Converting rabbit hybridoma into recombinant antibodies with effective transient production in an optimized human expression system

Transcription

1 Converting rabbit hybridoma into recombinant antibodies with effective transient production in an optimized human expression system Dr. Tim Welsink Molecular Biology Transient Gene Expression

2 OUTLINE Short introduction to InVivo BioTech Introduction Project goal / needs Methods developed Case study rab-mab conversion & production Summary

3 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

4 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

5 mg rab IgG / liter InVivo produced several rabbit hybridoma clones Culture with bad performances and/or productivity Introduction Antibody 1L spinner system Rabbit antibody from hybridoma produced at InVivo representative selection Occupation of up-scale capacity in production Much effort in downstream and purification High need of rabbit IgG for IVD

6 PROJECT GOAL Enhancing productivity of rabbit antibodies from hybridoma by converting into recombinant antibodies and expressed transiently. Needs AB sequence at least Fv Need Sequence engineering cdna synthesis Production platform High titer Need Scalable Proprietary Need Antibody purification & Need

7 Project Fv SEQUENCE outline 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

8 RNA extraction, reverse transcription, gene amplification from cdna, subcloning, sequencing Fv SEQUENCE Total RNA extraction Cryo vial >1 x 10 6 cells in culture RNA-Isolation Supplier 1 Supplier 2 Supplier Reverse transcription Total RNA into cdna Reverse transcription (RT) PCR Parameters for kit decision RNA yield Quality of cdna hgapdh Primer: 1 M, 2 H2O, 3 -RT, 4/5 sample, 6 +ctr Suppplier 1 Supplier 2

9 Fv SEQUENCE Amplification of Fv sequences using PCR AB 1 AB 2 AB 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 : M, 2 to 5: Subcloned Fv amplificons, RE cut

10 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 IgG gene synthesis by a subcontractor 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 Cloning in mammalian expression vector Delivery by transient transfection Transient gene expression

11 PRODUCTION PLATFORM Needs Production platform High titer Need Scalable Proprietary Need & 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

12 Transfection reagents Basic medium formulation Medium development and optimization MEDIA DEVELOPMENT Cooperation: Transfection tests in media variants Analysis of cell growth, substrates, etc.

13 TRANSFECTION REAGENT Cooperation: Transfection of HEK cells with GFP vector Productivity analysis in HEK cells

14 Medium PLASMID PREPARATION E.Coli host strain 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

15 Directed Evolution CELL LINE DEVELOPMENT Recovery Phenotype Analysis Selection

16 PROCESS OPTIMIZATION Feeding regime: Combining the new achievements

17 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

18 mg rab IgG total CASE STUDY RAB-MAB CONVERSION Hybridoma rab. AB yield monoclonal rabbit hybridoma AB recombinant rabbit AB produced by TGE ) cdna sequencing 2) Vector design, preparation 3) Transient production 4) Purification 5) QC

19 CASE STUDY Sequence alignment HC variable region: Complementarity Determining Regions (CDRs) are colored, mismatches are highlighted Primer sequences from Seeber et al., 2014 & Rader et al., 2000

20 CASE STUDY Sequence alignment LC variable region: Complementarity Determining Regions (CDRs) are colored, mismatches are highlighted Primer sequences from Seeber et al., 2014 & Rader et al., 2000

21 CASE STUDY cdna Design IgG gene synthesis Cloning into expression vector Low-endotoxin plasmid preparation Transient gene expression

22 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

23 Titer [mg/l] CASE STUDY RESULTS 4 Batches produced Different production scale 2 cell lines ml 750 ml 50 ml 1500 ml HEK Production scale, cell line HEK-INV

24 QC: capillary gel electrophoresis for purity CASE STUDY Reducing conditions, Agilent 2100-BioAnalyzer, Protein 230 Kit Purity 98% Non-reducing conditions, Agilent 2100-BioAnalyzer, Protein 230 Kit Purity 85%

25 CASE STUDY Analytical SEC for determination of aggregates Vitamin B Da Myoglobin Da Thyroglobulin Da Gamma Globulin Da Ovalbumin Da AppliChrom ABOA-ProteSep S-L 5µ, 300mm x 8mm

26 Recombinant rab-mab [kda] M red. n.red. CASE STUDY Hybridoma rabbit AB [kda] M red. n.red. Right: CGE Below: analyt. SEC

27 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 Summary Conclusion: New rab-mab production system allows to generate mg to g scales recombinant rabbit IgGs by transient gene expression within weeks.

28 ACKNOWLEDGEMENTS Cooperation partners: Dr. Wolfgang Weglöhner Sebastian Püngel Penélope Soto Villegas Vanessa Vater Dr. Sabrina Schindler Mohamad Eidi Molecular Biology Lab Downstream Processing Lab

29 QUESTIONS