2009
Annual Report
BERG | BioEngineering Research Group
Contents
04
About BERG
06 Executive Summary
07 The Research Group
08 Main Indicators
10
Research Activities
12 Bioprocess Engineering and
Biocatalysis Laboratory
14 Nucleic Acid Bioengineering
Laboratory
16 Stem Cell Bioengineering
Laboratory
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18
Research Highlights
20 Miniaturization in Biocatalysis
22 Bio-inspired Affinity Polymer Systems
for Antibody Recognition
24 Affinity-enhanced Extraction of Human
Antibodies
26 Designing Safer DNA Vaccines
28 Membrane Chromatography for DNA
Vaccine Purification
30 Ex-vivo Expansion of Mesenchymal
Stem Cells for Cellular Therapies: from
Lab to Clinic
32 Ex-vivo Expansion of mouse Embry-
onic Stem Cell-derived Neural Stem Cells
Under Low Oxygen
34
Scientific Output
36 Articles in Peer-reviewed Journals
38 Articles in Conference Proceedings
39 Book Chapters
39 Patents
39 Dissertations
40 Oral Communications
42 Poster Communications
46 Prizes
About BERG
20092009
BERG Annual ReportBERG Annual Report
20 5 09 Annual Report
Executive Summary
General Description
Main Indicators
The annual report of the BioEngineering Research Group (BERG) of Centre for Biological and Chemi-cal Engineering at IST highlights the activities of its three laboratories, within the Associated Labo-ratory Institute for Biotechnology and Bioengineer-ing (IBB). The major achievements in Bioprocess Engineer-ing include a novel separation process for the puri-fication of monoclonal antibodies based on multi-stage liquid-liquid extraction in aqueous two-phase systems, which led to a joint world patent with Bayer Technology Services (Leverkusen, DE). At the interface between Bioprocess Engineering and Nucleic Acid Bioengineering, unit operations (e.g. precipitation, tangential flow filtration, and aqueous two phase extraction) were evaluated, as alterna-tives in the intermediate recovery of plasmid DNA from E. coli lysates; and phenyl-boronate chroma-tography and hydrophobic interaction chromatog-raphy were successfully used for the purification of plasmid DNA directly from alkaline lysates. In Biocatalysis and Biotransformations, a new strategy for penicillin G acylase immobilization in sol-gel was successfully developed, allowing a remarkable improvement of enzyme activity in comparison with previously reported sol-gel tech-niques; and the production of androstenedione from sitosterol was scaled-up from multi-well plates to bench-scale stirred bioreactor using suit-able engineering parameters. The Nanobiotechnology research within the three BERG Laboratories include: (i) the development of portable magneto-resistive biochips for pathogen microorganism with femtomolar DNA limit detec-tion using specific antibodies immobilized on nano-magneto particles (in collaboration with INESC-MN); (ii) the use of electric field pulses to increase the speed of DNA immobilization and hybridization on chemically functionalized SiO2 thin-film chips by several orders of magnitude, with reaction time scaled down from 1 to 2 h to a range between 100 ns and 1 ms (in collaboration with INESC-MN); and (iii) the previously established 3-D cellular
microarray platform demonstrated to be a powerful tool for investigating cellular mechanisms underly-ing mouse ESC self-renewal versus differentiation, while providing the basis for rapid identification of signals and conditions that can be used to direct cell fate. With impact on the Healthcare sector, the major achievements on Stem Cell Bioengineering in-clude: (i) the consolidation of the consortium IBB-IST, Instituto Português de Oncologia de Lisboa Francisco Gentil and Centro de Histocompatibili-dade do Sul, focusing the isolation and ex-vivo expansion of Mesenchymal Stem Cells (MSC) under GMP conditions for Cellular Therapies, with three more patients being successfully infused with expanded MSC; (ii) the improvement of both human MSC and mouse Neural Stem Cells ex-vivo expansion under a low oxygen environment (i.e. hypoxia); and (iii) the successful establish-ment of a non-viral gene delivery platform using cationic liposomes for Bone Marrow MSC, allow-ing the expression of a target gene in a safe and transient way for potential use in Cell and Gene Therapy settings. A research network on Stem Cells, Tissue Engi-neering and Regenerative Medicine, “Stem Cell Engineering & Clinical Research net – StemCell-net” was launched in November, within the MIT-Portugal Program, under the coordination of BERG, to promote excellent quality research and advanced education programmes, ensuring an international competitiveness in the area of Re-generative Medicine.
Joaquim M.S. Cabral
BERG Head and
Director of IBB
Executive Summary
20 7 09 Annual Report
BEBL NABL SCBL
The Research Group
BERG
The BioEngineering Research Group (BERG) is a research unit in engineering and
life sciences at the Centre for Biological and Chemical Engineering (CEBQ). CEBQ is
the leading Centre of the Associated Laboratory Institute for Biotechnology and Bio-
engineering (IBB), a network of research centres across Portugal. IBB has been
identified by the Portuguese Ministry of Science, Technology and Higher Education
as a strategic infrastructure for the development of the Portuguese R&D and innova-
tion policies in the areas of Biotechnology, Bioengineering, Biomaterials and Life,
Biomedical and Agricultural Sciences. BERG activities within the Associated Labora-
tory IBB are focused on the Thematic Areas of Industrial Biotechnology and Health
Biotechnology.
BERG aims at excellence in research and advanced education in biotechnology and
bioengineering. The overall goal is to contribute for a better understanding of the
mechanisms that occur at the molecular and cellular levels, in order to translate them
into rational applications of biological systems relevant to the Industrial and Health
care sectors. BERG research priorities have special emphasis on Bioprocessing and
Biomolecular Engineering, Nucleic Acid Bioengineering, Nanobiotechnology and
Stem Cell Engineering, featuring an integrated cross-disciplinary approach through
three laboratories:
Bioprocess Engineering and Biocatalysis Laboratory (BEBL)
Nucleic Acid Bioengineering Laboratory (NABL)
Stem Cell Bioengineering Laboratory (SCBL)
BERG was established in 1991 as one of the initial three research groups of the Centre for Biological and
Chemical Engineering at IST, under the coordination of Prof. Joaquim Cabral. The research carried out
throughout the years has been considered of excellent level by the international committees, which regularly
evaluate the research units funded by the Portuguese Ministry of Science, Technology and Higher Educa-
tion. The main output of the activities performed by BERG members in 2009 are summarized in the following
tables.
Human Resources
With the creation of the Institute for Biotechnology and Bioengineering (IBB), a scientific policy has been
implemented to integrate complementary expertises in the group through the inclusion of new staff members
and Postdoctoral researchers in strategic scientific areas for the development of advanced biochemical engi-
neering research programs. In 2009, BERG had 66 researchers in three established programs, of which 21
have a PhD degree, 25 a master degree, 12 a five-year diploma degree, and 8 a bachelor degree.
Main Indicators
Human Resources Number
Faculty staff 10
Research scientists 5
Post-doctoral researchers 6
PhD students 28
Master students 9
Research assistants 7
Technicians 1
Male
Female
PhD
MSc
Diploma (5 years)
BSc
20 9 09 Annual Report
Publications
In 2009, the output of BERG‟s activities includes the publication of a patent in collaboration with Bayer Tech-
nology Services (Leverkusen, Germany), 2 book chapters and 50 scientific articles in peer-reviewed jour-
nals.
Scientific events
In 2009, the members of BERG have participate in organizing and scientific committees of national and in-
ternational conferences, research networks, working groups and sections of the European Federation of
Biotechnology.
Type of Event Name of Event Comittee
Conference 4th International Meeting of the Portuguese Society for Stem Cells and Cell Therapies (SPCE-TC), Lisbon, Portugal
Joaquim Cabral and Cláudia Lobato da Silva
Conference International Conference on Biopartitioning and Purification (BPP2009), Brunel, UK
Joaquim Cabral and Raquel Aires-Barros
Forum European Forum for Industrial Biotechnology (EFIB 2009), Lis-bon, Portugal
Joaquim Cabral
Network StemCellnet | Stem Cell Engineering and Research Network, Lisbon, Portugal
Joaquim Cabral
Workshop Workshop on Biosensors, Satellite event on 14th ECB - Barce-lona, Spain
Luís Fonseca
Workshop Global Network on Bioenergy/Biobased Economy Science (GNBES), Lisbon, Portugal
Joaquim Cabral
Workshop Nano09 Workshop “Grand challenges & New Trends in Nanosciences and Nanotecnologies”, Braga, Portugal
Joaquim Cabral
Type of Publication Number
Articles in international peer-reviewed journals 50
Proceedings in international peer-reviewed journals 13
Patents 1
Book chapters 2
Oral communications in international conferences 22
Poster communications in international conferences 44
Oral communications in national conferences 13
Poster communications in national conferences 12
PhD Thesis 5
MSc Thesis 3
Research Activities
20092009
BERG Annual ReportBERG Annual Report
20 11 09 Annual Report
Bioprocess Engineering and Biocatalysis
Laboratory BEBL
Nucleic Acid Bioengineering Laboratory NABL
Stem Cell Bioengineering Laboratory SCBL
Bioprocess Engineering and Biocatalysis
BEB
L
Objectives
Bioprocess Engineering and Biocatalysis aims to
design and develop value-added bioproducts with
potential application in the key areas of food and
feed, aroma, pharmaceutical industry and biofuels.
The current projects are focused on the develop-
ment of technological platforms for biocatalysis and
biomolecules purification organized in four major
areas: i) Bioseparation, ii) Biocatalysis and Biocon-
versions, iii) Biosensors and Miniaturization, and iv)
Bioenergy.
Research Topics
1. Bioseparations - Novel purification processes are
being developed to optimize and intensify the down-
stream processing of proteins and biopharmaceuti-
cals, with special emphasis on monoclonal antibod-
ies, including aqueous two-phase extraction (ATPE)
and affinity precipitation. Extraction separation units
using affinity materials and nano-magnetic particles
are being evaluated, characterized and validated by
modelling. Processes combining ATPE with chro-
matographic steps and using new biospecific
ligands are being exploited. Stimuli-responsive
beads based on PNIPAM are also being developed
as a strategy to produce novel bio-inspired affinity
polymer systems for antibody recognition.
2. Biocatalysis and Bioconversions - Rational strate-
gies for enzyme and whole cell bioconversions are
being developed and implemented, based on hy-
drolases (cutinase, penicillin acylase and inulinase)
and whole bacterial cells, where biocatalyst per-
formance is evaluated through a representative ar-
ray of reactions and operational conditions. Ester
biosyntheses (flavors, biodiesel and macrocyclic
esters) by cutinase and engineered mutants are
assessed in an enzymatic platform. The effect of
alternative media composition in biocatalyst per-
formance and cell wall integrity is being evaluated
using Rhodococcus and Mycobacterium strains as
model systems.
3. Biosensors and Miniaturization - Nano/micro-
biocatalysts (biocomposites) are being developed
based on hydrogels, protein/cell assemblies and
nano-magnetic particles. New protein-ionic-
conducting-based biocompatible materials (Ion
Jelly) with tailor-made properties have been de-
signed to build new planar amperometric biosen-
sors. Portable magneto-resistive biochip is in devel-
opment for pathogen microorganism detection (in
collaboration with INESC-MN). High throughput
systems are being used to assess the effects of
toxic compounds in cellular adaptation mecha-
nisms, to speed the optimization of biotransforma-
tion/fermentation systems and to establish scaling
strategies.
4. Bioenergy - Lipase and cutinase are being used
in non-conventional media for the sustainable pro-
duction of regular and jet biofuel. Oxido-reductases
are used in combination with an innovative material,
Ion Jelly, for structuring enzymatic biofuel cells.
Rhodococcus spp. are being used for the desulfuri-
zation of crude oil, mitigating acid rain and air pollu-
tion, as well as for the production of electricity using
physicochemical cell surface properties.
Main Achievements
A joint world patent with Bayer Technology Ser-
vices (Leverkusen, DE) was published describ-
ing a novel separation process for the purifica-
tion of monoclonal antibodies based on multi-
stage liquid-liquid extraction in aqueous two-
phase systems (ATPS).
Glutaric acid was identified as the most effective
ligand for the purification of human antibodies
from a CHO cell supernatant using PEG/dextran
ATPE. This extraction step was successfully
integrated with cation exchange chromatography
yielding a final product with 100% protein purity.
20 13 09 Annual Report
B
ioprocess E
ngin
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nd B
iocata
lysis
A new strategy for penicillin G acylase immobili-
zation in sol-gel was successfully developed
allowing a remarkable improvement of enzyme
activity in comparison with previously reported
sol-gel techniques.
Production of androstenedione from sitosterol
was scaled-up from multi-well plates to bench-
scale stirred bioreactor using suitable engineer-
ing parameters, namely volumetric power con-
sumption for resting cells and volumetric oxygen
transfer coefficient for growing cells.
Portable magneto-resistive biochip has been
developed for pathogen microorganism with
femtomolar DNA limit detection using specific
antibodies immobilized on nano-magneto parti-
cles.
High throughput systems were developed to
correlate the effect of toxic compounds with cel-
lular adaptation using Rhodococcus erythropolis
as model cells, to improve biotransformation
systems.
Selected Publications
Azevedo, A.M., Rosa, P.A.J., Ferreira, I.F., de
Vries, J., Visser, T.J., Aires-Barros, M.R., J. Chro-
matogr. B, 887: 50-58
Carvalho, F., Marques, M.P.C., de Carvalho,
C.C.C.R., Cabral, J.M.S., Fernandes, P., Biore-
source Technol., 100: 4050-4053
Bernardino, S.M.S.A., Fernandes P., Fonseca,
L.P., Biotechnol. J., 4: 695-702
Martins, V.C., Cardoso, F.A., Germano, J., Car-
doso, S., Sousa, L., Piedade, M., Freitas, P.P.,
Fonseca, L.P., Biosens. Bioelectron., 24: 2690-
2695
de Carvalho, C.C.C.R., Wick, L.Y., Heipieper, H.J.,
Appl. Microbiol. Biotechnol., 82: 311–320
Patent
Baecker, W., Sommerfeld, S., Mutter, M., Rosa,
P.A.J., Aires-Barros, M.R., Azevedo, A.M., World
Patent WO/2009/112149
NAB
L
Nucleic Acid Bioengineering
Objectives
Nucleic Acid Bioengineering is focused on: i) plas-
mid DNA vectors and their application in gene ther-
apy or DNA vaccination; and ii) microchips for DNA,
protein and cell detection. The specific objectives
are to address the scientific/technological chal-
lenges associated with plasmid biopharmaceuticals,
by combining biomolecular engineering studies with
bioprocess engineering, and to co-develop (with
INESC-MN) thin-film microchip and microfluidic plat-
forms for the manipulation/detection of DNA, anti-
bodies and cells.
Research Topics
In the case of plasmids, the following research top-
ics are pursued:
1. Structural stability of plasmids - Studies on the
nuclease barriers to gene expression during plas-
mid trafficking through the cytosol of mammalian
cells are performed with the goal of constructing
plasmid vectors with an increased resistance to
nucleases and less prone to recombination, and
thus with a higher transfection activity. Additionally,
the structure of plasmid vectors is probed for the
presence of sequences like direct and inverted re-
peats that promote recombination and other insta-
bility events.
2. Manufacturing of plasmid vectors - Processes for
the production of plasmids are conceptually de-
signed, developed, optimised and compared. The
impact of specific plasmid structural elements in the
performance of upstream and downstream proc-
esses (microfluidization, tangential flow filtration,
membrane and fixed-bed chromatography, aqueous
two-phase extraction) and in the quality of the final
product is evaluated. Analytical procedures to moni-
tor manufacturing and control product quality are
also developed.
3. DNA vaccine prototyping - DNA vaccine candi-
dates are constructed by cloning antigenic proteins
of the causing parasite of sleeping sickness disease
(Trypanosoma brucei brucei) and tested in mice
models for their ability to generate cellular and hu-
moral responses, and to provide protection against
infection.
In the case of microchips for DNA detection the fol-
lowing topics are addressed:
1. Immobilization and handling of DNA and cells -
Thin film technologies, chemical modification, mi-
crofluidics and electronic addressing are used to
develop microchips for the molecular recognition of
specific analytes via hybridization or cell process-
ing. The core of the chips is a flat surface with im-
mobilized probe molecules or entrapped cells.
Other features include the presence of micro-
electrodes to generate electric fields that accelerate
the kinetics of binding/recognition and promote di-
electrophoresis.
2. Photodetectors - Amorphous silicon photodetec-
tors are developed for the optoelectronic detection
of coloured, chemiluminescent and fluorescent
molecules in thin film chips. The presence of these
molecules ultimately reports specific biorecognition
events such as DNA hybridization or metabolic cell
activity.
Main Achievements
Mice immunized intramuscularly with a DNA
vaccine coding for trans-sialidase were able to
survive after a challenge with the infective form
of T. brucei brucei parasites.
The use of increased temperatures (42ºC) im-
proves pCIneo amplification (up to 4-fold) in E.
20 15 09 Annual Report
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ucle
ic A
cid
Bio
engin
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coli and decreases the ratio of plasmid recombi-
nation. The characteristic heterodimeric plasmid
recombinants are differently selected according
to the concentration of kanamycin used.
Unit operations (e.g. alcohol and salt precipita-
tion, tangential flow filtration and aqueous two
phase extraction) were evaluated in terms of
technical performance, economic viability and
environmental impact, as alternatives in the in-
termediate recovery of plasmid DNA from E. coli
lysates. The use of phenyl-boronate chromatog-
raphy and hydrophobic interaction membrane
chromatography for the purification of plasmid
DNA directly from alkaline lysates was demon-
strated.
The examination of the role of probe-to probe
interactions on the hybridization of double
stranded DNA in magnetic microparticles indi-
cated that the intensity of the hybridization sig-
nal is proportional to differences between the
average area available per probe and the pro-
jected surface area of each probe.
The use of electric field pulses was shown to
increases the speed of DNA immobilization and
hybridization on chemically functionalized SiO2
thin-film chips by several orders of magnitude,
with reaction time scaled down from 1 to 2 h to
a range between 100 ns and 1 ms (in collabora-
tion with INESC-MN).
Selected Publications
Silva, M.S., Prazeres, D.M.F., Lança, A., Atouguia,
J., Monteiro, G.A., Parasitol. Res., 105: 1223-1229
Oliveira, P.H., Prazeres D.M.F., Monteiro, G.A., J.
Biotechnol., 143: 231-238
Freitas, S.S., Canário, S., Santos, J.A.L., Prazeres,
D.M.F., Biotechnol. J., 4: 265-278
Martins, S., Prazeres, D.M.F., Fonseca. L.P., Mon-
teiro, G.A., Anal. Bioanal. Chem., 394: 1711-1716
Cabeça, R., Rodrigues, M., Prazeres, D.M.F., Chu,
V., Conde, J.P., Nanotechnology, 20: 015503
SCB
L
Stem Cell Bioengineering
Objectives
The Stem Cell Bioengineering Laboratory aims at
the development of highly controlled culture sys-
tems (e.g. bioreactors) for the ex-vivo expansion of
stem cells and their controlled differentiation into
specific cell types. As stem cells (SC) are rare, their
isolation and expansion/differentiation in vitro sig-
nificantly increases the cell population available for
cellular and gene therapy settings, high-throughput
drug screening, tissue engineering and stem cell
research. Human hematopoietic stem cells (HSC),
human mesenchymal stem cells (MSC), as well as
human and mouse embryonic stem cells (ESC) and
neural stem cells (NSC) are used as model sys-
tems.
Research Topics
1. Ex-vivo expansion of HSC in co-culture with MSC
under serum-free conditions - Current research is
focused on: (i) the definition of optimal culture con-
ditions namely concerning cytokine combinations,
enrichment procedures and initial cell concentra-
tions used to provide an amplification of HSC, espe-
cially those obtained from the umbilical cord blood
(UCB); and (ii) the understanding of the mecha-
nisms underlying the hematopoietic supportive ca-
pacity of MSC. These will have implications in terms
of bioreactor design towards the maximization of
human HSC expansion in vitro.
2. Clinical-scale production of MSC for Cellular
Therapies - Culture protocols are being optimized
for the isolation and expansion of MSC under se-
rum-/xeno-free conditions, while maintaining their
multilineage differentiation and immunosuppressive
capacities, as well as their karyotypic stability. MSC
are isolated from adult bone marrow (BM), adipose
tissue (AT) and UCB. Culture of MSC under condi-
tions more closely related to the in vivo environment
(i.e. hypoxia (2%O2), dynamic systems allowing for
3-D cultivation) is currently being exploited to maxi-
mize MSC yield.
3. Multi-scale strategies for the expansion and neu-
ral differentiation of ESC and NSC - The expansion
of ESC and ESC-derived NSC is studied towards
the definition of highly controlled bioreactor systems
to establish an efficient, reproducible and cost-
effective bioprocess to obtain the starting material
to generate mature cells (i.e. neurons) for potential
use in the treatment of neurological disorders, as
well as for drug screening. High-throughput microar-
ray systems are also being developed to elucidate
important microenvironmental factors (i.e. chemical,
physical) affecting cell self-renewal and differentia-
tion, while providing the basis for rapid identification
of signals and conditions that can be used to direct
cellular responses.
4. Gene delivery strategies to stem cells - The main
goal is to develop efficient and safe methodologies
to genetically-engineer SC, using non-viral vectors,
enhancing their therapeutic efficacy in different Re-
generative Medicine applications. Therapeutic
genes or specific genes involved in self-renewal/
lineage commitment of SC, are being targeted for
their transient and safe overexpression, allowing for
the direct use of gene-modified SC in Cellular and
Gene Therapy settings, or the maximization of the
SC yield or their progeny for application in Cellular
Therapies and Tissue Engineering, respectively.
5. Recombinant protein production for stem cell
research - A versatile platform for the efficient, cost-
effective and reproducible production and purifica-
tion of stable and biologically active recombinant
proteins has been developed based on mammalian
bioreactor systems. The main goal is to produce
cytokines/growth factors such as Leukemia Inhibi-
tory Factor (LIF) and Bone Morphogenetic Protein 4
(BMP-4) which can be used to supplement stem cell
cultures, but also as potential therapeutics
(e.g. BMP-4).
20 17 09 Annual Report
Ste
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Main Achievements
The consortium established between IBB-IST,
Instituto Português de Oncologia de Lisboa
Francisco Gentil and Centro de Histocompatibili-
dade do Sul, focusing the isolation and ex-vivo
expansion of MSC under GMP conditions for
Cellular Therapies was consolidated with three
more patients being successfully infused with
expanded MSC.
A divergent division kinetics was found between
BM and UCB HSC, elucidating the heterogeneity
underlying phenotypically identical HSC from
different sources. We also showed that the
beneficial effects on HSC expansion provided by
the MSC feeder layer used in our co-culture sys-
tem were mainly contact-mediated, rather than
based on soluble factor trafficking.
A low oxygen environment (i.e. hypoxia) was
demonstrated to improve both human MSC and
mouse NSC ex-vivo expansion. Hypoxia can
thus be considered a powerful tool towards the
maximization of cell yield from a reduced initial
population for potential application in clinical
settings.
Our previously established 3-D cellular microar-
ray platform demonstrated to be a powerful tool
for investigating cellular mechanisms underlying
mouse ESC self-renewal versus differentiation,
while providing the basis for rapid identification
of signals and conditions that can be used to
direct cell fate.
A non-viral gene delivery platform using cationic
liposomes was successfully established for BM
MSC, allowing the expression of a target gene in
a safe and transient way for potential use in Cell
and Gene Therapy settings.
Selected Publications
Fernandes, T.G., Diogo, M.M., Clark, D.S., Dordick,
J.S., Cabral, J.M.S., Trends Biotechnol., 27: 342-
349
Madeira, C., Estrela, N., Ferreira, J.A.B., Andrade,
S.M., Costa S.M.B., Melo E.P., J. Biomed. Optics,
14: 044035
Serro, A.P., Completo, C., Colaço, R., dos Santos,
F., da Silva, C.L., Cabral, J.M.S., Araújo, H., Pires,
E., Saramago, B., Surface & Coatings Technol.,
203: 3701-3707
da Silva, C.L., Gonçalves, R., Porada, C.D., Ascen-
são, J.L., Zanjani, E.D., Cabral, J.M.S., Almeida-
Porada, G., J. Cellular Physiol., 220: 102-111
Temtem, M., Silva, L.M.C., Andrade, P.Z., dos San-
tos, F., da Silva, C.L., Cabral, J.M.S., Abecasis,
M.M., Aguiar-Ricardo, A., J. Supercritical Fluids,
48: 269-277
Research Highlights
20092009
BERG Annual ReportBERG Annual Report
20 19 09 Annual Report
Designing Safer DNA Vaccines
Membrane Chromatography for DNA Vaccine Purification
Miniaturization in Biocatalysis
Bio-inspired Affinity Polymer Systems for Antibody Recognition
Affinity-enhanced Extraction of Human Antibodies
BEBL
NABL
Designing Ex-vivo Expansion of Mesenchymal Stem Cells for
Cellular Therapies: from Lab to Clinic
Ex-vivo Expansion of Mouse Embryonic Stem Cell-derived
Neural Stem Cells Under Low Oxygen
SCBL
Pedro Fernandes, Carla C.C.R. Carvalho, Marco Marques and Joaquim M.S. Cabral
The use of miniaturized devices in biocatalysis
has been gaining momentum in recent years and
the trend for a wider dissemination of this ap-
proach to be well established. Based in the use of
vessels with volumes ranging from some mL
down to tenths of microL, with a wide level of par-
allelization (viz. 24- to 384-multiwell plates) and
prone to automation, this concept provides the
high throughput capability required for speeding
up process development with significant cost re-
ductions, considering both chemicals and biologic
agents and manpower. Within the scope of this
methodology, substantial research efforts have
been made in order to establish the conditions
that allow for reproducibility and scalability of the
data generated. These efforts are anchored in a
focused engineering approach, which had been
missing up to roughly the dawn of the new millen-
nium, and had somehow hampered the success in
the implementation of bio-based processes,
namely due to faulty scale-up.
Bioconversion of steroids
Among bioconversion systems with easily fore-
seen or already established industrial application,
several require a cascade of enzyme reactions
and/or a non-conventional environment, given the
sparingly water solubility of plenty of relevant
compounds. The large number of variables in-
volved when the characterization and optimization
of one such system is aimed at, such as the mi-
crobial side-chain cleavage of (phyto)sterols to
androstenedione (AD), makes it a perfect fit for
the miniaturization approach. This particular bio-
conversion was thus selected as model system.
Throughout the research work performed, 24-well
plates were established as suitable platforms for
the detailed study of the model system. The rele-
vance of operational parameters (viz. chemical
compatibility, evaporation rate, filling volume, na-
ture of the substrate/product carrier, hydrody-
namic conditions) in product yield and productivity
BEB
L
Miniaturization in Biocatalysis
Figure 1: Effect of chain length and ramification of aliphatic on the side-chain cleavage activity of Mycobacterium sp. NRRL B-
3805 performed in covered (open bars) and rubber sealed (closed bars) multiwell plates. Activity data referred to the activity
obtained in the presence of methanol, given in the inset.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
meta
no
l
eta
no
l
pro
panol
buta
no
l
pen
tan
ol
hexan
ol
hep
tan
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octa
nol
no
nanol
decan
ol
un
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do
deca
nol
2-p
ropanol
2-b
uta
no
l
2-p
en
tanol
2-h
exan
ol
2-h
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2-o
cta
nol
2-n
onanol
2-d
ecan
ol
2-u
nd
ecanol
2-d
odecanol
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onanol
Solvent
Rela
tive A
cti
vit
y
0.0
0.5
1.0
1.5
2.0
2.5
Co
nvert
ed
Sit
oste
rol(
mM
)
Activity (mMAD/h) Specific activity (mmol AD/g h)
Open 1,78E-02 7,13E-04
Closed 9,49E-03 3,80E-04
Activity data for methanol
20 21 09 Annual Report
Figure 2: AD production at different oxygen mass transfer
coefficient (kLa) measured in 24-well OxoDish® microtiter
plate. Filling volumes of 500 L were used in tape-sealed
wells. The kLa at 300rpm was 0.05±0.01 s-1 in unsealed micro-
titer plate with a filling volume of 500 l.
was ascertained (Fig. 1) [1]. The behaviour and
performance of the steroid bioconversion systems
anchored in either growing or resting cells were
addressed, and engineering criteria for scale-up
were identified, based in AD yield (Fig. 2). The
use of multiwell plates equipped with sensor spots
for pH and dissolved oxygen monitoring allowed
for gaining further insight on the time course of the
bioconversion, to compare with the pattern ob-
served in stirred bioreactor, and to further validate
scaling criteria [2].
Bacterial adaptation studies
High throughput systems were developed for test-
ing simultaneously a high number of reactions and
conditions during the study of cell resistance and
adaptation mechanisms towards toxic compounds
(Fig. 3). The inhibition concentrations could be
determined and generational adaptation to in-
creasing concentrations of toxic compounds was
studied. The results obtained allowed the im-
provement of biocatalytic and bioremediation sys-
tems, in particular the biotransformation of ter-
penes and the degradation of extremely high con-
centrations of toluene [3]. It also showed the effect
of antineoplastic agents on bacterial cells and the
response mechanisms activated by the cells.
Microfluidic reactors
Aiming for further miniaturization, research has
recently expanded towards the use of microfluidic
reactors, which allows for continuous operation as
well as for easier implementation of production
scale by scaling out rather than scaling-up. The
enzymatic oxidation of cholesterol to choleste-
none, one of the reactions of the pathway leading
to the production of AD from sterols, was effec-
tively performed in an organic-aqueous two-liquid
phase system. Further insight in the system is
aimed at, in order to contribute for implementing
the whole cell bioconversion system leading to
AD.
The work produced so far has not only contributed
to a better knowledge of the model system used,
but has also provided considerable insight to the
requirements of multiphase biocatalytic whole cell
systems. It is also contributing to define the key
issues to be addressed for the design of represen-
tative biotransformation systems based in minia-
ture scale platforms [4].
References
[1] Marques, M.P.C., Carvalho, F., Magalhães, S.,
Cabral, J.M.S., Fernandes, P., Process Biochem.,
44: 556-561 (2009)
[2] Marques, M.P.C., Magalhães, S., Cabral, J. M.
S., Fernandes, P., J. Biotechnol., 141: 174-180
(2009)
[3] de Carvalho, C.C.C.R., Wick, L.Y., Heipieper,
H.J., Appl. Microbiol. Biotechnol. 82: 311-320
(2009).
[4] Carvalho, F., Marques, M.P.C., de Carvalho,
C.C.C.R., Cabral, J.M.S., Fernandes, P., Biore-
source Technol., 100: 4050-4053 (2009)
R
esearch H
ighlights
0
0.002
0.004
0.006
0.008
0.010
0 0.2 0.4 0.6 0.8 1.0
Relative k La
g A
D .(
g b
iom
as
s)-1
0 164 216 246 267 300
rpm
Figure 3: Bacterial cells growing in the presence of
different concentrations of toxic compounds in a micro-titre plate.
BEB
L
Cláudia Silva, Jaqueline Garcia and M. Ângela Taipa
Affinity-based strategies offer high-resolution
separation and are the most commonly used in
the purification of immunoglobulins and other high
-value proteins of pharmaceutical importance [1].
The affinity ligands utilised range from biological
to synthetic designed molecules with enhanced
chemical resistance and stability.
Recent progresses in polymer chemistry science
open a number of possibilities for new materials
by allowing the synthesis of bio-inspired (or biomi-
metic) polymeric materials with highly controlled
structures. These materials can combine advanta-
geous chemical properties like thermal and pH
sensitivity, magnetic or optical behavior, with the
specificity and selectivity characteristic of biologi-
cal systems. “Smart” polymers, such as thermo-
sensitive poly(N-isopropylacrylamide) (PNIPAM),
are particularly suitable for biological applications
[2] and constitute an interesting alternative for
developing novel affinity materials.
The main goal of this project is the synthesis and
application of bio-inspired affinity polymer systems
based on a “smart” thermosensitive polymeric
material (poly (N-isopropylacrylamide) (PNIPAM)),
conjugated to synthetic (Protein L mimic) affinity
ligands with high selectivity for immunoglobulins
or antibodies. The systems are designed and
studied in order to implement mild and cost-
effective affinity methodologies for the molecular
recognition/purification of antibodies and geneti-
cally engineered related molecules (Figure 1).
Cationic PNIPAM microgel particles containing
primary amino groups were obtained via precipita-
tion polymerization under surfactant-free condi-
t ions by co-po lymer isat ion with 2-
aminoethylmethacrylate (AEMH) or N-(3-
aminopropyl)methacrylamide (APMH). Both co-
monomers improve colloidal stability leading to
the formation of smaller and higher number of
particles, being this effect more pronounced for
AEMH (Figure 2).
Figure 1: Schematic representation of an affinity precipitation process of antibodies with a
biomimetic thermosensitive polymer: (A) capture of the target in solution; (B) precipitation of the affinity complex and recovery of the precipitate; (C) dissociation of the target molecule from the affinity complex (D) recovery of the target molecule; and (E) recovery of the macroli-
gand for reuse (adapted from [1]).
Bio-inspired Affinity Polymer Systems for Anti-
body Recognition
Pure Antibody
Soluble-Phase
Affinity Binding
Reversible
Precipitation
Dissociation
Crude Sample
Product
Recovery
T > LCST
T < LCST
BA
CD
E
Pure Antibody
Soluble-Phase
Affinity Binding
Reversible
Precipitation
Dissociation
Crude Sample
Product
Recovery
T > LCST
T < LCST
BA
CD
E
20 23 09 Annual Report
Figure 2: Reversible swelling/shrinking transition of microgels triggered by small changes in temperature within the physiologi-
cal range. The occurrence of this macroscopic event is due to the existence of a lower critical solution temperature (LCST) at ~32ºC for PNIPAM linear homopolymer, corresponding to a volume phase transition temperature (VPTT) for cross-linked parti-cles. The variation of particle size with temperature was evaluated by dynamic light scattering (DLS) for particles synthesis ed
without co-monomer (A) and in the presence of different concentrations of co-monomer either in batch (1% mol) or by shot-
addition (2% mol) (B).
Figure 3: General structure of biomimetic polymer systems obtained by conjugation of synthetic affinity ligands to microgel
particles (A). Adsorption studies of human IgG with lead ligand/polymer systems at 20ºC and in PBS (pH 7.0) (B). Controls
with non-conjugated particles.
The hydrophilic character of co-monomers affects
the thermo-sensitive profile of the particles: with
AEMH (more hydrophilic) the transition occurs
within a broader range and at higher tempera-
tures, while with APMH (more hydrophobic) the
transition is more defined and occurs at slightly
lower temperatures (Figure 2B).
Triazine-scaffolded synthetic affinity ligands with
high affinity and selectivity for immunoglobulins
were covalently grafted to amino-functionalised
PNIPAM microgel particles (Figure 3A). Lead bio-
inspired/biomimetic polymer systems were
screened for the adsorption of human immu-
noglobulin G (hIgG) as a model-protein. Selected
biomimetic particles were shown to be
„biologically‟ active, and specifically recognize and
capture hIgG (Figure 3B).
The project Biomimpolymers is developed in col-
laboration with the Centro de Química-Física Mo-
lecular (CQFM) of Instituto Superior Técnico and
the Laboratoire de Catalyse, Chimie, Polymères
et Procédés-C2P2/CNRS/CPE/UCBLyon1
(France). As a main output this work aims to con-
tribute to the generation of new bio-inspired mate-
rials with controlled structures, projected towards
the molecular recognition of antibody molecules.
Such materials are expected to have impact in
applications regarding separation science and
technology, diagnostics and biomedicine.
References
[1] Roque, A.C.A., Silva, C.S.O., Taipa, M.A., J.
Chromatogr. A, 1160: 44-55 (2007)
[2] Silva, C.S.O., Baptista, R.P., Santos, A.M.,
Martinho, J.M.G., Cabral, J.M.S., Taipa, M.A.,
Biotechnol. Lett., 28: 2019-2025 (2006)
R
esearch H
ighlights
T<VPTT T>VPTT
T
T<VPTT T>VPTT
TT
PNIPAM
100
300
500
700
900
1100
1300
1500
1700
1900
15 20 25 30 35 40 45 50 55
T (°C)
Hyd
rod
yn
am
ic d
iam
ete
r (n
m)
Co-polymers of PNIPAM
100
200
300
400
500
600
15 20 25 30 35 40 45 50 55
T (°C)
Hyd
rod
yn
am
ic d
iam
ete
r (n
m)
2%APMHsa
1%APMH
2%AEMHsa
1%AEMH
A B
A B
-0.3
0.2
0.7
1.2
1.7
2.2
conjugated non conjugated conjugated non conjugated
Ad
so
rbe
d Ig
G (
mg
/g a
ds
orb
en
t)
1mg/g 3mg/g 5mg/g 7mg/g 10mg/g
PNIPAM-AEMH PNIPAM-APMH
Initial IgG:
N
N
N
R1
R2
NH
CH3O
O
N
N
N
R1
R2
NH
CH3O
O
T<VPTT T>VPTT
T
T<VPTT T>VPTT
TT
PNIPAM
100
300
500
700
900
1100
1300
1500
1700
1900
15 20 25 30 35 40 45 50 55
T (°C)
Hyd
rod
yn
am
ic d
iam
ete
r (n
m)
Co-polymers of PNIPAM
100
200
300
400
500
600
15 20 25 30 35 40 45 50 55
T (°C)
Hyd
rod
yn
am
ic d
iam
ete
r (n
m)
2%APMHsa
1%APMH
2%AEMHsa
1%AEMH
A B
BEB
L
Ana Azevedo, Paula Rosa, Filipa Ferreira and Raquel Aires-Barros
Monoclonal antibodies are becoming a major
source of biopharmaceuticals for the treatment of
different type of diseases including cancer. As a
consequence, the design and production of anti-
bodies for therapeutic purposes has been consid-
erably improved in the last decade, with titres sur-
passing 10 g/l. Hence, with capacity bottlenecks
moving towards downstream purification areas,
the need for a broader strategic approach for the
purification of monoclonal antibodies is being in-
creasingly recognized in order to improve the
overall process performance.
The evidence supporting the success and effec-
tiveness of aqueous two-phase extraction as a
unit operation in the downstream processing of
antibodies is compelling [1]. The possibility of
modifying the affinity of a protein for one of the
phases by functionalization of the phase forming
component constitutes a major advance in this
area, by allowing an increase in both binding ca-
pacity and selectivity.
Single-stage extraction
PEG molecules with different molecular weights
have been modified with different type of ligands,
selected on the basis of their expected interaction
with antibodies through electrostatic, hydrophobic
and affinity effects (Fig. 1). The ability of these
ligands to bind to human IgG was assessed either
as phase-forming compounds or as free ligands
(attached to triethylene glycol - TEG molecules). It
was observed that systems containing polymer
molecules modified with glutaric acid, an amino-
acid mimetic ligand, showed the highest affinity for
human antibodies [2].
The selectivity of each ligand was also evaluated,
for the purification of human IgG from a Chinese
Hamster Ovary (CHO) cell supernatant and again
higher recovery yields in the top PEG-rich phase
were observed using glutaric acid ligands. Table 1
summarizes the performance parameters of each
ligand. The highest partition coefficient was ob-
tained in PEG/dextran systems with added
triethylene glycol glutaric acid (TEG-COOH).
The effect of pH, TEG-COOH concentration and
volume ratio on the partitioning of IgG and on the
different components of the CHO supernatant was
studied and it was further observed that the condi-
tions that favoured the selective extraction of IgG
were lower pH values and high TEG-COOH con-
centrations. The extraction of IgG could be en-
hanced using higher volume ratios, however with
a significant decrease in both purity and percent-
age of contaminants removal [3]. After optimiza-
tion, it was possible to recover 96% of IgG in the
Affinity-enhanced Extraction of Human Anti-
bodies
OH
O O
NH2
SONaO
O
N
N
H
N
NN
N
N
N
N
NH
NH
OH
OH
N
N
O
S
Ligands attached to PEG or TEG molecules
Glutaric acid (COOH)
Amino
Benzyl
Pyrimidine (Pym)
Sulfonate Modified Triazine (A2P)
Aminoquinuclidine (AQ1)
Mercaptoethylpyridine (MEP)
Figure 1: Ligands attached to PEG or TEG molecules.
20 25 09 Annual Report
top phase (TP) outlet, with a final concentration of
0.22 g/l (Fig. 2A).
Multi-stage extraction
In order to enhance simultaneously both recovery
yield and purity, a four stage cross-current opera-
tion was simulated and the corresponding liquid-
liquid equilibrium (LLE) data determined allowing
the prediction of an optimised counter current ex-
traction based on McCabe-Thiele diagrams [3].
Accordingly, IgG can be purified in the PEG-rich
top phase with a final recovery yield of 95%, a
final concentration of 1.04 g/l and a protein purity
of 93%, if a PEG/dextran ATPS containing 1.3%
TEG-COOH, 5 stages and volume ratio of 0.4 are
used (Fig. 2B). This represents a significant im-
provement specially in terms of purity in compari-
son to the single-stage extraction.
This project has been developed together with
Bayer Technology Services (Leverkussen, Ger-
many) and Syncom BV (Gronigen, NL).
References
[1] Azevedo, A.M., Rosa, P.A.J., Ferreira, I.F.,
Aires-Barros, M.R., Trends Biotecnhol., 27: 240-
247 (2009)
[2] Azevedo, A.M., Rosa, P.A.J., Ferreira, I.F.,
Pisco, A.M.M.O., de Vries, J., Korporaal, R., Vis-
ser, T.J., Aires-Barros, M.R., Sep. Purif. Tecnhol.,
65: 31-39 (2009)
[3] Rosa, P.A.J., Azevedo, A.M., Ferreira, I.F.,
Sommerfeld, S., Bäcker, W., Aires-Barros, M.R.,
J. Chromatogr. A, 1216: 8741-8749 (2009)
R
esearch H
ighlights
1
YTop = 96%
TP Outlet
[IgG] = 0.22 g/l
PProtein = 87%
PTotal = 43%
BP Inlet
[IgG] = 0.424 g/l
PProtein = 65%
PTotal = 29%
BP Outlet
[IgG] = 0.02 g/l
TP Inlet
[IgG] = 0 g/l
System log KP Yield PProtein PHPLC
PEG/dextran -0.46 23% 39% 29%
PEG-COOH/dextran 0.65 97% 94% 41%
PEG-NH2/dextran 0.09 76% 82% 32%
PEG-Triazine/dextran -1.00 8% - -
PEG-Benzyl/dextran 1.32 88% 87% 34%
PEG-MEP/dextran -0.24 58% 72% 25%
PEG-Pym/dextran -0.16 59% 82% 21%
PEG/dextran+TEG-COOH 1.32 96% 95% 41%
PEG/dextran+TEG-MEP -0.21 45% 61% 13%
PEG/dextran+TEG-SO3 -0.28 37% 52% 33%
PEG/dextran+TEG-AQ1 -0.29 15% - -
Table 1: Performance of PEG/dextran systems modified with different types of ligands for the purification of human
antibodies from CHO cell culture supernatant, including the partition coefficient (KP), recovery yield in the top phase,
protein purity (determined as the IgG/protein ratio - PProtein) and total purity determined by size-exclusion HPLC (PHPLC).
Figure 2. Optimized scheme of: (A) single-stage ATPE of IgG
using a PEG/dextran ATPS containing 1.3% TEG-COOH with a
volume ratio of 2.2 and (B) predicted counter-current multi-stage
ATPE of IgG using a PEG/dextran ATPS containing 1.3% TEG-
COOH, 5 stages and a volume ratio of 0.4; BP: dextran-rich
bottom phase (14.19% dextran and 2.60% PEG 3350); TP:
PEG-rich top phase (9.74% PEG 3350 and 0.004% dextran);
Y: IgG recovery yield in the top phase.
1
Y = 5%
2
Y = 11%
3
Y = 21%
4
Y = 42%
5
Y = 95%
BP Inlet
[IgG] = 0.44 g/l
PProtein = 65%
PTotal = 30%
BP Outlet
[IgG] = 0.02 g/l
TP Outlet
[IgG] = 1.04 g/l
PProtein = 93%
PTotal = 85%
TP Inlet
[IgG] = 0 g/l
[IgG] = 0.06 g/l [IgG] = 0.23 g/l
[IgG] = 0.12 g/l [IgG] = 0.46 g/l
A
B
NAB
L
Pedro H. Oliveira, Sofia C. Ribeiro, Geisa Gonçalves, Duarte Miguel F. Prazeres,
Gabriel A. Monteiro
Gene therapy and DNA vaccination have
emerged in the last two decades as promising
alternatives for the treatment and prevention of
genetic disorders and acquired diseases. Non-
viral vectors, such as naked plasmids, constitute a
safer gene delivery alternative to viral vectors due
to their lower toxicity and larger gene capacity.
Plasmid DNA (pDNA) vaccines also offer a credi-
ble alternative for the prevention and treatment of
infectious and acquired diseases. In order for a
DNA vaccine to be successfully developed, sev-
eral scientific and technological challenges asso-
ciated with the design of pDNA must be ad-
dressed [1]. In fact, one of the main obstacles
found during the developmental stages of a DNA
molecule involves tackling several instability
events, therefore assuring its structural integrity
[1]. The research program on “Nucleic Acid Bioen-
gineering” addresses some of these challenges.
The type and overall organization of the genetic
elements present in pDNA vaccines will directly
impact not only its bulk production, but also shelf-
stability, efficacy and ultimately its clinical ap-
proval. The mammalian expression vector and
DNA vaccine backbone pCIneo was used
throughout our studies as a model plasmid to gain
further insight into genetic instability. During
Figure 2: Plasmid pCIneo::IS2.
shown is the hybrid promoter
generated by the IS2 −35 and
pCIneo −10 hexamer sequen-
ces. Capital letters match the E.
coli promoter consensus
sequence TTGACA N16–18
TATAAT. The boxed region
represents the 5 bp duplicated
sequence generated upon IS2
insertion. The arrow indicates
the junction site between the
right inverted repeat (RIR) of
IS2 and pCIneo.
Designing Safer DNA Vaccines
3788 atcggaaT tcaAg cacctaaacggggatatAaAGgTctgt 3748
-35 hexamer-10 hexamer 17 bp
IS2neor
Ampr
SV40 enhancer and early promoter
Phage f1 region
CMV immediate early enhancer and promoter
Figure 1: A) Recombination between directly repeated sequences (blue arrows) in a parental (P) plasmid can
give rise to monomeric (M) and heterodimeric (1+2; 1+3) deletion products.
P M 1+2 1+3
pCIneo::IS2
6805 bp
20 27 09 Annual Report
1.E-10
1.E-08
1.E-06
1.E-04
1.E-02
0 1000 2000 3000 4000
FR
LS (bp)
18
50
352
800
LR (bp)
Figure 3: A) Recombination frequency (FR) as a function of spacer length (LS) for fix values of repeat length (LR) in recA+
strains. Lines correspond to model predictions while symbols correspond to experimental data. B) Distribution of direct repeats in plasmid vectors. Repeat distribution analysis shows that direct repeats are frequently found within antibiotic resistance genes, non-coding regions and regulator sequences in bacterial expression vectors while in mammalian expression vectors
they are predominantly found within eukaryotic promoters, non coding and regulator sequences.
pCIneo amplification in Escherichia coli cells, we
were faced with a recurrent contamination consist-
ing in aberrant monomeric and heterodimeric side
products (Fig. 1). The latter were the result of dele-
tion-formation events between two 28-bp direct
repeats of pCIneo, ultimately leading to the expres-
sion of a distant neomycin resistance (neoR) gene
[2]. Expression of this neoR gene was explained by
RNAII transcription beyond ori as a result of non-
hybridized RNAII-DNA molecules [3]. Interestingly,
we were able to lower the ratio of deletion forma-
tion products to total plasmid content by exploiting
the runaway properties of this vector at higher tem-
peratures (42ºC) and/or using a minimal growth
medium such as MM9 [4].
Besides deletion-formation, our group also ob-
served the occurrence of IS2-mediated transposi-
tion events in pCIneo. IS2 was found to transpose
upstream the neoR gene and was able to potenti-
ate the transcription of this gene through the gen-
eration of a hybrid promoter in the IS2-neoR junc-
tion (Fig. 2).
Aiming at a broader picture of both frequency and
abundance of instability events mediated by re-
peated sequences, our group has also performed i)
a meta analysis on plasmid recombination fre-
quency in order to develop mathematical functions
able to correlate this variable with repeat and
spacer lengths [5] (Fig. 3A); ii) an in silico search
for direct and inverted repeats using Repseek in a
large number of bacterial and mammalian expres-
sion vectors (Fig. 3B). Some of these sequences
are currently under evaluation for its removal or
modification.
DNA vaccination and gene therapy have matured
to the point where a number of products should be
hitting the market in the wake of the first veterinary
DNA vaccines. Furthermore, new developments
are likely to surface within the coming years be-
cause of increased investments from academia
and industry in the area. Our work is a contribution
to these efforts.
References
[1] Oliveira, P.H., Prather, K.J., Prazeres, D.M.F.,
Monteiro, G.A., Trends Biotechnol., 27: 503-511
(2009)
[2] Ribeiro, S.C., Oliveira, P.H., Prazeres, D.M.F.,
Monteiro, G.A., Mol. Biotechnol., 40: 252-262
(2008)
[3] Ribeiro, S.C., Prazeres, D.M.F., Monteiro, G.A.,
Appl. Microbiol. Biotechnol., doi: 10.1007/s00253-
009-2339-3 (2010)
[4] Oliveira, P.H., Prazeres, D.M.F., Monteiro, G.A.,
J. Biotechnol., 143: 231-238 (2009)
R
esearch H
ighlights
A B
NAB
L
Luis Raiado, Duarte Miguel F. Prazeres, Marília Mateus
The BioEngineering Research Group has for long
been recognized for its relevant experience in
downstream processing, namely, with membrane
technologies. In the framework of the research pro-
gram on “Nucleic Acid Bioengineering”, the appli-
cation of consolidated and design of new mem-
brane technologies, associated with fundamental
studies in areas of biomolecular engineering and
bioprocessing, have been supporting the develop-
ment of DNA vaccine prototypes.
Chromatography is one of the key operations in the
downstream processing of plasmid DNA (pDNA).
However, the increased demand for highly purified
pDNA experienced in recent years has made clear
the need for alternative processes capable of re-
taining the advantages of conventional chromatog-
raphy, such as selectivity, while providing in-
creased throughput at a lower cost. Recent
achievements have proven that alkyl-like chains in
functionalized membrane supports have the ability
to separate and resolve the model plasmid pVAX1-
LacZ (6050bp) from impurities in clarified Es-
cherichia coli cell lysates (specifically RNA) [1].
Characterization studies on derivatized mem-
branes evidenced the influence of reaction media
(Fig. 1) on reepoxydation yields. Membrane A was
at first suggested as the best candidate for the HIC
membrane process due to its higher epoxy surface
density (43% epoxy yield). Membrane C had the
lowest epoxy surface density (only 32% epoxy
yield), had the highest density of residual free
amines and was, therefore, figured as the worst
candidate for HIC. The presence of these residual
amines is undesirable since these moieties can
mediate non-specific interactions with pDNA, and
thus deteriorate HIC resolution [2].
Implementation of chromatography with these
membranes (Figs. 2 & 3) has shown that mem-
brane C presented the highest selectivity among
those tested (gel electrophoresis analysis showed
less contaminated pDNA peaks by RNA). Struc-
tural differences due to cross-linking in the mem-
brane matrix are probably conditioning the selectiv-
ity results. Optimizing elution profiles with the best
performing modified membrane (Fig. 4), led to a
4.7 purification factor with 73% plasmid yield, com-
petitive with its bead process counterpart [2].
Membrane Chromatography for DNA Vaccine
Purification
Figure 1: Derivatization of epoxy membrane by reaction in 3-steps: (I) amination,
(II) re-epoxidation, (III) blocking.
Figure 2: Modified 25 mm membrane
discs in an in-line membrane holder linked to a chromatographic system (ÄKTA Puri-
fier, GE Healthcare).
20 29 09 Annual Report
This project is in its early stages; however the on-
going work aims to perform the essential research
to fasten the development of membrane chroma-
tography for purification of plasmid-mediated DNA
vectors based on hydrophobic and affinity interac-
tions. The main strategy consists of three ap-
proaches: (i) rational design of membrane material
properties, based on chemical composition of func-
tional groups for bio-interaction with nucleic acids
and topography and internal structure of mem-
brane chromatographic matrices, to render high
resolution and high hydrodynamic capacity chro-
matographic media; (ii) the design of rapid, sensi-
tive and specific biological assays to monitor effec-
tiveness of biological interactions between model
plasmid DNA bio-therapeutics and functional
groups built-up on the surfaces of matrices and; (iii)
the improvement of atomic force microscopy proto-
cols for scanning interaction forces between de-
signed chromatographic membranes surfaces and
in-house developed plasmid-DNA probes.
The approach is innovative for tailoring membrane
materials properties and has the potential of be-
coming a high throughput screening technology to
expand membrane chromatography of DNA bio-
therapeutics. For a successful undertaking, the
“Nucleic Acid Bioengineering” research team is
working in close collaboration with the “Materials
Physics” group of the Institute of Materials and Sur-
faces Sciences and Engineering of Instituto Supe-
rior Técnico which has profound knowledge on the
methodological development of AFM applications.
References
[1] Raiado-Pereira, L., Prazeres, D.M.F., Mateus,
M., J. Sep. Sci. (2010) (accepted)
[2] Freitas, S.S., Santos, J.A.L., Prazeres, D.M.F.,
Sep. Purif. Technol., 65: 95-104 (2009).
R
esearch H
ighlights
0
20
40
60
80
100
120
140
160
180
200
220
0
200
400
600
800
1000
1200
1400
0 5 10 15 20 25 30 35
Co
nd
ucti
vit
y (m
S/c
m)
Ab
so
rban
ce @
260n
m (
mA
U)
Time (min)
UV Absorbance Lysate with pDNA
UV Absorbance Lysate without pDNA
Conductivity
(1)
(2,3) (4)
(5-10)
0
20
40
60
80
100
120
140
160
180
200
220
0
200
400
600
800
1000
1200
1400
0 5 10 15 20 25 30 35 40 45 50 55 60
Co
nd
ucti
vit
y (m
S/c
m)
Ab
so
rban
ce @
260n
m (
mA
U)
Time (min)
UV Absorbance injection1
UV Absorbance injection2
Conductivity
Figure 3: Chromatographic performance for feeds of 100 μL
diluted clarified lysate solutions of DH5α E. coli cells bearing
no plasmid (dashed lines) and bearing the pVAX1-LacZ
plasmid model (solid grey lines), over one membrane C disc,
at room temperature. Chromatography performed with 1.5 M
(NH4)2SO4 in 10 mM Tris pH 8.0 as binding buffer and 10mM
Tris pH 8.0 as elution buffer using isocratic elution at 0.5 mL/
min.
Fractions of the chromatographic runs of lysates bearing
pDNA were analysed by agarose gel electrophoresis. Only
those fractions numbered as (2,3) contained significant
amounts of pDNA with a slight contamination by RNA impu-
rities, while increasingly more hydrophobic RNA fractions
were separated (fraction 4 and higher numbered).
Figure 4: Superimposed chromatograms of
repeated chromatographies performed over 2
staked membrane-C specimens at room tempe-
rature using each time 400 μL of lysate volume
to be separated.
Buffers: 1.8 M of (NH4)2SO4 in 10mM Tris pH
8.0 used for equilibration and binding and 10
mM Tris pH 8.0 used in elution.
Elution profile: 20 min long gradient elution from
0% up to 40% elution buffer followed by a step
up to 100% of elution buffer for 20 min; at
2.5MV/min of flowrate.
SCB
L
Stem Cell Bioengineering Science aims to contrib-
ute for a better knowledge of the ex-vivo expan-
sion of stem cells and/or their controlled differen-
tiation into specific cell types in bioreactor sys-
tems. In particular, human mesenchymal stem
cells (MSC) have become one of the most promis-
ing candidates for Tissue Engineering and Regen-
erative Medicine applications, mostly due to the
differentiative potential and immunologic proper-
ties of these multipotent cells. By combining a
cross-disciplinary approach of Stem Cell Bioengi-
neering and Experimental Hematology, our goal at
BERG-SCBL is the optimization of MSC ex-vivo
expansion for application in Cellular Therapy,
namely for supplementation in hematopoietic stem
cell transplantation settings.
A low oxygen environment - Hypoxia
In their bone marrow (BM) niche, self-renewal
and/or differentiation of MSC are governed by a
complex microenvironment signaling that involves
cell-to-cell interactions, soluble factors, mechani-
cal forces and oxygen tension.
A 2% O2 hypoxic environment was found to im-
prove BM MSC expansion levels by inducing an
early start of the exponential growth phase, with
cells starting cell division earlier in culture com-
pared to normoxia. In addition, we observed an
increase of cellular metabolism efficiency, associ-
ated to the adaptation of BM MSC to the low oxy-
gen tension environment. These results gave im-
portant insights on how hypoxia culture favors
human BM MSC ex-vivo expansion, being advan-
tageous towards the maximization of cell yield in a
clinical-scale MSC expansion process (Fig. 1) [1].
Culture under stirred conditions
MSC cultures have been performed in traditional
culture flasks under static conditions, which are
limited in terms of cell productivity, their non-
homogeneous nature, resulting in concentration
gradients (pH, nutrients, metabolites,...), difficulty
of monitoring and an extensive handling require-
ment for feeding/harvesting procedures and lim-
ited productivity. Therefore the development of a
reproducible and robust process is required for
the production of clinical relevant human MSC
numbers.
We demonstrated the feasibility of using a micro-
carrier-based stirred culture system for the effi-
cient expansion of both BM and adipose tissue
(AT)-derived MSC in low serum conditions (Fig.
2).
Francisco dos Santos, Pedro Z. Andrade, Cláudia Lobato da Silva and Joaquim MS
Cabral
Figure 1: Ex-vivo expansion of human BM MSC under hypoxia (2% O2) and normoxia (20% O2).Total cell numbers (A) were
determined throughout the culture for both hypoxia (black circles) and normoxia (white squares) and the fold increase values in total cell number (B) obtained for hypoxia (black bars) and normoxia (gray bars) are presented as mean ± SEM (*p < 0.05)
[1].
Ex-vivo Expansion of Mesenchymal Stem Cells
for Cellular Therapies: from Lab to Clinic
A B
20 31 09 Annual Report
Clinical-scale production of MSC for Cellular
Therapies
The consortium between IBB-IST, Instituto
Português de Oncologia de Lisboa Francisco
Gentil and Centro de Histocompatibilidade do Sul,
focusing the isolation and ex-vivo expansion of
MSC under GMP conditions for Cellular Therapies
was established in 2007. Since then, 8 patients
have already benefited from this pioneer treat-
ment. MSC were used in the treatment or preven-
tion of graft-versus-host disease (GVHD) and also
to facilitate allogeneic hematopoietic stem cell
engraftment and decrease regimen-related toxic-
ity. The clinical cases under this project for the
last 2 years include:
Acute GVHD
Extensive chronic GVHD
Hurler‟s syndrome
Familial hemophagocytic lymphohistiocytosis
Aplastic anemia
References
[1] dos Santos, F., Andrade, P.Z., Boura, J.S.,
Abecasis, M.M., da Silva, C.L., Cabral, J.M.S., J.
Cell Physiol., 223: 27-35 (2010)
Figure 2: Expansion of BM (A) and AT (B) MSC in spinner flasks using low serum (2% Fetal Bovine Serum) media. We com-pared Cultispher S gelatin-microcarriers (blue line) with the animal-free plastic microcarriers (red line) as support for MSC
adhesion and proliferation. Results are presented as mean ± SEM.
Figure 3: Images of an ex-vivo expansion of human MSC for cellular therapy under GMP-conditions in a clean room at Centro
de Histocompatibilidade do Sul, Lusotransplante, Lisboa.
R
esearch H
ighlights
0.0E+00
5.0E+06
1.0E+07
1.5E+07
2.0E+07
2.5E+07
3.0E+07
3.5E+07
0 1 2 3 4 5 6 7 8 9
Ce
ll n
um
be
r
Time (days)
0.E+00
1.E+07
2.E+07
3.E+07
4.E+07
5.E+07
6.E+07
1 3 5 7 9
Ce
ll n
um
ber
Time (days)
Cell n
um
ber
Time (days) Time (days)
Cell n
um
ber
A B
SCB
L
Neural stem (NS) cells can provide a source of
material with potential applications in different
settings. The Stem Cell Bioengineering Labora-
tory at BERG-SCBL aims at the large-scale ex-
vivo expansion of NS cells, as starting material to
generate mature cells (e.g. neurons) for potential
use in regenerative medicine (e.g. treatment of
neurological disorders), as well as for neural drug
testing or developmental studies. The ultimate
goal is to define an efficient, reproducible and cost
-effective process for the production of NS cells in
large scale under highly controlled conditions,
while maintaining their multipotency.
The in vitro derivation of cultures of NS cells grow-
ing under adherent conditions, with no significant
decline in potential over time, was recently dem-
onstrated, from several sources, such as mouse
ES cells. These cells show long-term self renewal
capacity and maintain the capacity to generate
neurons, astrocytes and oligodendrocytes. Under
the presence of epidermal growth factor (EGF)
and basic fibroblast growth factor (bFGF), these
cells expand under adherent conditions, which
may be advantageous compared to the usual NS
cell culture as floating aggregates (neurospheres).
In neurospheres, besides oxygen and nutrients
diffusion limitations to the cluster centre, NS cells
are not directly identifiable and co-exist with com-
mitted progenitors and differentiated cells. Conse-
quently, this heterogeneity can lead to non-
reproducible results amongst different laborato-
ries. In this work, the continuous ex-vivo expan-
sion of the model mES cell-derived NS cell line
CGR8-NS was characterized and optimized in
serum-free conditions [1].
Oxygen and NS cells
Oxygen is an important energy source for cell me-
tabolism and its concentration is tightly regulated
in the central nervous system, where the pO2 val-
ues are similar among different mammalian spe-
cies and range from 2-5% in the cortex. Lower
concentrations of oxygen have been demon-
strated to have a positive effect on the prolifera-
tion of NS cells from different origins, both in
terms of species or tissue and had influence in
their fate, when induced to differentiate. The effect
of low oxygen tension on one mES cell-derived
NS cell line was studied.
mES cell-derived NS cell growth under differ-
ent oxygen tensions
The influence of oxygen on the optimization of
mouse ES cell-derived NS cell culture was evalu-
ated by testing different O2 tensions: 20%, 10%,
Carlos Rodrigues, Margarida Diogo, Cláudia Lobato da Silva and Joaquim MS Cabral
Ex-vivo Expansion of mouse Embryonic Stem Cell-
derived Neural Stem Cells Under Low Oxygen
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
1.E+09
1.E+10
0 3 6 9 12
Cu
mu
lati
ve C
ell
Nu
mb
er
Time (days)
0.6
0.65
0.7
0.75
0.8
0.85
0.9
0.95
0 5 10 15 20
Sp
ecif
ic G
row
th R
ate
(d
ay
-1)
O2 Tension (%)
Figure 1: Effect of O2 on CGR8-NS cell expansion. (A) Cumulative number of cells during successive passaging under 20%
(), 10% (), 5% () and 2% (▲) O2. Cells were passaged every 3 days during a maximum of 12 days. (B) Specific growth
rate determined from the slopes of the cumulative cell number curves plotted in Figure 1A [1].
A B
20 33 09 Annual Report
5% and 2% (Fig. 1). Cells were plated with an
optimal initial cell density and passaged succes-
sively under an atmosphere with the different O2
concentrations mentioned above. It is clear that,
when compared to the standard 20% atmospheric
O2 tension, low oxygen tension strongly enhances
the expansion of CGR8-NS cells. This positive
effect was observed after the first passage and
was consistently maintained throughout the fol-
lowing passages. These results indicate that, to
obtain the best performance in terms of cell prolif-
eration, CGR8-NS cells must be cultured under an
optimum O2 tension range between 2-5%, ap-
proximately the physiological concentration of
oxygen in the brain (Fig. 2). These conditions led
to fold increase values in total cell number about
twice higher than observed under 20% oxygen
being this effect more pronounced when cells
were plated at low density.
Hypoxia does not affect mES-derived NS cell
multipotency
Before selecting 2% as the optimum oxygen ten-
sion value for the expansion of CGR8-NS cells,
we verified that the expression of Nestin, a typical
marker of neural stem/progenitor cells was not
impaired by this condition. The differentiative po-
tential of NS cells expanded under 2% O2 was
also studied in order to assess the multipotency of
these cells. In the case of astrocyte differentiation,
after 3 days, the typical “star-like” cell morphology
was observed and cells were positive for the ex-
pression of astroglial markers. In terms of neu-
ronal differentiation, cells were harvested after 6
days, when expression of early neuronal markers
is expected, and analyzed by flow cytometry. The
results suggest similar efficiency to undergo neu-
ronal differentiation for the cells previously ex-
panded under both conditions. Further studies
were then performed under 2% O2.
Hypoxia leads to increased proliferation of
mES-derived NS cells
The positive effect of low oxygen in the expansion
of CGR8-NS cells may be the result of: (i) en-
hanced proliferation of the cells under 2% O2; (ii)
lower levels of cell death/apoptosis under low oxy-
gen; or (iii) a combined effect of both factors. To
test these hypotheses, cell divisional history, cell
cycle and apoptosis/necrosis studies were per-
formed for both culture conditions. We observed
that although the number of apoptotic and necrotic
cells is comparable under both oxygen tensions,
cells divide faster under hypoxic conditions. A
larger population in a quiescent G0 phase was
observed in normoxic conditions when compared
to cells cultured under 2% O2.
References
[1] Rodrigues, C.A.V., Diogo, M.M., Lobato da
Silva, C., Cabral, J.M.S., Biotechnol. Bioeng., doi:
10.1002/bit.22648 (2010)
Figure 2: Growth kinetics of CGR8-NS cell expansion. (A) Number of viable cells experimentally determined under 2% O2 (♦)
and 20% O2 (■) and determined from a model for hypoxia (green line) and normoxia (blue line). (B) Specific growth rate val-
ues (day-1) during expansion of CGR8-NS cells under hypoxic (green line) and normoxic conditions (blue line). Data pre-
sented are means ± standard error of the mean for 2 determinations from 4 independent experiments.
*, p< 0.05
R
esearch H
ighlights
0.E+00
2.E+05
4.E+05
6.E+05
8.E+05
1.E+06
1.E+06
1.E+06
0 1 2 3 4
Nu
mb
er
of
Cell
s
Time (days)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 0.5 1 1.5 2 2.5 3 3.5 4
Sp
ecif
ic G
row
th R
ate
(d
ay
-1)
Time (days)
*
*
*
A B
Scientific Output
20092009
BERG Annual ReportBERG Annual Report
20 35 09 Annual Report
Articles in Peer-reviewed Journals
Articles in Conference Proceedings
Patents
Book Chapters
Dissertations
Oral Communications
Poster Communications
Prizes
Articles in Peer-reviewed Journals
Azevedo, A.M., Gomes, A.G., Rosa, P.A.J.,
Ferreira, I.F., Pisco, A.M.M.O., Aires-Barros, M.R.,
“Partitioning of human antibodies in polyethylene
glycol - sodium citrate aqueous two-phase sys-
tems”, Sep. Purif. Technol., 65, 14-21
Azevedo, A.M., Rosa, P.A.J., Ferreira, I.F., Aires-
Barros, M.R., “Chromatography-free recovery of
biopharmaceuticals through aqueous two-phase
processing”, Trends Biotechnol., 27, 240-247
Azevedo, A.M., Rosa, P.A.J., Ferreira, I.F., Pisco,
A.M.M.O., de Vries, J., Korporaal, R., Visser, T. J.,
Aires-Barros, M.R., “Affinity-enhanced purification of
human antibodies by aqueous two-phase extrac-
tion”, Sep. Purif. Technol., 65, 31-39
Azevedo, A.M., Rosa, P.A.J., Ferreira, I.F., de
Vries, J., Visser, T.J., Aires-Barros, M.R.,
“Downstream processing of human antibodies inte-
grating an extraction capture step and cation ex-
change chromatography”, J. Chromatogr. B, 887,
50-58
Bernardino, S.M.S.A., Fernandes, P., Fonseca,
L.P., "A new biocatalyst: Penicillin G acylase immo-
bilized in sol-gel micro-particles with magnetic prop-
erties", Biotechnol. J., 4, 695-702
de Barros, D.P.C., Fonseca, L.P., Cabral, J.M.S.,
Weiss, C.K., Landfester, K., “Synthesis of alkyl es-
ters by cutinase in miniemulsion and organic sol-
vent media”, Biotechnol. J., 4, 674-683
de Barros, D.P.C., Fonseca, L.P., Fernandes, P.,
Cabral, J.M.S., Mojovic L., “Biosynthesis of ethyl
caproate and other short ethyl esters catalyzed by
cutinase in organic solvent”, J. Mol. Catal. B: En-
zym., 60, 178-185
Cabeça, R., Rodrigues, M., Prazeres, D.M.F., Chu,
V., Conde, J.P., “The effect of the shape of single,
sub-ms voltage pulses on the rates of surface im-
mobilization and hybridization of DNA”, Nanotech-
nology, 20: 015503
Calado, C.R.C., Brandão, M., Biscaia, J., Cabral,
J.M.S., Fonseca, L.P., “Effect of Tween-80 on sta-
bility and secretion of hydrophobic tagged-
cutinases”, Chem. Biochem. Eng. Q., 23, 411-417
Cardoso, M.A.T., Antunes, S., Van Keulen, F.,
Ferreira, B.S., Geraldes, A., Cabral, J.M.S., Pa-
lavra, A.M.F., “SAS micronization of synthetic all
trans-beta-carotene with tetrahydrofuran as solvent
and carbon dioxide as antisolvent”, J. Chem. Tech-
nol. Biotechnol., 84, 215-222
Carvalho, F., Marques, M.P.C., de Carvalho
C.C.C.R., Cabral, J.M.S., Fernandes, P., “Sitosterol
bioconversion with resting cells in liquid polymer
based systems”, Bioresource Technol., 100, 4050
-4053
Carvalho, J., Prazeres, D.M.F., Monteiro, G.A.,
“Bringing DNA vaccines closer to commercial use”,
iDrugs, 12, 642-647
de Carvalho, C.C.C.R., Wick, L.Y., Heipieper, H.J.,
“Cell wall adaptations of planktonic and biofilm
Rhodococcus erythropolis cells to growth on C5 to
C16 n-alkane hydrocarbons”, Appl. Microbiol. Bio-
technol., 82, 311-320
Cattorini, S., Marques, M.P.C., Carvalho, F.,
Chheub, V., Cabral, J.M.S., Fernandes, P.,
“Lentikat®-based Biocatalysts: Effective Tools for
Inulin Hydrolysis”, Chem. Biochem. Eng. Q., 23,
429-434
Costa, L., Brissos, V., Lemos, F., Ribeiro, F.R.,
Cabral, J.M.S., “Enhancing the thermal stability of
lipases through mutagenesis and immobilization on
zeolites”, Bioprocess Biosyst. Eng., 32, 53-61
Costa, L., Coelho, A., Lemos, F., Ribeiro, F.R.,
Cabral, J.M.S., “Modulating the acid strength of
zeolite H-ZSM-5 to increase the selectivity in the
racemization of 1-phenylethanol”, Appl. Catal. A:
Gen., 354, 33-37
Fernandes, T.G., Diogo, M.M., Clark, D.S., Dordick,
J.S., Cabral, J.M.S., “High-throughput cellular mi-
croarray platforms: applications in drug discovery,
toxicology and stem cell research”, Trends Bio-
technol., 27, 342-349
Fernandes, P., Marques, M.P.C., Carvalho, F.,
Cabral, J.M.S., “A simple method for biocatalyst
immobilization using PVA-based hydrogel particles”,
J. Chem. Technol. Biotechnol., 84, 561-564
Publications
20 37 09 Annual Report
Freitas, S.S., Canário, S., Santos, J.A.L., Prazeres,
D.M.F., “Alternatives for the intermediate recovery
of plasmid DNA: Performance, economic viability
and environmental impact”, Biotechnol. J., 4, 265-
278
Freitas, S.S., Santos, J.A.L., Prazeres, D.M.F.,
“Plasmid purification by hydrophobic interaction
chromatography using sodium citrate in the mobile
phase”, Sep. Purif. Technol., 65, 95-104
Gomes, A.G., Azevedo, A.M., Aires-Barros, M.R.,
Prazeres, D.M.F., “Purification of plasmid DNA with
aqueous two phase systems of PEG 600 and so-
dium citrate/ammonium sulphate”, Sep. Purif.
Technol., 65, 22-30
Guerrero-Germán, P., Prazeres, D.M.F., Guzmán,
R., Montesinos-Cisneros, R.M., Tejeda-Mansir, A.,
“Purification of plasmid DNA using tangential flow
filtration and tandem anion-exchange membrane
chromatography”, Bioprocess Biosyst. Eng., 32,
615-623
Henriques, A.M., Madeira, C., Fevereiro, M., Praz-
eres, D.M.F., Aires-Barros, M.R., Monteiro, G.A.,
“Effect of cationic liposomes/DNA charge ratio on
gene expression and antibody response of a candi-
date DNA vaccine against Maedi-Visna virus”, Int.
J. Pharm., 377, 92-98
Madeira, C., Estrela, N., Ferreira, J.A.B., Andrade,
S.M., Costa S.M.B., Melo, E.P., “Fluorescence life-
time imaging microscopy and fluorescence reso-
nance energy transfer from cyan to yellow fluores-
cent protein validates a novel method to cluster pro-
teins on solid surfaces”, J. Biomed. Opt., 14:
044035
Marques, M.P.C., Cabral, J.M.S., Fernandes, P.,
“High throughput in biotechnology: from shake-
flasks to fully instrumented microfermentors”, Re-
cent Pat. Biotechnol., 3, 124-140
Marques, M.P.C., Carvalho, F., Magalhães, S.,
Cabral, J.M.S., Fernandes, P., “Screening for suit-
able solvents as substrate carriers for the microbial
side-chain cleavage of sitosterol using microtitre
plates”, Process Biochem., 44, 174-180
Marques, M.P.C., Magalhães, S., Cabral, J.M.S.,
Fernandes, P., “Characterization of 24-well micro-
titer plate reactors for a complex multi-step biocon-
version: from sitosterol to androstenedione”, J. Bio-
technol., 141, 174-180
Martins, S.A.M., Prazeres, D.M.F., Fonseca, L.P.,
Monteiro, G.A., “The role of probe-probe interac-
tions on the hybridization of double stranded DNA
targets onto DNA-modified magnetic microparti-
cles”, Anal. Bioanal. Chem., 394, 1711-1716
Martins, S.A.M., Prazeres, D.M.F., Fonseca, L.P.,
Monteiro, G.A., “Application of central composite
design for DNA hybridization onto magnetic mi-
croparticles”, Anal. Biochem., 391, 17-23
Martins, V.C., Cardoso, F.A., Germano, J., Cardo-
so, S., Sousa, L., Piedade, M., Freitas, P.P., Fon-
seca, L.P. “Femtomolar limit of detection with a
magnetoresistive biochip”, Biosens. Bioelectron.,
24, 2690-2695
Oliveira, P.H., Prather, K.J., Prazeres, D.M.F., Mon-
teiro, G.A., “Structural instabilities of plasmid bio-
pharmaceuticals: Challenges and implications”,
Trends Biotechnol., 27, 503-509
Oliveira, P.H., Prazeres, D.M.F., Monteiro, G.A.,
“Deletion-formation mutations in plasmid expression
vectors are unfavoured by runaway amplification
conditions and differentially selected under kanamy-
cin stress”, J. Biotechnol., 143, 231-238
Paixão, L., Rodrigues, L., Martins, M., Couto, I.,
Fernandes P., de Carvalho, C.C.C.R., Monteiro, G.,
Sansonetty, F., Amaral, L., Viveiros, M.,
“Fluorometric determination of ethidium bromide
efflux kinetics in Escherichia coli”, J. Biol. Eng., 3:
18
Pereira, A.T., Pimentel, A.C, Chu, V., Prazeres,
D.M.F., Conde, J.P., “Chemiluminescence detection
of horseradish peroxidase using an integrated using
an amorphous silicon thin film photosensor”, IEEE
Sensors, 9, 1282-1290
Pimentel, A.C, Pereira, A.T., Prazeres, D.M.F., Chu,
V., Conde, J.P., “Detection of fluorescently labeled
biomolecules immobilized on a detachable sub-
strate using and integrated amorphous silicon
photodetector”, Appl. Phys. Lett., 94: 164106
Pinotti, L.M., Fonseca, L.P., Prazeres, D.M.F., Rod-
rigues, D.S., Nuccia, E.R., Giordano, R.L.C.,
“Recovery and partial purification of penicillin G acy-
lase from E. coli homogenate and B. megaterium
culture medium using an expanded bed adsorption
column”, Biochem. Eng. J., 44, 111-118
Ribeiro, S.C., Monteiro, G.A., Prazeres, D.M.F.,
“Evaluation of the effect of non-B DNA structures on
plasmid integrity via accelerated stability studies”, J.
Pharm. Sci., 98, 1400-1408
Rosa, P.A.J., Azevedo, A.M., Ferreira, I.F., Som-
merfeld, S., Bäcker, W., Aires-Barros, M.R.,
“Downstream processing of antibodies: Single-
stage versus multi-stage aqueous two-phase ex-
traction”, J. Chromatogr. A, 1216, 8741-8749
Rosa, P.A.J., Azevedo A.M., Sommerfeld, S., Mut-
ter, M., Aires-Barros, M.R., Bäcker, W., “Application
of aqueous two-phase systems to antibody purifica-
tion: A multi-stage approach”, J. Biotechnol., 139,
306-313
Serro, A.P., Completo, C., Colaço, R., dos Santos,
F., da Silva, C.L., Cabral, J.M.S., Araújo, H., Pires,
E., Saramago, B., “A comparative study of titanium
nitrides, TiN, TiNbN and TiCN, as coatings for bio-
medical applications”, Surf. Coat. Technol., 203,
3701-3707
Silva, A.I., Mateus, M., “Development of a polysul-
fone hollow fiber vascular bio-artificial pancreas
device for in vitro studies”, J. Biotechnol., 139, 236
-249
da Silva, C.L., Gonçalves, R., Porada, C.D., Ascen-
são, J.L., Zanjani, E.D., Cabral, J.M.S., Almeida-
Porada, G., “Differences amid bone marrow and
cord blood hematopoietic stem/progenitor cell divi-
sion kinetics”, J. Cell. Physiol., 220, 102-111
Silva, M.S., Prazeres, D.M.F., Lança, A., Atouguia,
J., Monteiro, G.A., “Trans-sialidase from Trypano-
soma brucei as a potential target for DNA vaccine
development against African Trypanosomiasis”,
Parasitol. Res., 105, 1223-1229
Silva, T., Lima, P., Hageman, S., Fonseca, L.P.,
Calado, C.R.C., “Prediction of dynamic plasmid pro-
duction by recombinant Escherichia coli fed-batch
cultivations with a generalized regression neural
network”, Chem. Biochem. Eng. Q., 23, 419-427
Sousa, A., Sousa, F., Prazeres, D.M.F., Queiroz,
J.A., “Histidine affinity chromatography of homo-
oligonucleotides. Role of multiple interactions on
retention”, Biomedical Chromatogr., 23, 745-753
Sousa, F., Prazeres, D.M.F., Queiroz, J.A.,
“Improvement of transfection efficiency by using
supercoiled plasmid DNA purified from a clarified
lysate with arginine affinity chromatography”, J.
Gene Med., 11, 79-88
Sousa, F., Prazeres, D.M.F., Queiroz, J.A., “Binding
and elution strategy for improved performance of
arginine affinity chromatography in supercoiled
plasmid DNA purification”, Biomedical Chroma-
togr., 23, 160-165
Sousa, I.T., Ruiu, L., Lowe, C.R., Taipa, M.A.,
“Synthetic affinity ligands as a novel tool to improve
protein stability”, J. Mol. Recognit., 22, 83-90
Temtem, M., Silva, L.M.C., Andrade, P.Z., dos San-
tos, F., da Silva, C.L., Cabral, J.M.S., Abecasis,
M.M., Aguiar-Ricardo, A., “Supercritical CO2 gener-
ating chitosan devices with controlled morphology.
Potential application for drug delivery and mesen-
chymal stem cell culture”, J. Supercritical Fluids,
48, 269-277
Wu, M.L., Freitas, S.S., Monteiro, G.A., Prazeres,
D.M.F., Santos, J.A.L., “Stabilization of naked and
condensed plasmid DNA against degradation in-
duced by ultrasounds and high shear vortices”, Bio-
technol. Appl. Biochem., 53, 237-246
Articles in Conference Proceedings
Andrade, P.Z., dos Santos, F., Almeida-Porada, G.,
da Silva, C.L., Cabral, J.M.S., “Non-viral gene deli-
very to human mesenchymal stem Synergistic cy-
tokine effects modulate ex-vivo expansion of he-
matopoietic stem/progenitor cells in co-culture
with mesenchymal stem cells ” Exp. Hematology,
37, S97
de Barros, D.P.C., Fonseca, L.P., Cabral, J.M.S.,
Weiss, C.K., Landfester, K., “Biosynthesis of fatty
acids alkyl esters in miniemulsion as a reaction me-
dia”, New Biotechnol., 25, S116-S117
Bernardino, S., Fernandes, P., Fonseca, L.,
“Penicillin G acylase stabilization by immobilization
in sol-gel micro-particles. Increase in hydrolase and
synthetase activity using Tris buffer”, New Biotech-
nol., 25, S120-S121
20 39 09 Annual Report
Cordas, C.M., Lourenço, N.M.T., Vidinha, P.,
Afonso, C.A.M., Barreiros, S., Fonseca, L.P.,
Cabral, J.M.S., “New conducting biomaterial based
on Ion Jelly technology for development of a new
generation of biosensors”, New Biotechnol., 25,
S138-S139
Madeira, C., Ribeiro, S., Boura, J., da Silva, C.L.,
Cabral, J.M.S., “Microporation of human mesenchy-
mal stem cells promotes high cellular recoveries
and efficient plasmid gene delivery”, Hum. Gene
Ther., 20, 1490-1491
Madeira, C.P., Ribeiro, S.C., dos Santos, F., Mon-
teiro, G.A, da Silva, C.L, Cabral, J.M.S.
“Comparision of polyadenylation signals for use in
transient genetic modification of human mesenchy-
mal stem cells”. Exp. Hematology, 37, S97.
Madeira, C.P., Mendes, R.D., Ribeiro, S.C., Aires-
Barros, M.R., dos Santos, F., da Silva, C.L., Cabral,
J.M.S. “Non-viral gene delivery to human mesen-
chymal stem cells using cationic liposomes”, Exp.
Hematology, 37, S97
Martins, S.A., Prazeres, D.M.F., Fonseca, L.P.,
Monteiro, G., “DNA biosensors: towards a micropar-
ticle based-platform for genomic DNA detection”,
New Biotechnol., 25, S19-S20
Martins, V., Cardoso, F., Freitas, P., Germano, J.,
Cardoso, S., Souse, L., Piedade, M., Fonseca, L.,
“Magneto resistive biochip-based portable platform
for biomolecular recognition detection”, New Bio-
technol., 25, S358-S359
Pereira, A.T., Chu, V., Prazeres, D.M.F., Conde,
J.P., "Enzymatic biosensors with integrated thin film
a-Si:H photodiodes", Mat. Res. Soc. Symp. Proc.,
1153, A08-04
Pereira, A.T., Chu, V., Prazeres, D.M.F., Conde,
J.P., "Miniaturization of immunoassays using optical
detection with integrated amorphous silicon photo-
diodes", Mat. Res. Soc. Symp. Proc., 1191, OO08-
04
Toledo, M.A.S., Monteiro, G.A., Prazeres, D.M.F.,
Souza, A.P., Azzoni, A.R., “Development of recom-
binant proteins for non viral gene delivery taking
advantage of molecular motor proteins”, Hum.
Gene Ther., 20, 1438-1438
Toledo, M.A.S., Monteiro, G.A., Prazeres, D.M.F.,
Souza, A.P., Azzoni, A.R., “The use of recombinant
dynein light chains to mediate enhanced plasmidial
DNA transfection proteins for non viral gene deli-
very taking advantage of molecular motor proteins”,
Hum. Gene Ther.,20, 1441
Book chapters
de Carvalho, C.C.C.R., “Biotransformation of ter-
penes by Fungi”, in Advances in Fungal Biotech-
nology (Mahendra Rai, Editor), I.K. International
Publishing House Pvt. Ltd, New Delhi, pp. 528-542
Fernandes, P., “Enzymes in sugar industries”, in
Enzymes in Food Processing: Fundamentals
and Potential Applications (Parmjit S. Panesar,
Satwinder S. Marwaha, Harish Kumar, Editors), I.K.
International Publishing House Pvt. Ltd, New Delhi,
pp. 165-198
Patents
Baecker, W., Sommerfeld, S., Mutter, M., Rosa,
P.A.J., Aires-Barros, M.R., Azevedo, A.M., “Method
for purifying therapeutic proteins” World Patent
WO/2009/112149
Dissertations
Ph.D. Thesis
Ana Isabel Quinta dos Santos Silva, “Study of a
membrane bioreactor as a model of bioartificial pan-
creas”, PhD in Chemical Engineering, IST
(Supervisor: M. Mateus)
Pedro Nuno de Sousa Sampaio, “Processos Inte-
grados para produção e recuperação in situ da
ciprosina por estirpes transformadas de Saccharo-
myces cerevisiae”, PhD in Biotechnology, IST
(Supervisors: L.P. Fonseca - IST, M.S. Soares Pais
- FCUL)
Ricardo Manuel Marques Caleiro Cabeça,
“Electric-Field assisted DNA surface immobilization
and hybridization”, PhD in Biotechnology, IST
(Supervisors: J.P. Conde, D.M.F. Prazeres)
Tiago Paulo Gonçalves Fernandes, "Cell-based
microarray systems for stem cell expansion and
differentiation", PhD in Biotechnology, IST
(Supervisor: J.M.S. Cabral)
Verónica Cristina Baião Martins Romão, “A mag-
netoresistive biochip platform: detection of DNA
hybridization and antibody/cell recognition”, PhD in
Biotechnology, IST (Supervisor: L.P. Fonseca)
M.Sc. Thesis
Laura Coutinho de Almeida Santos, MSc in bio-
logical engineering, “Utilização de Quitosano em
Aplicações Biomédicas” (Supervisors: M.R. Aires
Barros and I.A. Pires)
Meritxell Zurita Turk, MSc in Biotechnology,
“Evaluation of inducible vs. constitutive protein ex-
pression in HEK293Tcells” (Supervisors: J.M.S.
Cabral, T.C.P. Madeira)
Rui Daniel Mendes, MSc in Human Molecular Biol-
ogy from Faculdade de Ciências da Universidade
de Lisboa, “Optimization of differentiation protocolos
for human mesenchymal stem cells (hMSC)”,
(Supervisors: C.L. da Silva - IST, M.G.G.F. Rodri-
gues - FCUL).
Oral Communications
International Conferences
Botelho-Cunha, V., Mateus, M., Petrus, J.C.C., de
Pinho, M.N, "Nanofiltration process in fractionation
of galacto-oligosaccharides continuously produced
in bioreactors", EUROMEMBRANE 2009 Confer-
ence, Montpellier, France, September
de Carvalho, C.C.C.R., “Adaptation of bacterial cells
to antineoplastic agents may increase the risk of
post-chemotherapy infections”, BIT's 2nd World
Cancer Congress, Beijing, P.R. of China, June
(Invited speaker)
de Carvalho, C.C.C.R., “The remarkable adaptabil-
ity of Rhodococcus erythropolis cells”, III Interna-
tional Conference on Environmental, Industrial and
Applied Microbiology | BioMicroWorld2009, Lisbon,
Portugal, December
Carvalho, J., Monteiro, G.A., Atouguia, J., Prazeres,
D.M.F., Rodgers, J., “The challenges of developing
a DNA vaccine for African Trypanosomiasis”, Joint
Malaria and Spring Meeting, British Society for
Parasitology, Edinburgh, UK, April
Ferreira, I.F., de Carvalho, C.C.C.R., Wang, D.I.C.,
Aires-Barros, M.R., “Desulfurization of crude oil by
Rhodococcus erythropolis cells”, III International
Conference on Environmental, Industrial and Ap-
plied Microbiology | BioMicroWorld2009, Lisbon,
Portugal, December
Fernandes, P., Marques, M.P.C., Carvalho, F.,
Cabral, J.M.S., “Carbohydrate hydrolysis with com-
mercial inulinase entrapped in PVA-based beads”,
29th International Exhibition-Congress on Chemical
Engineering, Environmental Protection and Biotechnol-
ogy | ACHEMA 2009, Frankfurt, Germany, May
(Invited speaker)
Fonseca, L.P., “Modern methods for assessment of
enzyme activity in microcapsules”, Spring Work-
shop on Bioencapsulation, Luxembourg, April
Fonseca, L.P., “From conventional biosensors to
new analytical devices for a better quality of life:
Magneto-Resistive Biosensors”, Biosensor Work-
shop, University of Ljubljana, Ljubljana, Slovenia,
April
Fonseca, L.P., “New conducting biomaterial based
on Ion Jelly® technology for development of a new
generation of Biosensors”, Biosensors Workshop,
14th European Congress on Biotechnology, Barce-
lona, Spain, September
Gomes, A.G., Azevedo, A.M., Aires-Barros, M.R.,
Prazeres, D.M.F., "Exploring the interaction of nu-
cleic acids with phenyl boronate ligands as an alter-
native for the purification of pharmaceutical grade
pDNA", 15th International Conference Biopartitioning
and Purification | BPP2009, Uxbridge, UK, June
20 41 09 Annual Report
Marques, M.P.C., Carvalho, F., Fernandes, P.,
Cabral, J.M.S., “Steroids bioconversion: towards
green processes”, 29th International Exhibition-
Congress on Chemical Engineering, Environmental
Protection and Biotechnology | ACHEMA 2009,
Frankfurt, Germany, May
Oliveira, P.H., Prather, K.J., Prazeres, D.M.F., Mon-
teiro, G.A., “Structural instability in plasmid vectors
for DNA vaccination”, III International Conference
on Environmental, Industrial and Applied Microbiol-
ogy | BioMicroWorld2009, Lisbon, Portugal, Decem-
ber
Pereira, A.T., Chu, V., Prazeres, D.M.F., Conde,
J.P., "Enzymatic biosensors with integrated thin film
a-Si:H photodiodes", Spring Meeting of the Material
Research Society | MRS, San Francisco, USA, April
Pereira, A.T., Chu, V., Prazeres, D.M.F., Conde,
J.P., "Miniaturization of immunoassays using optical
detection with integrated amorphous silicon photo-
diodes", Spring Meeting of the Material Research
Society | MRS, San Francisco, USA, April
Raiado-Pereira, L., Prazeres, D.M.F. Mateus, M.,
"Effect of ligand type on membrane hydrophobic
interaction chromatography for the purification of
plasmid DNA vaccines", 15th International Confer-
ence on Biopartitioning and Purification | BPP2009,
Uxbridge, UK, June
Raiado-Pereira, L., Prazeres, D.M.F. Mateus, M.,
"Preparation, characterization and testing of ad-
sorption membranes for chromatographic separa-
tion of therapeutic plasmids", EUROMEMBRANE
2009 Conference, Montpellier, France, September
Rosa, P.A.J., Ferreira, I.F., Azevedo, A.M., Aires-
Barros, M.R., "Affinity partition and purification of
therapeutic proteins, namely antibodies", 15th Inter-
national Conference on Biopartitioning and Purifica-
tion | BPP2009, Uxbridge, UK, June (invited key-
note lecture)
Rosa, P.A.J., Azevedo, A.M., Sommerfeld, S.,
Bäcker, W., Aires-Barros, M.R., "Continuous aque-
ous two-phase extraction process for the purifica-
tion of antibodies: Performance and economical
feasibility", 15th International Conference on Biopar-
titioning and Purification | BPP2009, Uxbridge, UK,
June
Santa, G.L.M., Bernardino, S.M.S.A., Chheub V,
Fonseca, L.P., Fernandes, P., “From inulin to fruc-
tose syrups: evaluation of the sol-gel approach for
inulinase immobilization”, 8th International Meeting
of the Portuguese Carbohydrate Group | Glupor 8,
Braga, Portugal, September
Santos, R., Marques, M., Oliveira, P., Carvalho, F.,
de Carvalho, C.C.C.R., Monteiro, G., Cabral,
J.M.S., Frade, R., Silva, M., Fernandes, P., “The
sunny side of Mycobacteria”, European Society of
Mycobacteriology, Porto, Portugal, July
Silva, A.I., Mateus, M., "Construction and perfor-
mance assessment of an in vitro testing device for
the development of vascular bio-artificial pancreas",
Membranes in Medicine, Workshop in the frame-
work of the of the European Network of Excellence
on Nanoscale-based Membrane Technologies, Lis-
bon, Portugal, February
Sousa, I.T., Taipa, M.A., “Triazine-scaffolded syn-
thetic affinity ligands as a novel tool to improve pro-
tein stability”, 8th International Conference on Pro-
tein Stabilisation | ProStab2009, Graz, Austria, April
National Conferences
Andrade, P.Z., dos Santos, F., Almeida-Porada, G.,
da Silva, C.L., Cabral, J.M.S., “Ex-vivo expansion of
hematopoietic stem/progenitor cells in co-culture
with mesenchymal stem cells: optimizing the cyto-
kine cocktail", 4th International Meeting of the Portu-
guese Society of Stem Cells and Cell Therapies |
SPCE-TC, Lisbon, April
de Carvalho, C.C.C.R., “Rhodococcus: aplicações
em Biotecnologia”, V Dia de Biologia Marinha e
Biotecnologia, Escola Superior de Turismo e Tec-
nologia do Mar, Peniche, May
Eibes, G., dos Santos, F., Andrade, P.Z., da Silva,
C.L., Cabral, J.M.S., “A microcarrier-based stirred
culture system for the expansion of human mesen-
chymal stem cells", 4th International Meeting of the
Portuguese Society of Stem Cells and Cell Thera-
pies | SPCE-TC, Lisbon, April
Fernandes, P., Marques, M.P.C., Cattorini, S., Car-
valho, F., Cabral , J.M.S., “A dual purpose immobi-
lized biocatalyst for inulin and sucrose hydrolysis”,
Carbohydrates as Organic Raw Materials V (CORM
V), Lisbon, January
Fernandes-Platzgummer, A.M., Diogo, M.M., Bap-
tista, R.P., da Silva, C.L., Cabral, J.M.S. “Scale-up
of mouse embryonic stem cell expansion: from spin-
ner-flask to a fully controlled bioreactor”, 4th Interna-
tional Meeting of the Portuguese Society of Stem
Cells and Cell Therapies | SPCE-TC, Lisbon, April
Fonseca, L.P., “Biocatálise enzimática, dos reacto-
res industriais aos micro-reactores”, Jornadas Bio-
tecnologia ESB-UCP, Porto, October
Fonseca, L.P., “Biocatálise no desenvolvimento de
processos industriais mais sustentáveis e amigos
do ambiente”, Jornadas Tecnológicas da UNL-FCT
| JORTEC 2009, Lisboa, May
Fonseca, L.P., “New trends in biocatalysisin indus-
trial biotechnology”, Joint Meeting of the Portuguese
Society of Microbiology and of the Portuguese Society
of Biotechnology | MicroBiotec09, Vilamoura,
November
Lourenço, N.M.T., Vidinha, P., Cordas, C.M., Afon-
so, C.A.M., Barreiros, S., Cabral, J.M.S., Fonseca,
L.P., “Real-time pathogens bio-sensing based on
Ion Jelly® technology”, MIT-Portugal Program, Can-
tanhede, March
Oliveira, P.H., Prather, K.J., Prazeres, D.M.F., Mon-
teiro, G.A., “Structural instability in plasmid vectors
for DNA vaccination”, Joint Meeting of the Portuguese
Society of Microbiology and of the Portuguese Society
of Biotechnology | MicroBiotec09, Vilamoura,
November
Rodrigues, C.A.V., Diogo, M.M., da Silva, C.L.,
Cabral, J.M.S., “Low oxygen tension enhances pro-
liferation of mouse neural stem cells", 4th Interna-
tional Meeting of the Portuguese Society of Stem
Cells and Cell Therapies | SPCE-TC, Lisbon, April
Teixeira, M.C., Mira, N.P., Raposo, L.R., Sintra,
E.R., de Carvalho, C.C.C.R., Lourenço, A.B., Sá-
Correia, I., “Global analysis of yeast determinants of
resistance to ethanol: the important role of FPS1”,
Joint Meeting of the Portuguese Society of Microbiol-
ogy and of the Portuguese Society of Biotechnology |
Microbiotec09, Vilamoura, November
Poster Communications
International Conferences
Andrade, P.Z., dos Santos, F., Almeida-Porada, G.,
da Silva, C.L., Cabral, J.M.S., " Ex-vivo expansion
of hematopoietic stem/progenitor cells in co-culture
with mesenchymal stem cells: optimizing the cyto-
kine cocktail", 7th Annual Meeting of International
Society of Stem Cell Research | ISSCR, Barcelona,
Spain, July
Andrade, P.Z., dos Santos, F., Almeida-Porada, G.,
da Silva, C.L., Cabral, J.M.S., “Synergistic cytokine
effects modulate ex-vivo expansion of hemato-
poietic stem/progenitor cells in co-culture with
mesenchymal stem cells”, 38th Annual Scientific
Meeting of the ISEH-Society for Hematology and
Stem Cells, Athens, Greece, September
Azevedo, A.M., Rosa, P.A.J., Aires-Barros, M.R.,
“Purification of antibodies by phenyl boronate affin-
ity chromatography”, 15th International Conference
on Biopartitioning and Purification | BPP2009, Ux-
bridge, UK, June
Azevedo, A.M., Rosa, P.A.J., Sommerfield, S.,
Baecker, W., Aires-Barros, M.R., “Affinity extraction
of human therapeutic antibodies in aqueous two-
phase systems”, 18th Biennial Meeting of the Inter-
national Society for Molecular Recognition | Affinity
2009, Reykjavik, Iceland, July
Azevedo, A.M., Rosa, P.A.J., Borlido, L., Aires-
Barros, M.R., “Purification of antibodies by phenyl
boronate affinity chromatography”, 18th Biennial
Meeting of the International Society for Molecular
Recognition | Affinity 2009, Reykjavik, Iceland, July
Azzoni, A., Monteiro, G.A., Prazeres, D.M.F.,
Souza, A.P., “Development of recombinant proteins
for non viral gene delivery. Taking advantage of
molecular motor proteins”, XXXVIII Annual Meeting
of the Brazilian Society for Biochemistry and Mo-
lecular Biology, Águas de Lindóia, São Paulo, Bra-
zil, May
Badenes, S.M., Lemos, F., Cabral, J.M.S., “Bio-
catalytic process using microencapsulated recombi-
nant cutinase in AOT-isooctane reversed micelles
for the synthesis of biodiesel”, 14th European Con-
gress on Biotechnology | ECB 14, Barcelona,
Spain, September
20 43 09 Annual Report
Badenes, S.M., Lemos, F., Cabral, J.M.S., “Bio-
diesel production by an enzymatic transesterifica-
tion using microencapsulated recombinant cutinase
in AOT-isooctane reversed micelles”, Enzyme Engi-
neering XX, Groningen, Netherlands, September
de Barros, D.P.C., Fonseca, L.P., Cabral, J.M.S.,
“Fed-batch operation mode as a tool for successful
biosynthesis of aroma esters catalyzed by cuti-
nase”, 9th International Symposium on Biocatalysis
and Biotransformations | BIOTRANS 2009, Berne,
Switzerland, July
de Barros, D.P.C., Fonseca, L.P., Cabral, J.M.S.,
Weiss, C.K., Landfester, K., “Great potential of mini-
emulsion system as a green chemistry reaction me-
dia for biosynthesis of aroma esters”, 9th Interna-
tional Symposium on Biocatalysis and Biotransfor-
mations | BIOTRANS 2009, Berne, Switzerland,
July
de Barros, D.P.C., Fonseca, L.P., Cabral, J.M.S.,
Weiss, C.K., Landfester, K., “Biosynthesis of fatty
acids alkyl esters in miniemulsion as a reaction me-
dia”, 14th European Congress on Biotechnology,
Barcelona | ECB 14, Spain, September
de Barros, D.P.C., Fonseca, L.P., Cabral, J.M.S.,
Weiss, C.K., Landfester, K., “Miniemulsion as a
green chemistry reaction media in biosynthesis of
fatty acids alkyl esters”, Enzyme Engineering XX,
Groningen, Netherlands, September
Bernardino, S.M.S.A., Fernandes, P., Fonseca,
L.P., “Penicillin G acylase stabilization by immobili-
zation in sol-gel micro-particles: increase in hy-
drolase and synthetase activity using tris buffer”,
14th European Congress on Biotechnology, Barce-
lona | ECB 14, Spain, September
Bernardino, S.M.S.A., Fernandes, P., Fonseca,
L.P., “Optimization of Penicillin G Acylase Immobili-
zation by Entrapment in Silica Xerogel with Mag-
netic Properties”, 9th International Symposium on
Biocatalysis and Biotransformations | BIOTRANS
2009, Bern, Switzerland, July
Bernardino, S.M.S.A., Fernandes, P., Fonseca,
L.P., “Penicillin G Acylase: Tris or phosphate
buffer? What is the best option?”, 8th International
Conference on Protein Stabilisation | ProStab2009,
Graz, Austria, April
Borlido, L., Azevedo, A.M., Aires-Barros, M.R.,
“Studies on IgG stability in aqueous two-phase ex-
traction process”, 15th International Conference on
Biopartitioning and Purification | BPP2009, Ux-
bridge, UK, June
Chheub, V., Cattorini, S., Fonseca, L.P., Fernan-
des, P., “Immobilization in Lentikat® based parti-
cles: towards the production of fructose syrups”,
Enzyme Engineering XX, Groningen, Netherlands,
September
de Carvalho, C.C.C.R., Caramujo, M.J., “Tumor
metastasis as an adaptation of tumor cells to fulfill
their phosphorus requirements”, BIT's 2nd World
Cancer Congress, Beijing, P.R. of China, June
Cordas, C.M., Lourenço, N.M.T., Vidinha, P.,
Afonso, C.A.M., Barreiros, S., Fonseca, L.P.,
Cabral, J.M.S., “New conducting biomaterial based
on Ion Jelly technology for development of a new
generation of biosensors”, 14th European Congress
on Biotechnology | ECB 14, Barcelona, Spain, Sep-
tember
Eibes, G., dos Santos, F., Andrade, P.Z., da Silva,
C.L., Cabral, J.M.S., "A microcarrier-based stirred
culture system for the expansion of human mesen-
chymal stem cells", 7th Annual Meeting of Interna-
tional Society of Stem Cell Research | ISSCR, Bar-
celona, Spain, July
Fernandes, T.G., Fernandes-Platzgummer, A.M.,
Diogo, M.M., da Silva, C.L., Dordick, J.S., Cabral,
J.M.S., "Effects of low oxygen tension on mouse
embryonic stem cell expansion", 7th Annual Meeting
of International Society of Stem Cell Research |
ISSCR, Barcelona, Spain, July
Fernandes, T.G., Fernandes-Platzgummer, A.M.,
Diogo, M.M., da Silva, C.L., Dordick, J.S., Cabral,
J.M.S., "Effects of low oxygen tension on mouse
embryonic stem cell expansion", 3rd International
Stem Cell Meeting: Biology and Clinical Applica-
tions, Tel-Aviv, Israel, June
Fernandes-Platzgummer, A.M., Diogo, M.M., Bap-
tista, R.P., da Silva, C.L., Cabral, J.M.S. “Scale-up
of mouse embryonic stem cell expansion: from spin-
ner-flask to a fully controlled bioreactor”, 7th Annual
Meeting of International Society of Stem Cell Re-
search | ISSCR, Barcelona, Spain, July
Fonseca, L.P., Bernardino, S.M.S.A., Fernandes,
P., “Biocatalysts prepared by the entrapment of
inulinase and penicillin G acylase in sol-gel”, XVII
International Conference on Bioencapsulation,
Groningen, Netherlands, September
Gomes, A.G., Azevedo, A.M., Aires-Barros, M.R.,
Prazeres, D.M.F., “RNA clearance from plasmid-
containing lysates by phenyl boronate adsorption”,
18th Biennial Meeting of the International Society for
Molecular Recognition | Affinity 2009, Reykjavik,
Iceland, July
Lourenço, N.M.T., Monteiro, C.M., Afonso, C.A.M.,
“Enzymatic kinetic resolution of sec-alcohols based
on ionic-acylating- agents”, 9th International Sympo-
sium on Biocatalysis and Biotransformations | BIO-
TRANS 2009, Bern, Switzerland, July
Madeira, C.P., Ribeiro, S.C., dos Santos, F., Mon-
teiro, G.A., da Silva, L., Cabral, J.M.S.,
“Comparison of polyadenylation signals for use in
transient genetic modification of human mesenchy-
mal stem cells”, 38th Annual Scientific Meeting of
the ISEH-Society for Hematology and Stem Cells,
Athens, Greece, September
Madeira, C., Ribeiro, S., Boura, J., da Silva, C.L.,
Cabral, J.M.S., “Microporation of human mesenchy-
mal stem cells promotes high cellular recoveries
and efficient plasmid gene delivery”, XVII Annual
Congress of the European Society of Gene and Cell
Therapy | ESGCT, Hannover, Germany, Novem-
ber
Madeira, C.P., Ribeiro, S.C., dos Santos, F., Mon-
teiro, G.A., da Silva, L., Cabral, J.M.S., “Non-viral
gene delivery to human mesenchymal stem cells
using cationic lipossomes”, 38th Annual Scientific
Meeting of the ISEH-Society for Hematology and
Stem Cells, Athens, Greece, September
Martins, S.A., Prazeres, D.M.F., Fonseca, L.P.,
Monteiro, G., “DNA biosensors: towards a micropar-
ticle based-platform for genomic DNA detection”,
14th European Congress on Biotechnology | ECB
14, Barcelona, Spain, September
Martins, V., Cardoso, F., Freitas, P., Germano, J.,
Cardoso, S., Souse, L., Piedade, M., Fonseca, L.,
“Magneto resistive biochip-based portable platform
for biomolecular recognition detection”, 14th Euro-
pean Congress on Biotechnology | ECB 14, Barce-
lona, Spain, September
Nascimento, K.S., Yelo, S., Azevedo, A.M.,
Cavada, B.S., Aires-Barros, M.R., “Liquid-liquid
equilibrium data for aqueous two-phase system-
scomposed by ethylene oxide propylene oxide co-
polymers”, 15th International Conference on Biopar-
titioning and Purification | BPP2009, Uxbridge, UK,
June
Nascimento, K.S., Azevedo, A.M., Cavada, B.S.,
Aires-Barros, M.R., “Optimization of aqueous two-
phase extraction of Canavalia brasilienses lectin by
response surface methodology”, 15th International
Conference on Biopartitioning and Purification |
BPP2009, Uxbridge, UK, June
Oliveira, P.H., Prazeres, D.M.F., Monteiro, G.A.,
“Hotspots for recombination are widespread among
plasmid vectors”, FEMS 2009, 3rd Congress of
European Microbiologists, Gothenburg, Sweden,
June-July
Raiado-Pereira, L., Prazeres, D.M.F., Mateus, M.,
"Exploring liposome affinity membrane for chroma-
tographic separation of plasmid DNA vaccines",
EUROMEMBRANE 2009 Conference, Montpellier,
France, September
Ribeiro, S., Mendes, R., Madeira, C., dos Santos,
F., da Silva, C.L., Cabral, J.M.S., “Establishment of
a protocol for the evaluation of mesenchymal stem
cell transfection using real-time PCR”, 7th Annual
Meeting of International Society of Stem Cell Re-
search | ISSCR, Barcelona, Spain, July
Rodrigues, C.A.V., Diogo, M.M., da Silva, C.L.,
Cabral, J.M.S., “Low oxygen tension enhances pro-
liferation of mouse neural stem cells", 7th Annual
Meeting of International Society of Stem Cell Re-
search | ISSCR, Barcelona, Spain, July
Rodrigues, C.A.V., Diogo, M.M., da Silva, C.L.,
Cabral, J.M.S., “Low oxygen tension enhances pro-
liferation of mouse neural stem cells", 3rd Interna-
tional Stem Cell Meeting: Biology and Clinical Appli-
cations, Tel-Aviv, Israel, June
dos Santos, F., Andrade, P.Z., da Silva, C.L., Abe-
casis, M., Cabral, J.M.S., "Ex-vivo expansion of
human mesenchymal stem cell (MSC) under hy-
poxia: effects on proliferation kinetics and metabo-
lism", 7th Annual Meeting of International Society of
Stem Cell Research | ISSCR, Barcelona, Spain,
July
20 45 09 Annual Report
dos Santos, F., Andrade, P.Z., da Silva, C.L., Abe-
casis, M., Cabral, J.M.S., "Ex-vivo expansion of
human mesenchymal stem cell (MSC) under hy-
poxia: effects on proliferation kinetics and metabo-
lism", Regenerative Medicine and Adult Stem Cell
Therapy | MSC2009, Cleveland, USA, August
da Silva, C.L., Eibes, G., dos Santos, F., Andrade,
P.Z., da Silva, C.L., Cabral, J.M.S., "A microcarrier-
based stirred culture system for the expansion of
human mesenchymal stem cells", Regenerative
Medicine and Adult Stem Cell Therapy | MSC2009,
Cleveland, USA, August
Silva, C.S.O., Lansalot, M., Afonso, C.A.M., Marti-
nho, J.M.G., Taipa, M.A., �”Bio-inspired affinity-
polymer systems for antibody recognition”, 18th Bi-
ennial meeting of the International Society for Mo-
lecular Recognition | Affinity 2009, Reykjavik, Ice-
land, July
Toledo, M.A.S., Monteiro, G.A., Prazeres, D.M.F.,
Souza, A.P., Azzoni, A.R., “Development of recom-
binant proteins for non viral gene delivery taking
advantage of molecular motor proteins”, XVII Con-
gress of the European Society of Gene and Cell
Therapy | ESGCT, Hannover, Germany, November
Toledo, M.A.S., Monteiro, G.A., Prazeres, D.M.F.,
Souza, A.P., Azzoni, A.R., “The use of recombinant
dynein light chains to mediate enhanced plasmidial
DNA transfection proteins for non viral gene deli-
very taking advantage of molecular motor proteins”,
XVII Congress of the European Society of Gene
and Cell Therapy | ESGCT, Hannover, Germany,
November
National Conferences
Andrade, P.Z., dos Santos, F., Almeida-Porada, G.,
da Silva, C.L., Cabral, J.M.S., “Ex-vivo expansion of
hematopoietic stem/progenitor cells in co-culture
with mesenchymal stem cells: optimizing the cyto-
kine cocktail", 4th International Meeting of the Portu-
guese Society of Stem Cells and Cell Therapies |
SPCE-TC, Lisbon, April
de Barros, D.P.C., Fonseca, L.P., Cabral, J.M.S.,
Weiss, C.K., Landfester, K., “Synthesis of flavor
esters by cutinase in miniemulsion and organic sol-
vent media”, Joint Meeting of the Portuguese Society
of Microbiology and of the Portuguese Society of Bio-
technology | Microbiotec09, Vilamoura, November
Boura, J.S., dos Santos, F., da Silva, C.L., Abe-
casis, M.A., Vale, P., Cabral, J.M.S., “Culture me-
dium optimization for the ex-vivo expansion of adi-
pose tissue and bone marrow-derived mesenchy-
mal stem cells", 4th International Meeting of the Por-
tuguese Society of Stem Cells and Cell Therapies |
SPCE-TC, Lisbon, April
Eibes, G., dos Santos, F., Andrade, P.Z., da Silva,
C.L., Cabral, J.M.S., “A microcarrier-based stirred
culture system for the expansion of human mesen-
chymal stem cells", 4th International Meeting of the
Portuguese Society of Stem Cells and Cell Thera-
pies | SPCE-TC, Lisbon, April
Fernandes, T.G., Fernandes-Platzgummer, A.M.,
Diogo, M.M., da Silva, C.L., Dordick, J.S., Cabral,
J.M.S., "Effects of low oxygen tension on mouse
embryonic stem cell expansion", 4th International
Meeting of the Portuguese Society of Stem Cells
and Cell Therapies | SPCE-TC, Lisbon, April
Fernandes-Platzgummer, A.M., Diogo M.M., Bap-
tista, R.P., da Silva, C.L., Cabral, J.M.S. “Scale-up
of mouse embryonic stem cell expansion: from spin-
ner-flask to a fully controlled bioreactor”, 4th Interna-
tional Meeting of the Portuguese Society of Stem
Cells and Cell Therapies | SPCE-TC, Lisbon, April
Gomes, A.G., Azevedo, A.M., Aires-Barros, M.R.,
Prazeres, D.M.F., “RNA clearance from plasmid-
containing lysates by phenyl boronate adsorption”,
Joint Meeting of the Portuguese Society of Microbiol-
ogy and of the Portuguese Society of Biotechnology |
Microbiotec09, Vilamoura, November
Mendes, R., Madeira, C., Ribeiro, S., dos Santos,
F., da Silva, C.L., Cabral, J.M.S. “Non-viral gene
delivery to human mesenchymal stem/progenitor
cells using cationic liposomes”, 4th International
Meeting of the Portuguese Society of Stem Cells
and Cell Therapies | SPCE-TC, Lisbon, April
Oliveira, P.H., Prather, K.J., Prazeres, D.M.F. and
Monteiro, G.A. “Rational engineering of E. coli
strains and vectors for improved manufacturing of
plasmid biopharmaceuticals”, 1st MIT-Portugal An-
nual Conference: Engineering for Better Jobs. Lis-
bon, July
Ribeiro, S., Madeira, C., dos Santos, F., Monteiro,
G., da Silva, C.L.L., M.S. Cabral, J.M.S.
“Comparison of polyadenylation signals for use in
transient genetic modification of human mesenchy-
mal stem cells”, 4th International Meeting of the
Portuguese Society of Stem Cells and Cell Thera-
pies | SPCE-TC, Lisbon, April
Rodrigues, C.A.V., Diogo, M.M., da Silva, C.L.,
Cabral, J.M.S., “Low oxygen tension enhances
proliferation of mouse neural stem cells", 4th Inter-
national Meeting of the Portuguese Society of
Stem Cells and Cell Therapies | SPCE-TC, Lisbon,
April
dos Santos, F., Andrade, P.Z., da Silva, C.L., Abe-
casis, M., Cabral, J.M.S., "Ex-vivo expansion of
human mesenchymal stem cell (MSC) under hy-
poxia: Effects on proliferation kinetics and metabo-
lism", 4th International Meeting of the Portuguese
Society of Stem Cells and Cell Therapies | SPCE-
TC, Lisbon, April
Prizes
UTL/Deloitte Young Researcher Award
Cláudia Lobato da Silva received the UTL/Deloitte
Jovens Investigadores 2009 award in the scientific
domains of Biological Engineering, Biochemistry
and Biotechnology sponsored by the Technical
University of Lisbon and Deloitte (23rd June 2009).
Paula Rosa was distinguished with a Honourable
Mentions in UTL/Deloitte Jovens Investigadores
2009 award in the scientific domains of Biological
Engineering, Biochemistry and Biotechnology
sponsored by the Technical University of Lisbon
and Deloitte (23rd June 2009).
Filipa Ferreira was distinguished with a Honourable
Mentions in UTL/Deloitte Jovens Investigadores
2009 award in the scientific domains of Biological
Engineering, Biochemistry and Biotechnology
sponsored by the Technical University of Lisbon
and Deloitte (23rd June 2009).
UTL/Santander Totta Scientific Award
Miguel Prazeres was distinguished with a Honour-
able Mention in the Technical University of Lisbon
(UTL) scientific award competition in the Biological
Engineering, Biochemistry and Biotechnology area,
for the recognition of his scientific work and career,
sponsored by Santander Totta bank (16th Novem-
ber 2009).
Best Poster Award
Nuno Lourenço was named for best poster pre-
sented by a Young Researcher, at the 9th Interna-
tional Symposium on Biocatalysis and Biotransfor-
mation (Biotrans 2009) held in Bern, Switzerland
(9th July 2009).
Roche Younger Investigator Award
Cláudia Silva was distinguished with a Honourable
Mention of the Roche Younger Investigator Award
2009 at the Affinity 2009, the 18th biennial meeting
of the International Society for Molecular Recogni-
tion, in Reykjavik, Iceland (16th July 2009).
World Economic Forum's Annual Meet-
ing of the New Champions "Summer
Davos"
Cláudia Lobato da Silva was nominated as an out-
standing young scientist by the InterAcademy
Panel/World Economic Forum to participate in the
Annual Meeting of the New Champions "Summer
Davos" (10-12th September 2009).
20 47 09 Annual Report
Staff
Faculty
Joaquim M. S. Cabral
M. Raquel Aires-Barros
Luís P. Fonseca
D. Miguel F. Prazeres
Marília Mateus
Gabriel A. Monteiro
José A. L. Santos
Cláudia Lobato da Silva
M. Ângela Taipa
Frederico Ferreira
Research Scientists
Ana M. Azevedo
Carla C.C.R. de Carvalho
M. Margarida Diogo
Pedro Fernandes
Catarina Madeira
Post-doctoral Fellows
Ricardo Baptista
Cristina Cordas
Raquel Frade
Gemma Eibes
Nuno Lourenço
Sofia Ribeiro
PhD Students
Pedro Andrade
Susana Bernardino
Sara Badenes
Dragana Barros
Luís Borlido
Joana Carvalho
Nídia Estrela
Ana Fernandes
Tiago Fernandes
I. Filipa Ferreira
José Forte
Gabriela Gomes
Geisa Gonçalves
João Guerreiro
David Malta
Marco Marques
Sofia Martins
Verónica Martins
Patricia Soares
Kelany Nascimento
Pedro Oliveira
Carlos Rodrigues
Paula Rosa
Francisco Santos
Cláudia Silva
Ana Isabel Silva
Isabel Sousa
Jonathan de la Vega
Master Students
David Barata
Margarida Caras Altas
Filipe Carvalho
Rui Mendes
Johannes Österreicher
Sara Pereira
Irina Pinheiro
Ana Paula Sousa
Meritxell Turk
Research Assistants
Joana Biscaia
Joana Boura
Sofia Duarte
Jaqueline Garcia
João Martins
Luís Raiado-Pereira
Sofia Rebelo
Technicians
Ricardo Pereira
BERG
BioEngineering Research Group
Institute for Biotechnology and Bioengineering
Instituto Superior Técnico
Av. Rovisco Pais
1049-001 Lisbon
Portugal
www.ibb.pt/berg