
for fresh and dry biomass, respectively. However, the proportion of fresh biomass composed
of hyperhydric (vitrified) shoots increased from 47.5% to 63% from day 50 to 80.
A B
Figure 6.3 Visual demonstration of shoot morphogenesis in large- and small-scale temporary
immersion bioreactors. (A) 60 l mechanical bioreactor; (B) 0.5 l RITA® TIB.
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Figure 6.4 Increase in fresh and dry biomass accumulation in the mechanical temporary
immersion bioreactor after 50 and 80 days culture
6.1.3.2 Comparison of biomass accumulation between the mechanical bioreactor
and RITA
Figure 6.5 shows a comparative analysis of fresh and dry biomass accumulation in the 60 l
mechanical and 0.5 l RITA bioreactors. Although comparatively, the mechanical bioreactor
produces 9.8- and 19.3-fold higher fresh biomass yields at days 50 and 80, respectively, than
that in the RITA® temporary immersion bioreactor, 120.1 g (Figure 6.5 (A)), this distinction
is superficial due to the size difference in culture vessel. For a more reliable analysis, the
biomass yields should be normalised. Figures 6.5 (B) and (C) show fresh and dry biomass
accumulation normalised against medium volume and the ‘floor space’ area of the vessels.
The volumes of medium used in the RITA® system and mechanical bioreactor system are 0.3
l and 16 l respectively. The estimated space footprints of the RITA® and mechanical
bioreactor are 0.015 m2 and 0.502 m2, respectively. Although the external diameter and actual
cross-sectional area of the RITA® is 0.124 m and 0.015 m2, since the cross-section is
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circular, the area was adjusted to that of a square to account for ‘lost’ space between stacked
vessels. The footprint of the mechanical bioreactor is based on the larger 60 l box. After
normalisation against medium volume or area, the adjusted biomass yield of the RITA® is
greater than that in the mechanical bioreactor. The fresh and dry biomass yields per litre of
medium are 2.8- and 2.6-fold greater in the RITA® bioreactor than the mechanical
bioreactor. The fresh and dry biomass yields per square metre of ‘floor space’ are 1.7- and
1.6-fold greater in the RITA® bioreactor than the mechanical bioreactor.
5.1.3.3 Comparative analysis of TetC expression in the mechanical bioreactor
SDS-PAGE and immunoblot analysis confirms the expression of TetC in shoot biomass
regenerated in the large mechanical bioreactor, in vitrified and non-vitrified shoots (Figure
6.6). Densitometric quantification of TetC yields was undertaken to estimate intrinsic yields
and ‘absolute’ yields in the large bioreactor (Figure 6.7 (A, B)). The absolute yield (per
bioreactor vessel) was normalised to volume of medium and ‘floor space’ area, just as for
biomass yields (Figure 6.7 (C, D)).
Figure 6.5 Comparison of fresh
and dry biomass accumulation in
large bioreactor and RITA® culture
vessels. (A) biomass accumulation per
vessel (non-normalised); (B) biomass
accumulation normalised to medium
volume; (C) biomass accumulation
normalised to space footprint (area).
A B
C
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Estimated intrinsic yields of TetC in the large TIB were 24.5 and 10.4 ng µg-1 TSP at days 50
and 80, compared to 7.1 ng µg-1 TSP in standard 40-day RITA culture. Hence in the large
TIB, at 50 and 80 days, there is a 3.4-fold and 46% increase, representively, compared to in
RITA culture. The estimated absolute yields of TetC per vessel at days 50 and 80 in the large
TIB are 28.0 and 23.3 mg, representing a 34- and 28- fold difference compared to in the
RITA, though this is largely due to the difference in size between these vessels. When the
absolute TetC yields are normalised to volume of medium or area, the yields in the large TIB
and RITA are comparable.
Figure 6.6 SDS-PAGE and immunoblot demonstrating TetC expression in large
mechanical TIB. Comparison of TetC yields between large mechanical TIB (50 days and 80 days
culture), 40-day RITA® TIB and young leaves of a soil-grown plant. 12% acrylamide; LWM marker
(A); Precision Plus marker (B); 14 µg protein loading per well; Coomassie staining.
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6.1.3.4 Discussion on the development of a large-scale bioreactor
Scaling of transgenic plant-based bioprocesses will be necessary to providing mass quantities
of inexpensive vaccines and other biotherapeutics, as well as deriving ‘economies of scale’
(Xu et al., 2012b). When scaling in vitro plant tissue culture processes for molecular farming
applications, there are two approaches that can be taken, scale-out and scale-up. Scaling-out
of field-grown plants is straightforward, by simply devoting more agricultural land to
cultivation, whereas for in vitro micropropagation it would involve multiplication of vessels
(and possibly growth facilities). Scaling-up is appropriate for in vitro cell suspension and
tissue cultures undertaken in bioreactors (Akita et al., 1994; Hellwig et al., 2004; Reuter et
Figure 6.7 Comparison of TetC yield in large bioreactor and RITA® culture vessels. (A) TetC
intrinsic yield (ng TSP / µg TSP) determined from densitometric analysis of immunoblots (B) Estimated
‘absolute’ TetC yield per bioreactor (mg TetC) (non-normalised) (C) Estimated TetC yield normalised to
medium volume (mg/l) (D) Estimated TetC yield normalised to space footprint (area). Error bars denote
standard errors.
A B
C D
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al., 2014), and approaches can be adapted from conventional microbial and mammalian cell
bioprocesses (Glacken et al.; Junker, 2004; Thiry and Cingolani, 2002). This study describes
the scale-up of shoot regeneration for TetC expression from 0.5 l (RITA® TIB) to 60 l
(mechanical bioreactor) and the implications on biomass growth and heterologous protein
yields.
In this study, the mechanism of medium immersion was altered upon scale-up. In the small-
scale RITA®, immersion is pneumatically-driven (Watt, 2012), whereas in the large
bioreactor, immersion was hydraulic, involving physical displacement of the medium. The
change in mode of operation resulted in no loss of TetC yield. In fact there was a 3.4-fold
increase in TetC intrinsic yield between the RITA® and large TIB at 50 days culture. This
may be associated with reduced imposition of abiotic stresses related to scaling of the culture
environment (Chaves et al., 2002; Ciarmiello et al., 2011). The large TIB presumably
experienced reduced humidity as a result of a larger headspace and the increased space
promoted organogenesis (Figure 5.3). When TetC yield is normalised to medium volume or
‘floor space’, the large TIB and RITA system give comparable yields. Relative to the
‘floorspace’ footprint, the actual TetC yield, after 50 days culture in the large TIB, was
roughly equivalent to that in the RITA. The 57% reduction in TetC intrinsic yield in the large
mechanical TIB between days 50 and 80 (Figure 5.7 (A)) may be related to age-related
proteolysis in the chloroplast. However, this is largely offset by exponential biomass growth,
so in terms of absolute yields, there is a 17% decrease in TetC yield between days 50 and 80.
Scale-up of RITA®-type bioreactors may be difficult due to the high energy requirements
required for gas-powered suspension of liquid media (Majumdar, 1996). This study
demonstrates the straightforward construction and operation of a large bioreactor using
simple, easily obtainable components. Although this is a prototype, similar inexpensive low-
tech micropropagation approaches may be adopted in developing countries for large-scale
production of plant-made vaccines (Savangikar, 2004). Alternatively, a scale-out approach
involving multiplication of small-scale RITA® or similar vessels may be used.
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6.2 The influence of pre-culture preservation of encapsulated callus on
temporary immersion morphogenic potential and TetC expression
6.2.1 Synthetic seed technology
The concept of a “synthetic seed” was first proposed in 1977 (Murashige, 1977), and has
since evolved into a field of intense research and experimentation within the field of
micropropagation. A synthetic (artificial) seed is defined as artificially encapsulated somatic
embryo, shoot bud or any other meristemic tissue that can be used as a functionally mimic
seed for sowing and possesses the ability for conversion to a plant under in vitro or ex vitro
conditions and that can retain this potential even after storage (Hicks, 1994; Munetaka, 1999).
Originally, synthetic seeds were used for encapsulation of somatic embryos, though has since
been extended to other non-embryonic vegetative propagules such as apical shoot buds, nodal
segments as well as calli (Babaoglu and Yorgancilar, 2000; Choffe et al., 2000; Park et al.,
2004a; Standardi and Piccioni, 1998). Despite great advances in the development of synseed
technology, a true analog to natural seeds is yet to be realised (Kumar et al., 2005). Synthetic
seed technology could provide an efficient, cost-effective means for mass clonal propagation
of plant material, if proliferation, rooting and conversion are well controlled (Piccioni, 1997;
Standardi and Piccioni, 1998). Encapsulation of in vitro-derived plant tissues can have
several applications in micropropagation, such as cultivation independent of natural and
seasonal conditions, conservation of germplasm, long-term storage of plant material through
cryopreservation and exchange of sterile material between laboratories (Teng, 1999).
There are two main types of synthetic seed, hydrated and dessicated. A number of coating
agents such as sodium alginate, potassium alginate, carrageenan, sodium alginate with
gelatin, sodium pectate, carboxymethyl cellulose are used for encapsulation, though sodium
alginate is most extensively used (Teng, 1999). An alginate hydrogel is frequently selected as
a matrix due to characteristics including moderate viscosity and low spinnability of the
solution, low toxicity and quick gelation (Onishi et al., 1994). Alginate beads can be made by
the ‘droplet hardening’ method, through dropping propagules with sodium alginate solution
(0.5-5.0% w/v) into CaCl2 solution (30-100 mM) (Onishi et al., 1994; Redenbaugh et al.,
1993). It has been observed that the gel capsule provides hindrance to the emergence of the
shoot and root.
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This study described below explains the application of ‘synthetic seed’ technology to the
long-term storage of Nicotiana tabacum callus germplasm of the Nt-pJST12 transplastomic
line and the subsequent in vitro regeneration of shoots in RITA® TIBs for biosynthesis of
transplastomic TetC protein.
6.2.2 Design of experiment
A major application of synthetic seed technology in micropropagation is the long-term
storage of germplasm prior to tissue culture, such as hairy roots, somatic embryos
(Vdovitchenko and Kuzovkina, 2011).
0.5 g callus aggregates were encapsulated in sodium alginate according to the method
described in 2.2.2.4 and stored at 4°C or 25°C, for 0, 9, 18, 40 or 138 days. Subsequently,
encapsulated callus propagules were inoculated into RITA temporary immersion bioreactors
and shoot regeneration cultures were undertaken, as duplicates. After 40-day temporary
immersion cultures, biomass was weighed, and SDS-PAGE and immunoblots of TSPs were
undertaken to assess the influence of germplasm storage time and temperature.
6.2.3 Results
6.2.3.1 Influence of duration and temperature of encapsulated callus preservation on
growth and morphogenesis in temporary immersion culture
Encapsulation of calli within an alginate matrix without storage had no apparent effect on its
capacity for expansion and morphogenesis in temporary immersion culture. The
Figure 6.8 Callus aggregates
encapsulated in a sodium
alginate matrix.
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morphogenesis dynamics exhibited by the encapsulated callus inocula were similar to those
without having undergone encapsulation. After a lag phase of approximately 10 days in
which little expansion is observed, expansion of the callus mass and development of
meristemic nodules was observed between days 10-25. Between days 20 and 25, formation of
shoot buds and differentiation of leaves were observed.
Although alginate encapsulation had little effect on the morphogenesis dynamics, the extent
of growth was affected by storage duration and temperature. The fresh and dry biomass
accumulation of temporary immersion regenerated shoots is indicative of the shoot
regeneration potential of the encapsulated callus germplasm (Figure 6.9). There is a decline
in temporary immersion culture regeneration potential, with increasing germplasm storage
time. Moreover, the regeneration potential is greater after storage at 4°C than at 25°C. After
storage for 18 days at 4°C and 25°C respectively, the fresh biomass accumulation is 72% and
70% of that having undergone no storage (day 0). After storage for 138 days at 4°C and 25°C
respectively, the fresh biomass accumulation is 37% and 5% of that having undergone no
storage (day 0).
Figure 6.9 Effect of callus encapsulation duration and
temperature on fresh and dry biomass accumulation in
temporary immersion shoot regeneration cultures. Error bars
denote standard error.
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6.2.3.2 Influence of Influence of duration and temperature of encapsulated callus
preservation on TetC expression in regenerated shoots
SDS-PAGE and immunoblot analysis was undertaken to assess the influence, if any, of callus
encapsulation duration and temperature on TetC intrinsic yield (Figure 5.10). No significant
differences were observed between the various treatments. The TetC intrinsic yields under all
storage durations and temperatures are comparable to that in biomass grown in the control
culture (inoculum was not encapsulated).
6.2.3.3 Discussion on the influence of alginate encapsulation on temporary
immersion regeneration and TetC expression
With alginate encapsulation for callus preservation, there is a trade-off in terms of the gradual
loss of regeneration potential with increased germplasm storage time, which is heightened at
room temperature compared to 4°C, as these results have shown. This is due to reduced
Figure 6.10 SDS-PAGE and
immunoblot demonstrating
TetC expression in shoot
biomass regenerated from
encapsulated callus stored for
various durations and
temperatures. TetC control
culture was inoculated with non-
encapsulated callus. Negative
control is TIB-grown WT biomass.
12% acrylamide; LWM marker
(A); Precision Plus marker (B); 8
µg protein loading per well; Sypro
orange staining.
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viability of callus germplasm with prolonged storage. However, alginate encapsulation may
still be useful as a viable method for short-term germplasm preservation, in spite of reduced
growth potential. For example, 40 days alginate preservation of callus at 4°C will result in
fresh biomass accumulation of 178.6 g l-1, compared to 277.8 g l-1 if no storage was
undertaken. Although this represents a 36% loss in fresh weight, the 40-day decoupling of
inoculum generation and biopharmaceutical manufacture may be invaluable in satisfying
unpredicted increases in demand. This reduction in growth potential of encapsulated
germplasm with increased storage time is consistent with previous studies (Naik and Chand,
2006; Perveen and Anis, 2014; Rai et al., 2008; Zych et al., 2005).
6.3 Discussion on described studies and how they relate to new
developments in in vitro molecular farming
These studies have highlighted two areas of intense research and innovation in the
micropropagation field, the development of large-scale cell and tissue culture capabilities,
and the preservation of germplasm. (Dodds, 1988; George et al., 2007; Murashige, 1977;
Patel et al., 2000). Such developments in micropropagative technology can be easily adapted
to the high throughput biosynthesis of plant-produced biopharmaceuticals.
The first study focussed on the development of a large-scale hydraulically-driven temporary
immersion bioreactor for the biosynthesis of transplastomic vaccines. Although there has
been notable progress in the development and scale-up of plant cell suspension cultures for
recombinant protein and small molecule synthesis over the last 30 years (Boivin et al., 2010;
Eibl and Eibl, 2008; Kieran et al., 1997; Kwok et al., 1992; Magy et al., 2014; Moon et al.,
1999; Scragg et al., 1988; Srinivasan et al., 1995; Terrier et al., 2007), there has been little
equivalent innovation in large-scale culture of differentiated plant tissues (Huang and
McDonald, 2012; Steingroewer et al., 2013). Most projects involving recombinant protein
expression in whole tissues have focussed on field or greenhouse seedling propagation
(Artsaenko et al., 1998; Busse et al., 2002; Ko and Koprowski, 2005), although as an
exception, there has been moderate progress in the development of systems for culturing
hairy roots / rhizosecretion (Drake et al., 2003; Drake et al., 2009; Wongsamuth and Doran,
1997) and mosses (Decker and Reski, 2004; Decker and Reski, 2007; Hohe and Reski, 2002).
It is likely that in the near future, tissue culture scale-up will become an intense field of
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research, as the need for high-throughput in vitro differentiated biomass growth systems for
biopharmaceutical synthesis will increase. This is especially relevant for transplastomic
protein expression, which is highly dependent on the development of mature chloroplasts in
differentiated leafy tissue. This study demonstrates a very straightforward strategy for the
scale-up of temporary immersion culture, which provides transplastomic protein yields
comparable to that in 0.5 l RITA cultures. Such a system does not require sophisticated
equipment, instrumentation or ‘stainless steel’ infrastructure associated with microbial
fermentation plants and is amenable to a disposable single-use bioprocessing approach (Eibl
et al., 2010; Huang and McDonald, 2012; Kwon et al., 2013). Single-use approaches confer
technical benefits relating to simplified bioprocess set-up such as ease of validation, less
capital investment for stainless steel vessels, reduced turnover time between each run, and the
potential use of integrated processes for more robust processes with shorter development time
and increased throughput (Eibl et al., 2010; Huang and McDonald, 2012).
The short-term to mid-term preservation of germplasm is an important part of any in vitro
micropropagation programme. Cryopreservation and alginate encapsulation are two
important methods for preservation of elite plant lines in commercial micropropagation and
plant conservation, though the latter is the simpler and cost-effective option (Perveen and
Anis, 2014). Hence alginate encapsulation protocols for vegetative propagules have been
established for a number of woody and non-woody species (Gardi et al., 1999; Hung and
Trueman, 2012; Kim and Park, 2002; Nagamori et al., 1999; Perveen and Anis, 2014). In
terms of in vitro tissue culture for biopharmaceutical production, preservation of germplasm
may confer several technical benefits. Alginate encapsulation may be employed in the
banking of stable transformant lines, after genetic stability has been established. Alginate
encapsulation can be used in the maintenance of a stable inventory of germplasm. Hence,
when demand for a biotherapeutic peaks, a source of tissue culture inoculum is readily
available to scale-up biosynthesis to satisfy demand. In practical terms, encapsulated
propagules may be easily transported between labs and culture facilities with minimal loss of
viability.
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Chapter 7 Summary and future directions
7.1 In vitro plant tissue culture as an alternative platform for
biosynthesis of biopharmaceuticals
The ‘molecular farming’ paradigm started in the early 1980s, when the first successful higher
plant transformation was reported, (Fraley et al., 1983), soon followed by the earliest reports
of expression of antibodies in plants (During, 1988; Hiatt et al., 1989). Since then, an intense
global programme of research and development into plants as suitable hosts for foreign
protein expression has been underway (Faye and Gomord, 2010; Ritala et al., 2014;
Schillberg et al., 2013; Spök et al., 2008a; Xu et al., 2012b). However, despite the
demonstrated advantages over conventional host technologies (Xu et al., 2012b), 30 years
later, only a small number of plant-produced therapeutics have been licensed, notably a
poultry vaccine against Newcastle disease (though this was never marketed) (Dow
Agrosciences, USA) (Katsnelson et al., 2006; Ritala et al., 2014), ELELYSO™ enzyme
replacement therapy for Gaucher disease (Protalix BioTherapeutics, Israel, in collaboration
with Pfizer) (Zimran et al., 2011) and ZMapp monoclonal antibody treatment against Ebola
(Qiu et al., 2014). A number of reasons behind the apparent decoupling of research and
commercialisation of plant-made biopharmaceuticals have been cited, including low initial
yields of foreign proteins, recalcitrance of industry leaders to replace firmly-established
microbial or mammalian bioprocesses with plant-based platforms, biosafety concerns related
to open field cultivation, and regulatory frameworks tailored to non-plant host systems
(Fischer et al., 2013; Martine et al., 2009; Soria-Guerra et al., 2011; Spök et al., 2008a; Spök
et al., 2008b; Xu et al., 2012b; Xu et al., 2011). It must be remembered that many of these
apparent bottlenecks hampering the adoption of molecular farming approaches are directly
related to the host tissue and cultivation method (Doran, 2013).
Historically, conventional soil-based cultivation of whole plants has been implicated as being
pertinent for the high-yield production of plant-made pharmaceuticals, as standard
agricultural procedures represent a very straightforward, low-tech approach and field
cultivation is highly scalable (Doran, 2000; Fischer et al., 2012; Rybicki, 2009; Stoger et al.,
2002; Xu et al., 2012b). Soil cultivation is the standard and most intuitive approach to both
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leafy and seed-based biomass growth (Xu et al., 2012b). The preference for soil-grown
seedlings appears to be reflected in much of the ‘molecular farming’ research zeitgeist over
the last 30 years (Chargelegue et al., 2005; Ma et al., 1994; Villani et al., 2009). In principle,
the case for agriculturally cultivated transgenic plants is compelling, though in reality, a
number of practical issues persist. Long development times, dependence of biomass yields on
prevailing environmental (biotic and abiotic) conditions and season, and genetic variation as
a result of sexual reproduction can affect product quality and consistency in accordance with
GMP principles (Fischer et al., 2012). Moreover transformational biosafety strategies, based
on transplastomic maternal inheritance (Daniell et al., 2002), or male sterility through RNA
silencing or barnase-induced (Commandeur et al., 2003; Gleba et al., 2004), are not absolute
guarantees against transgene pollution. Indeed, recalcitrance from both the public and
regulatory bodies have hindered large-scale field cultivation of transgenic biopharmaceutical
crops, especially in the European Union (Sang et al., 2013; Spök et al., 2008a). It is hoped
that the proliferative global adoption of genetically modified food crops (also known as
‘biotech’ crops) since the 1990s, especially USA, Brazil, Argentina, India, Canada, and
China, (James, 2013) will set the precedence for a similar adoption of biopharmaceutical
crops.
The strength of in vitro plant growth compared to agricultural propagation is in the ability to
control environmental conditions. Suspension culture has been pursued as a viable alternative
to agricultural plant propagation by researchers and industrialists (Hellwig et al., 2004;
Schillberg et al., 2013; Weathers et al., 2010; Xu et al., 2011). Suspension culture combines
the benefits of whole plant systems with those of microbial or mammalian cells (Hellwig et
al., 2004; Xu et al., 2011). Dedifferentiated callus aggregates can be cultured under scalable
tightly controlled conditions in a similar manner to industrial microbial fermentations, and
the same approaches to bioprocess optimisation can be applied (Xu et al., 2011). Moreover,
suspension cultures have fewer regulatory and environmental compliance hurdles for
ensuring product quality and safety than soil-grown plants (Xu et al., 2011). This must be
balanced against considerably higher capital costs than for soil-grown plants (Weathers et al.,
2010). Cell suspension culture has a relatively long history of over 50 years, initially as a
means for commercial high-value metabolite synthesis (Georgiev et al., 2009), and in recent
years as a platform for recombinant protein expression (Hellwig et al., 2004; Holland et al.,
2010; Schillberg et al., 2013; Vasilev et al., 2013; Weathers et al., 2010). Despite decades of
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bioprocess optimisation, low recombinant protein yields are still a major limitation of cell
suspensions (Twyman et al., 2013).
While agriculturally produced whole plants have been the host system of choice for
preclinical and clinical development of plant-made biopharmaceuticals (Karg and Kallio,
2009), and cell suspension technologies are emerging as a technically superior alternative
(Xu et al., 2011), there has been relatively little development in the development and scale-up
of plant tissue and organ cultures for biopharmaceutical production (Steingroewer et al.,
2013). While micropropagation techniques for multiplication and ex vitro transfer of plantlets
have been developed and optimised for decades, these have been applied mainly to
commercial horticulture and conservation of rare plants (Akin-Idowu et al., 2009; Dubranszki
and da Silva, 2010; Escalona et al., 1999; Kumar et al., 2006; Zych et al., 2005). There has
been some precedence of in vitro differentiated plant tissue culture for synthesis of bioactive
metabolites, though this has been largely limited to hairy roots and adventitious roots
(Steingroewer et al., 2013; Weathers et al., 2010). There has been considerable investigation
of hairy root culture for synthesis of bioactive metabolites, though scaled-up and
commercially feasible technologies are currently lacking (Bourgaud et al., 2001; Choi et al.,
2006; Georgiev et al., 2008; Steingroewer et al., 2013). Adventitious roots have been
particularly successful for commercial production of several metabolites, with over 45 tonnes
fresh weight per year reported by CBN Biotech Company (South Korea) for ginseng
production (Baque et al., 2012; Steingroewer et al., 2013). Although the in vitro culture of
differentiated explants such as shoots, plantlets, bulbs, microtubers and embryos have been
undertaken in commercial micropropagation for decades (George et al., 2007), and more
recently for the synthesis of bioactive metabolites (Steingroewer et al., 2013), there are few
reports of these used for the expression of recombinant proteins. Recent studies undertaken
by the Nixon group demonstrated that in vitro shoots regenerated from callus in temporary
immersion bioreactors could result in the overexpression of transplastomic proteins,
including TetC tetanus antigen, GFP+ (Michoux et al., 2011) and Lyme disease vaccine
antigen (Michoux et al., 2013). These were the first reports of in vitro shoots regenerated in
bioreactors being used for the expression of foreign proteins and the studies presented in this
PhD dissertation follow on from these pioneering studies.
Perhaps an explanation for the limited exploitation of in vitro tissue and organ cultures for
molecular farming lies in the intrinsic features of in vitro differentiated plant tissues (Huang
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and McDonald, 2012; Steingroewer et al., 2013). Suspension cultures, being composed of
undifferentiated callus aggregates have been relatively simple to grow in accordance with
GMP principles, borrowing heavily from bioprocessing strategies of established microbial
fermentations and mammalian cell cultures (Hellwig et al., 2004; Schillberg et al., 2013;
Weathers et al., 2010; Xu et al., 2011). In contrast, tissue cultures are morphologically and
biochemically complex. Traditional bioreactor configurations used for cell suspensions such
as stirred-tank reactors are not suited to differentiated tissues, as mechanical agitation,
hydrodynamics and reduced mass transfer and oxygen supply limitations in liquid culture can
be damaging to plantlet morphology at high biomass concentrations (Huang and McDonald,
2012; Steingroewer et al., 2013; Weathers et al., 2010). However, with modifications to
design and operation of stirred tank reactors, stirred tank bioreactors may be adapted to
differentiated tissues (Steingroewer et al., 2013). For example, low impeller speeds of 30-100
min-1 are recommended and tolerable tip speeds vary between 1 and 2 m s-1 (Steingroewer et
al., 2013). It is recommended to keep power input per unit volume under 1000 W m-3 for
reduced shear damage (Steingroewer et al., 2013). Pneumatic bioreactors, such as bubble
column reactors, have simple designs and operations, and may be used to alleviate the issues
associated with mechanical agitation, though there may be mass transfer limitations at high
tissue densities, caused by gas channelling among dense organs (Choi et al., 2006;
Sivakumar, 2006; Steingroewer et al., 2013; Vlaev and Fialova, 2003; Wang and Zhong,
2007). For metabolite production in hairy root culture, ‘unorthodox’ designs such as liquid-
dispersed and gas-phase bioreactor configurations have been used to alleviate such issues
(Weathers et al., 2010). Avoidance of stress conditions related to permanent submersion,
through the use of temporary immersion micropropagation has also been applied for a variety
of explants (Watt, 2012), though research undertaken in the Nixon group represents the first
application of this for biopharmaceutical production (Michoux et al., 2013; Michoux et al.,
2011).
The studies presented in this dissertation demonstrate the feasibility of in vitro tissue culture
via temporary immersion culture as an alternative to both agricultural propagation and cell
suspension, providing the advantages of differentiated tissue cultivation with the ease of
manipulating production conditions associated with suspension culture in bioreactors. In this
system, biomass and recombinant protein yields can easily be modulated through
manipulation of various culture parameters. The temporary immersion organogenesis system
can be a viable platform for large-scale GMP production of biopharmaceuticals, in a
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technically competent and economical manner. This system is especially suitable for the
biosynthesis of transplastomic proteins, as intrinsic protein expression is approximately 15-
fold higher in differentiated shoots than callus suspension in the case of TetC, reflecting
chloroplast maturation during morphogenesis. Moreover, 500-fold fresh weight biomass
accumulation is observed in this system.
7.2 The influence of temporary immersion shoot regeneration on
biosynthesis of transplastomic proteins
Chapters 3, 5 and 6 of this PhD dissertation have focussed on the biosynthesis of
transplastomic proteins in temporary immersion culture of N. tabacum shoots. In particular,
Chapter 3 focussed on the effects of various culture parameters on yield of TetC.
In the early development of the molecular farming paradigm, low intrinsic yields of foreign
proteins rendered transgenic plants uneconomical host systems compared to established
mammalian and microbial systems, and hindered the adoption of the new technology by
industry (Sabalza et al., 2014). Currently, these low yields have been addressed a number of
genetic strategies, transplastomics for overexpression in plastids (Bock, 2007; Bock, 2014;
Clarke and Daniell, 2011; Maliga and Bock, 2011), transient expression (Boivin et al., 2010;
Davies, 2010; Joh et al., 2005), inclusion of genetic regulatory elements in transplastomic and
nuclear transformation constructs for more efficient transcription and translation (Parra et al.,
2011; Ruhlman et al., 2010; Sharma et al., 2008). Plant growth conditions are also important
in determining yields of foreign protein (Sabalza et al., 2014).
As was emphasised in Chapter 3, ‘absolute’ foreign protein yield is the multiplication of
intrinsic yield (target protein as percentage of total soluble protein or per unit biomass) by
total biomass (expressed as fresh weight or soluble protein equivalent). Both depend on the
complex interplay of several genetic, biochemical, physiological and environmental factors.
The intrinsic yield is a measure of accumulation of protein, which depends on the rate of
protein synthesis balanced against protein degradation (Sabalza et al., 2014). In vitro biomass
accumulation depends on several metabolic and physiological factors including nutrient
163
assimilation, photosynthetic capacity and cytokinin-induced morphogenesis (George et al.,
2007).
Various culture treatments and factors were found to affect transplastomic protein
accumulation (both intrinsic and absolute), to various degrees. Transplastomic protein yields
were especially sensitive to nitrogen source ratio, sucrose concentration, irradiance, MS basal
medium concentration, hydrodynamics and protease activity. Other treatments were found to
have little or no influence on transplastomic protein accumulation, including water stress-
induced hyperhydricity, medium pH, mechanical immersion (as opposed to pneumatic
immersion) and scale-up, and encapsulation of callus inocula in alginate.
It is possible that protein localisation in the chloroplast stroma may isolate the foreign protein
from certain cellular phenomena which would otherwise adversely affect protein
accumulation. For example, hyperhydricity, a predominantly apoplastic phenomenon, was
demonstrated to have no effect on TetC expression in the plastids. Encapsulation of callus in
alginate presumably had no influence on chloroplast development, and therefore did not
affect transplastomic protein expression (though it did influence the extent of shoot
regeneration). Transplastomic protein expression was enhanced at reduced sucrose
concentrations, which is directly related to elevated photosynthetic capacity and development
of chloroplast thylakoids (Arigita et al., 2002). The requirement of both nitrate and
ammonium for biomass morphogenesis and transplastomic protein expression is related to the
assimilation of nitrogen for synthesis of amino acid and proteins and nitrogen metabolism in
the chloroplasts.
The experiments investigating the influence of hydrodynamics on biomass accumulation and
transplastomic protein were particularly intriguing, with implications for scale-up of
pneumatic temporary immersion culture. This was the first reported attempt to quantify
biological responses against hydrodynamics for in vitro shoot regeneration, although similar
studies have been undertaken for plant suspension cultures (Dunlop et al., 1994;
MacLoughlin et al., 1998; Scragg et al., 1988; Sowana et al., 2001). For 40-day temporary
immersion cultures, a critical air flow rate of 440 ml min-1 was identified, corresponding to
an average shear rate of 96.7 s-1 and energy dissipation rate of 8.82 mW kg-1 and total energy
dissipation of 127 J kg-1 (over first 20 days), which resulted in significantly reduced biomass
accumulation, mitochondrial activity and transplastomic protein yields. If scale-up of
164
pneumatic temporary immersion regeneration is to be undertaken, it is recommended not to
reach these values. The steady decline in transplastomic protein intrinsic and volumetric
yields even at moderate air flow rates suggests that operating at low air flow rates is
advisable.
These studies investigating the influence of various parameters on transplastomic protein
yields can serve as “prior art” for further optimisation of culture conditions for maximisation
of protein yields. These studies involved deviations from standard culture conditions,
involving the classical approach of alteration of one parameter at a time. The limitation of
this approach is that it does not account for interactions between factors tested (Vaidya et al.,
2009). However, these studies can be the starting point for medium optimisation by
‘statistical experimental design’ techniques, such as fractional factorial design or response
surface methodology, which can give insight into synergistic effects between medium
components (Dinarvand et al., 2013; Jeon et al., 2014; Vasilev et al., 2013). A combination of
fractional factorial design and response surface methodology was applied to the optimisation
of culture medium for tobacco BY-2 cells producing a secretory antibody (Vasilev et al.,
2013).
7.3 Scale-up of callus-to-shoot regeneration for biopharmaceutical
expression
Chapter 6 of this PhD dissertation described the scale-up of temporary immersion shoot
morphogenesis for transplastomic biopharmaceutical expression in a hydraulically-driven
mechanical bioreactor, from 0.5 l (standard bench-scale RITA® culture) to 60 l (volume of
the biomass chamber, though the volume of the entire set-up was 145 l). This is the first
reported attempt of the scale-up of in vitro callus-to-shoot morphogenesis, and could be the
basis of large-scale biopharmaceutical synthesis in differentiated tissues. Importantly, it was
observed that biomass accumulation and transplastomic protein yields were comparable to
that in small-scale RITA® bioreactors.
While suspension cultures have been successfully scaled up from shake flasks to working
volumes of 50 – 100 l or greater, with little impact on growth kinetics (Terrier et al., 2007),
tissue and organ cultures are known to be difficult to scale-up (Steingroewer et al., 2013;
165
Weathers et al., 2010). This has not been a pertinent issue in commercial horticultural
micropropagation, in which multiplication of small-scale culture vessels has been common
(George et al., 2007). To date, little progress has been made in the scale-up of shoot cultures.
Shoot propagules are sensitive to shear damage and form dense clumps in which mass
transfer may be limited. Mass propagation of Stevia rebaudiana shoots to 65 kg fresh weight
were reported in a 500 l bioreactor without mechanical agitation (Akita et al., 1994;
Takayama and Akita, 1994; Takayama and Akita, 2006). More modest attempts to scale-up
micropropagation, such as producing high quality orchid plantlets in medium-scale rocker
boxes, have been reported (Adelberg, 2006; Weathers et al., 2010).
The choice of explant is also important in scale-up of tissue culture for biopharmaceutical
expression and can affect product quality and consistency. The 500 l culture of Stevia used
shoots as inocula, though this may be inadvisable for biopharmaceutical expression. Direct
organogenesis using shoots as inocula may be difficult to scale-up for a number of reasons.
Shoots have only a limited number of meristems, thereby limiting regenerative potential.
Shoots have complex morphologies, which complicates scale-up approaches as well as
handling and inoculation (Akita et al., 1994; Takayama and Akita, 1994; Takayama and
Akita, 2006). Moreover, the competence to form shoot buds can be highly variable and
depend on length of leaves (Sreedhar et al., 2008), and regenerative potential decreases with
age of leaves (Ibrahim and Debergh, 2001; Sreedhar et al., 2008). As a compromise,
primordia can be used as inocula, being more convenient for inoculation and able to form
many shoots (Akita et al., 1994; Takayama and Akita, 1994). In the studies outlined in this
PhD thesis, dedifferentiated callus was used as inocula. Callus tissue is an advisable choice of
explant for molecular farming purposes. Callus is a relatively homogeneous, unorganised
tissue with high regenerative potential, containing multiple meristemic nodes (George et al.,
2007; Ikeuchi et al., 2013), and therefore better suited to GMP production of
biopharmaceuticals.
7.4 Biosynthesis and assembly of functional monoclonal antibodies in
temporary immersion shoot regeneration
30 years of previous studies have confirmed that plants possess the relevant cellular
‘machinery’ to express, assemble and post-translationally modify antibodies with antibody-
166
binding function (Artsaenko et al., 1998; Brodzik et al., 2006; Drake et al., 2003; Drake et al.,
2009; Hiatt et al., 1989; Hiatt and Pauly, 2006; Holland et al., 2010; Khoudi et al., 1999; Ko
and Koprowski, 2005; Ko et al., 2003; Ma et al., 1998; Ma et al., 1994; Magy et al., 2014;
Qiu et al., 2014; Tavladoraki et al., 1993; Vasilev et al., 2013). However, the studies
described in Chapter 4 describe the first reported attempt to express functional monoclonal
antibodies in in vitro regenerated shoots, using expression of nuclear-encoded Guy’s 13
antibody in N. tabacum as a model system. These studies demonstrate the potential
application of temporary immersion regeneration of shoots from callus for monoclonal
antibody expression, giving antibody titres comparable to soil-grown plants. Until now,
studies involving expression of mAbs in plants have involved soil cultivation of whole plants
(Artsaenko et al., 1998; Busse et al., 2002; Ko et al., 2009; Stoger et al., 2000), although there
have been some innovations in the development of root rhizosecretion systems (Drake et al.,
2003; Drake et al., 2009) and cell suspensions (Holland et al., 2010; Vasilev et al., 2013).
Unexpectedly, hyperhydricity (vitrification) of shoots resulted in increased antibody titres,
which presents an opportunity to enhance titres through imposition of water stress.
The expression of antibodies in transgenic plants (“plantibodies”) is one of the most exciting
technological developments of the molecular farming field (Ko et al., 2009; Ko and
Koprowski, 2005). This study presents an alternative in vitro biomanufacturing platform
which is amenable to GMP standards of quality and consistency. Glycosylation, the covalent
linkage of sugar moieties to proteins, is an important post-translational modification of
immunoglobulins in mammals (Wright and Morrison, 1997). In plant cells, glycosylation
occurs in the secretory pathway in the ER and Golgi. However, the mechanisms of N-linked
glycosylation differ in plants and mammals (Schoberer and Strasser, 2011). Plants add α(1,3)
fucose and β(1,2) xylose residues to the N-glycan of their glycoproteins, whereas mammals
add α(1,6) fucose moieties, glucose and sialic acid residues to the N-glycan (Obembe et al.,
2011). Plant glycans are immunogenic in several mammals, although their role in human
allergies has not been clarified (Bardor et al., 2003; Sabalza et al., 2014; van Ree et al.,
2000). Nonetheless, the potential impact of plant glycans on immunogenicity and adverse
allergic reactions, there is a need to engineer plants to emulate human N-glycosylation
(Gomord et al., 2010; Sabalza et al., 2014). One strategy is to include the fusion of the ER-
retention signal KDEL, to ensure glycosylation is only of the universal ‘high mannose type’
(Petruccelli et al., 2006; Saint-Jore-Dupas et al., 2007; Triguero et al., 2005). Another
approach is ‘glycoengineering’, in which host plants have been engineered to prevent
167
addition of plant-type glycans or add human-type glycans (Fischer et al., 2012; Ko et al.,
2008). However, plant glycosylation can even be beneficial for enhanced antibody avidity, as
is the case of Protalix’s Gaucher’s disease therapeutic, ELELYSO™ (taliglucerase alfa), a
recombinant glucocerebrosidase produced in carrot cells (Protalix BioTherapeutics, Israel)
(Fischer et al., 2012; Sabalza et al., 2014). The glucocerebrosidase is targeted to localise in
vacuolar compartments for terminal addition of plant-specific mannose for improved uptake
(Sabalza et al., 2014; Shaaltiel et al., 2007). The challenges associated with glycosylation of
plant-produced antibodies are considered one of the major regulatory bottlenecks of the
“plantibody” paradigm (Obembe et al., 2011). In our study, the impact of temporary
immersion shoot regeneration on glycosylation of Guy’s 13 antibody was not characterised.
As a future work, the glycosylation pattern and glycan quantitative ratio can be determined
by exoglycosidases digestion followed by HPLC analysis or GC-MS (Shaaltiel et al., 2007).
7.5 Implementation of robust bioprocesses for biopharmaceutical
synthesis
The studies outlined in this dissertation have investigated the feasibility of temporary
immersion shoot regeneration from callus as the basis for a bioprocess platform for high-
yield synthesis of biotherapeutics. Temporary immersion culture of N. tabacum biomass in
small-scale pneumatic systems (RITA® bioreactors) and a large-scale mechanical system
was undertaken. Such systems can be implemented with little capital equipment,
instrumentation or expertise, as opposed to conventional ‘stainless steel’ microbial
fermentation facilities. Standard temperature-controlled micropropagation or greenhouse
facilities can be used, such as those used in commercial horticulture, though laminar flow
hoods and extensive light and space would be required. Power requirements would be
considerably less than standard fermentation facilities. A simple inexpensive low-power air
pump can provide pneumatic suspension of liquid media for approximately 25 RITA®.
Although lighting would be a major energy sink, low power light-emitting diodes (LED)
could be used; these have been shown to effectively stimulate growth in a number of
micropropagative processes (Nhut et al., 2003; Okamoto et al., 1996; Tan Nhut et al., 2001).
Low-tech micropropagative facilities can be set up easily in developing countries, for
example, to provide cheap vaccines for the populace.
168
Much of the focus of molecular farming research has been optimisation of recombinant
protein yields, through molecular or growth strategies (Twyman et al., 2013). Unfortunately,
relatively little development of downstream processes for product extraction and purification
has been undertaken (Fischer et al., 2012). Downstream processing routes must conform to
GMP standards and result in a sufficiently pure and homogeneous pharmaceutical product,
according to regulatory requirements. Robust downstream processing technologies are well-
established for endotoxin removal from bacteria and viruses from mammalian cells (Fischer
et al., 2012). Since plants do not contain endotoxins or mammalian viruses, it is expected that
downstream processes for plant-based systems can be simplified from established
technologies. Downstream processing can account for 80% of the cost of plant-based
bioprocesses, so there are significant savings to be gained from optimisation (Evangelista et
al., 1998; Kusnadi et al., 1997).
169
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