converting rabbit hybridoma into recombinant antibodies ... · hah belfast 2016 dr. tim welsink...
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HAH Belfast 2016
Dr. Tim Welsink Molecular Biology
Transient Gene Expression
Converting rabbit hybridoma into recombinant antibodies with effective transient production in an optimized human
expression system
HAH Belfast 2016
OUTLINE
Short introduction to InVivo BioTech
Introduction
Project goal / needs
Methods developed
Case study rab-mab conversion & production
Summary
HAH Belfast 2016
COMPANY
CMO for R&D, diagnostics and pre-clinic
Founded 1998
>18 years experience in protein production
Staff – 50 persons
ISO9001 certified since 2004
Hybridoma Cultivation
More than 3000 different hybridoma cell lines
Up to 100 batches of one product
Complete serum-free production
Batches from 10 mg to 500 g
Recombinant Protein Production
More than 500 successful projects
Mainly HEK and CHO
Own proprietary vector and cell systems
Transient gene expression
HAH Belfast 2016
Introduction
Research and diagnostic standard:
mouse monoclonal AB (mAB)
Several advantages of mAB, but:
Lack of affinity, sensitivity and specificity
Small epitopes; non rodent immunogenic antigens
Rabbit monoclonal antibodies (rab-mabs) show up to 100 times higher affinities than mAB
Important in clinical and non-clinical diagnostic
rab-mabs are advantageous
But: very limited by low productivity
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Introduction
InVivo produced several rabbit hybridoma clones
Culture with bad performances and/or productivity
Occupation of up-scale capacity in production
Much effort in downstream and purification
High need of rabbit IgG for IVD
0
10
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891 1160 1163 1164 1796 1816 2675-1
mg
ra
b I
gG
/ l
ite
r
Antibody 1L spinner system
Rabbit antibody from hybridoma produced at InVivo – representative selection
HAH Belfast 2016
PROJECT GOAL
Needs
AB sequence – at least Fv Need
Sequence engineering
cDNA synthesis
Production platform & Need
High titer Need
Scalable
Proprietary Need
Antibody purification
Enhancing productivity of rabbit antibodies from hybridoma by converting into recombinant
antibodies and expressed transiently.
HAH Belfast 2016
cDNA sequencing
IgG cDNA design and IgG gene synthesis
Cloning in mammalian expression vector
Transient gene expression in serum-free suspension
culture
Purification by commonly used techniques
Fv SEQUENCE Project outline
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Fv SEQUENCE
RNA extraction, reverse transcription, gene amplification from cDNA, subcloning, sequencing
RNA-Isolation
Supplier 1 Supplier 2 Supplier 3
Reverse transcription (RT) PCR
Suppplier 1 Supplier 2
Total RNA extraction
Cryo vial
>1 x 106 cells in culture
Reverse transcription
Total RNA into cDNA
Parameters for kit decision
RNA yield
Quality of cDNA
1 2 3 4 5 6
hGAPDH Primer: 1 M, 2 H2O, 3 -RT, 4/5 sample, 6 +ctr
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Fv SEQUENCE
Amplification of Fv sequences using PCR
AB 1 AB 2 AB 3
500 bp 250 bp
Agarose gels of amplified Fv regions of 3 antibodies using 3 pairs of primers
Subcloning using TA- or blunt-method in cloning vectors
Transformation in E.coli
Plasmid preparation
Sequencing with standard primers
1 2 3 4 5
1: M, 2 to 5: Subcloned Fv amplificons, RE cut
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IgG GENE SYNTHESIS and CLONING
cDNA design including
Appropriate heavy and light chain constant region IgG sequence
Addition of RE sites, KOZAK, further modifications if appropriate
Codon optimization
GGC GGC CGC GAT ATC CCT AGG GCC ACC ATG AAG TGG GTC ACC TTT ATC TCC CTG CTG TTC CTG TTC
TCC TCC GCC TAC TCC GAA CTG GAC ATG ACC CAA ACC CCT GCC TCC GTG GAA GCT GCT GTT GGT GGT
ACT GTC ACC ATT AAG TGT CAA GCG AGT CAG AGC ATA TCC AAC CTG TTG GCC TGG TAT CAG CAG AAA
CCT GGG CAA AGA CCG AAA CTG CTC ATC TAT TAC GCC TCT AAT CTT GCT TCA GGC GTT TCC AGT CGG
TTC AAA GGG TCA GGA AGC GGT ACC GAG TAT ACA CTG ACC ATT TCT GGC GTC CAG TGT GCA GAT GCT
GCC ACC TAT TAC TGC CAG TCC TAC TAC TAC TCT AGC ACT TCC AAC TAT GCG TTC GAC TTT GGA GGA
GGG ACA GAA GTG GTG GTA AAG GGC GAT CCA GTG GCA CCC ACA GTC CTC ATC TTT CCT CCA GCA GCA
GAC CAG GTA GCC ACC GGT ACT GTC ACA ATC GTG TGT GTG GCC AAC AAG TAC TTT CCC GAC GTT ACG
GTG ACT TGG GAG GTT GAC GGC ACA ACT CAG ACG ACA GGC ATT GAG AAC AGC AAG ACA CCC CAG AAT
AGT GCC GAT TGC ACC TAT AAC CTG TCA AGC ACC CTT ACC CTG ACT TCA ACC CAG TAC AAT AGC CAC
AAG GAG TAC ACA TGC AAA GTG ACT CAA GGA ACC ACG TCT GTC GTG CAG AGC TTC AAT AGG GGC GAT
TGC TAA TAG GTT TAA ACT TAA TTA AAA GCT T
IgG gene synthesis by a subcontractor
Cloning in mammalian expression vector
Delivery by transient transfection
Transient gene expression
HAH Belfast 2016
PRODUCTION PLATFORM
Needs
Production platform & Need
High titer Need
Scalable
Proprietary Need
Several years of experience and research & development in transient gene expression
New developments needed
Customized CD-ACF media platform
Novel polycationic transfection reagent
Vector generation and large-scale plasmid preparation
Optimized host cell line
HAH Belfast 2016
MEDIA DEVELOPMENT
Medium development and
optimization
Transfection tests in media
variants
Analysis of cell growth,
substrates, etc.
Transfection reagents
Basic medium formulation
MEDIA DEVELOPMENT
Cooperation:
HAH Belfast 2016
TRANSFECTION REAGENT TRANSFECTION REAGENT
Cooperation:
Productivity analysis in HEK cells
Transfection of HEK cells with GFP vector
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PLASMID PREPARATION Medium
E.C
oli
ho
st
str
ain
1 2 3
A 11 mg/L 32 mg/L 17 mg/L
B 15 mg/L 28 mg/L 11 mg/L
C 17 mg/L 17 mg/L 27 mg/L
D 8 mg/L 28 mg/L 12 mg/L
PLASMID PREPARATION
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CELL LINE DEVELOPMENT Directed Evolution
Phenotype Analysis
Selection
Recovery
CELL LINE DEVELOPMENT
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PROCESS OPTIMIZATION PROCESS OPTIMIZATION
Combining the new achievements Feeding regime:
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TRANSIENT GENE EXPRESSION Summary
InVivos Expression System for transient Transfection (InVEST)
Customized CD-ACF media platform: XellVivoTM
Novel polycationic transfection reagent: INVect
Vector generation and large scale plasmid preparation
Optimized host cell line: HEK-INV
HAH Belfast 2016
CASE STUDY
RAB-MAB CONVERSION
monoclonal rabbit hybridoma AB
recombinant rabbit AB produced by TGE
1) cDNA sequencing
2) Vector design, preparation
3) Transient production
4) Purification
5) QC
Hybridoma rab. AB yield
0102030405060708090
100
mg
ra
b I
gG
to
tal
HAH Belfast 2016
CASE STUDY
Sequence alignment HC variable region:
Primer sequences from Seeber et al., 2014 & Rader et al., 2000
Complementarity Determining Regions (CDRs) are colored, mismatches are highlighted
HAH Belfast 2016
CASE STUDY
Sequence alignment LC variable region:
Primer sequences from Seeber et al., 2014 & Rader et al., 2000
Complementarity Determining Regions (CDRs) are colored, mismatches are highlighted
HAH Belfast 2016
CASE STUDY
cDNA Design
IgG gene synthesis
Cloning into expression vector
Low-endotoxin plasmid preparation
Transient gene expression
HAH Belfast 2016
CASE STUDY
Transient gene expression
Cell line: HEK or HEK-INV
Transfection protocol: optimized
Culture protocol: optimized
Scale: 50 to 1,500 ml
Production for 7 days
Harvest by centrifugation
Purification
AF-rProtein A-650F (Tosoh Bioscience)
Äkta chromatography system
Elution by low pH
Dialysis to PBS pH 7.4
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CASE STUDY
RESULTS
4 Batches produced
Different production scale
2 cell lines
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500
600
700
800
150 ml 750 ml 50 ml 1500 ml
HEK HEK-INV
Tit
er
[mg
/L]
Production scale, cell line
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CASE STUDY QC: capillary gel electrophoresis for purity
Reducing conditions, Agilent 2100-BioAnalyzer, Protein 230 Kit
Non-reducing conditions, Agilent 2100-BioAnalyzer, Protein 230 Kit
Purity ≥ 98%
Purity ≥ 85%
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CASE STUDY
Analytical SEC for determination of aggregates
Vitamin B12 1.350 Da
Myoglobin 17.000 Da
Ovalbumin 44.000 Da
Gamma Globulin 158.000 Da Thyroglobulin
670.000 Da
AppliChrom® ABOA-ProteSep S-L 5µ, 300mm x 8mm
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CASE STUDY Recombinant rab-mab
[kDa] M red. n.red.
Hybridoma rabbit AB
[kDa] M red. n.red.
Right: CGE
Below: analyt. SEC
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Summary
We had to address various needs to aim the goal
Improvement of established methods
New methods developed
Sequencing of Fv, production platform
Case study rab-mab conversion & production
Production and purification of 4 independent batches
Outstanding:
Detailed results upon activity of the recombinant vs. hybridoma rab-mab
Conclusion:
New rab-mab production system allows to generate mg to g scales recombinant rabbit IgGs by transient gene expression within weeks.
HAH Belfast 2016
ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS
Dr. Wolfgang Weglöhner
Sebastian Püngel Penélope Soto Villegas
Vanessa Vater
Dr. Sabrina Schindler Mohamad Eidi
Molecular Biology Lab
Downstream Processing Lab
Cooperation partners:
HAH Belfast 2016
QUESTIONS