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Integration of Cell Line and Process Development to Expedite Delivery of Bispecifics to the Clinic

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Authored and Presented by: Dane A. Grismer, Yogender K. Gowtham, Srivatsan Gopalakrishnan, David. W. Chang,
Niket Bubna, Ph.D., and Sigma S. Mostafa, Ph.D.

Published in: Health & Medicine
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Integration of Cell Line and Process Development to Expedite Delivery of Bispecifics to the Clinic

  1. 1. 12 Titer(g/L) Day 12 Viability(%) Day Peak Day PeakVCC(x106 cells/mL) 100 L 50 L 12 Lactate(g/L) Day Dane A. Grismer, Yogender K. Gowtham, Srivatsan Gopalakrishnan, David. W. Chang, Niket Bubna, and Sigma S. Mostafa KBI Biopharma, Durham, NC 10th Annual World Bispecific Summit 17 Sep – 19 Sep 2019 • Waltham, MA www.bispecific.com Integration of Cell Line and Process Development to Expedite Delivery of Bispecifics to the Clinic Abstract Challenges with low productivity and product quality for bispecific antibodies often lead to lengthy timelines for generating clinical material. Implementation of the Selexis-KBI Integrated (SKI) workflow—cell line development, process development, and clinical manufacturing— addresses these hurdles. Stable pools of clones are used to assess product titer and product quality using a platform process designed for bispecifics. Clone selection is performed in parallel with other process development activities. The SKI workflow results in completion of the first FIH clinical manufacturing batch in 12 months. We have demonstrated successful use of the SKI project paradigm for more than 10 bispecifics. Three case studies, each highlighting an improvement in titer or product quality will be presented. The first molecule successfully employed a platform cell culture process with minor modifications, leading to over 2-fold increase in product titer. High titers during development enabled reduction of the manufacturing scale from 2,000 L to 1,000 L. The second case study will demonstrate successful remediation of product degradation in clarified harvest. The third case study will show results for significant homodimer reduction (more than 85%). Together, these case studies establish that a strong foundational platform shortens process development timelines, which allows focus to be placed on resolution of product challenges. Preventing Homodimer Generation High Titer Leading to Scale Reduction Inhibiting Product Fragmentation Acknowledgements Selexis-KBI Integrated (SKI) Workflow Conclusions A strong foundational cell culture and harvest platform process has removed the need for extensive development and optimization studies. Focus was maintained on resolving product challenges that were known before the start of the project or appeared during process development activities. Experience on numerous SKI projects and overcoming an array of product challenges provides an extensive toolkit to develop robust and scalable manufacturing processes for a variety of bispecifics. • Partnership between Selexis and KBI provides synergies that shorten the time between transfection and BDS fill. • Selexis has an established track record in cell line development and variant screening. • KBI Biopharma brings process and analytical development and clinical and commercial cGMP manufacturing expertise to the partnership. We would like to thank Stewart McNaull and Tim Kelly for their guidance. Additional thanks to Carl Zhang, James Hamlin, and Jake Kim for contributions to analysis and discussions. • A platform cell culture process with minor modifications performed consistently well across numerous scales. • A more than 2-fold increase in product titer during process development enabled reduction of the manufacturing scale from 2,000 L to 1,000 L while still achieving project benchmarks. 0 2 4 6 8 10 12 14 Day ambr15 (n=6) 3 L Scale 50 L Clone PD 200 L PD 1000 L cGMP 0 2 4 6 8 10 12 14 Day ambr15 (n=6) 3 L Scale 200 L PD 1000 L cGMP 6 8 10 12 14 Day ambr15 (n=6) 3 L Scale 50 L Clone PD 200 L PD 1000 L cGMP Acidic Basic Main Product Quality ambr15 (n=6) 3 L Scale 50 L Clone PD 200 L PD • The generation of significant levels of scFV homodimer was present at the beginning of the project. • A more than 85% reduction of homodimer levels was achieved during process development. • Three approaches were utilized together to reduce homodimer generation: 1. Optimization of the feeding scheme 2. Temperature downshift triggered by a VCC threshold 3. Reduction of the culture duration from 14 to 12 days • High titer for SKI processes allows for modulation of product quality attributes through a variety of methods, including shortening the process to ensure low levels of homodimers and high levels of heterodimers at harvest. References • Brinkmann, U. & Kontermann, R. E. (2017). The making of bispecific antibodies. mAbs, 9(2), 182–212. • Chung, W. K., et al. (2017) Effects of antibody disulfide bond reduction on purification process performance and final drug substance stability. Biotechnol. Bioeng., 114(6), 1264-1274. • Labrijn, A. F., et al. (2013) Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. P. Natl. Acad. Sci. USA, 110(13), 5145-50. • Liu, H., et al. (2017) Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds. Front. Immunol., 8(38). • Trexler-Schmidt, M., et al. (2010). Identification and prevention of antibody disulfide bond reduction during cell culture manufacturing. Biotechnol. Bioeng., 106(3), 452-61. • Wang, Q. (2019) Design and Production of Bispecific Antibodies. Antibodies, 8(3), 43. Harvest operations at process temperature LMW Main HMW PeakArea(%) 100 L Day 12 Bioreactor 100 L Day 14 Bioreactor 100 L Day 14 Clarified Harvest Bioreactor cooled prior to harvest operations LMW Main HMW %PeakArea 50 L Day 12 Bioreactor 50 L Day 12 Clarified Harvest • Process was shortened from 14 to 12 days. • Prior to harvest initiation: • DO setpoint was increased to 60-70%, • Bioreactor was cooled to 18-20 °C. • Clarified harvest was well-mixed and aerated before transfer to pre-inflated bags. • Clarified harvest was stored at 2-8 °C and quickly processed through downstream purification. SKI Transfection to BDS • The duration from transfection at Selexis to BDS fill at KBI is targeted at 12 months for bispecifics. • Flexibility built into the timeline allows for studies to investigate challenges unique to each molecule. Drug Substance GLP Tox Material in 11 mo cGMP (up to 2,000 L)200 L IND Tox Run MCB MCB Release Research Cell BankTransfect DNA Cell Line Development Upstream Development Downstream Development Analytical Development & Qualification Formulation Development Stable Pools RCBs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 SKI Performance Across Molecules • Final cell culture processes routinely achieve titers of 3-6 g/L for bispecifics. • Cell viability at the time of harvest is above 80% for most molecules, maintaining productivity through the end of the process.0 20 40 60 80 100 HarvestViability(%) Bispecific Molecules 0 2 4 6 8 10 Titer(g/L) Bispecific Molecules 10+ g/L 88% Avg. Fab Homodimer Heterodimer scFv Homodimer PeakArea(%) 3 L Day 10 3 L Day 12 3 L Day 14 50 L Day 10 50 L Day 12 50 L Day 14 cGMP - Day 12 ProA Reduction in homodimer levels achieved 4 6 8 10 12 14 3 L 50 L Titer(g/L) Day • During scale-up of harvest operations, reduction of the product led to high LMW species. SCC2 Generation 0 weeks 1 week 3.5 weeks 7 weeks Gene Synthesis & Vector Construction 4.5 weeks 12.5 weeks SCC1 Generation Super-Transfection SCC0 Generation Transfection + Pool Generation 2000 L SUB cGMP Manufacturing Run 50 L SUB Harvest Supply Run + ambr15 (24 vessels) Screening Study 200 L SUB Demonstration Run: IND Tox 50 L SUB Harvest Supply Run 3 L Glass Bioreactors (4-8 vessels) Aging Study ambr250 (12 vessels) Clones Evaluation DownstreamDevelopment,AnalyticalDevelopment,AnalyticalMethodQualification MCBProduction • Once available, SCC2 clones are utilized to scale-up the cell culture process. • Upstream and Downstream Process Development studies occur in parallel with Analytical Development activities. • Following tech transfer from process development, clinical manufacturing can be performed at scales up to 2,000 L. Selexis Activities KBI Activities • Process development at KBI starts with either stable pools, SCC0 candidates, or an enriched pool, depending on a project’s timeline.

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