Evolving of Biological Product Expression Systems with Host Cell Engineering

Transcription

1 Evolving of Biological Product Expression Systems with ost Cell Engineering Lianchun Fan Bristol-Myers Squibb CMC Strategy Forum Jan. 23rd, 2017

2 Outline Choice of expression systems New technologies and ost cell engineering (CE) Power of CE: case studies Improve cell line productivity to ensure robust drug supply Improve drug product quality to ensure safer and better drugs Shorten timeline to provide patient new medicine sooner Conclusion remarks and Acknowledgements

3 CLD: Foundation of CMC Development Starting point of CMC development Critical short window step with significant CMC impact Measurement of success: Shorter timeline to provide patient new medicine sooner Acceptable productivity to ensure robust drug material supply Acceptable product qualities to ensure the safety To start Cell line development Expression systems Transgene DNA Facility/equipment/people

4 Choice of Expression Systems Expression System Producti vity PTM* Glycosa lysion Culture cost Culture condition Endotoxin Scale up Doubling time(rs) Folding Secretion Bacteria low simple /? Yeast /- low simple Insect cells /- igh complex - Large vol. of virus Mammalian Cells igh complex Plant Cells igh Complex Trangenic Animal high complex - - NA *PTM: post translational modification

5 A Typical Mammalian ost CLG Process Overview DNA QC/QA Transfection Bulk Selection SCC T Screening Top Clones ID Clone selection Banking releasing (Automated processes) (Accumulated weeks) ~36 Early development material supply Tox material supply FI material supply

6 Evolving of CLD process: over the past 25 years Months s Amplification, Limiting dilution, Master Well Development 2000s 2010s Newer selection markers, FACS, T screening Targeted integration, FACS 9 PRODUCTIVITY 8 Shortened Timeline 50-70% Increased productivity 8-10 folds >50% improvement from CLD (Matthew Croughan) g/l s 2000s 2010s

7 New Technologies Enable ost Cell Changes Plasmid engineering Gene editing, Targeted integration Robust growth/behavior ighly productive Acceptable product qualities Energy efficient Downstream friendly Stable expression Cell marker engineering Metabolic engineering Gene editing Sequencing Analytical T scale down bioreactors New ost Cells Business need Better Faster More Genomics Transcriptomics Proteomics Metabolomics Lipidomics Diversified portfolio pipeline with more challenging molecules mab, multi-functional, protein, IgG, IgM, ADC Deliver commercial cell line at FI Faster clinical material supply Avoid switching cell line Root cause problem fixing

8 Case Study #1: CE Enable afucosylated Mab Production Fucose C 3 O O O O O Extracellular space Plasma membrane a-fucosidase Lysosome C 3 O Fucose O O O O ATP Fucose kinase ADP O Fucose-1- phosphate O O O GDP-fucose pyrophosphorylase Adapted from Becker and Lowe, Glycology 2003 C 3 O O C 3 O O O OPO 3 GDP PPi Fucose C 2 O O O O O O-GDP GMD (GDP-mannose NADP 4,6-dehydratase) GDP-mannose FX O C 3 O 3,5-Epimerase O C 3 O NADP 4-Reductase NADP C 3 O O O O O-GDP O O O-GDP O O O-GDP GDP-fucose GDP-4-keto-6- deoxymannose GDP-4-keto-6- deoxygalactose RMD Cytosol GDP-rhamnose Golgi lumen O C 3 O O-GDP O O C 3 O O O O O-GDP GDP-Fucose mabs PTM fucosylated mabs Knockout

9 Case Study #1: Express of RMD Led to afucosylated mabs Design mab Minipools RMD vector G418 or Puro selection Results Lane # 1 Neg Description 2 mab RMD G1 Lane # 35-50kDa RMD-FLAG Western MA/qPCR Genetic analysis 3 mab RMD G2 4 mab RMD P1 5 mab RMD P2 14d Fed batch Glycan analysis Sample Name Description Total NF-Glycans Total Fucosylated glycans NF% CTRL CTRL mab-rmd-g mab-rmd-g mab-rmd-p mab-rmd-p Ref. std Pos. Ctrl

10 ADCC activity Significantly enhanced with afucosylated mab Comparable cell culture growth, productivity Afucosylated monoclonal antibodies improved FcγIIIa binding mabs produced from engineered host cells are more potent and efficacious

11 Case Study #2: Transition from DG44 to BMSCO1 Platform BMS strong biological portfolio need a faster and robust CLD platform BMS lagged behind industry leaders in Cell Line Development (CLD) which affects time to FI Completed platform transition within one year Productivity improvement Shortened FI timeline Increased CLD capacity

12 New Platform New Technology Driving Productivity Gains Fed-Batch Titers of Top RCB Clones (non-optimized) Better understanding new platform New platform implementation igh throughput process with automation Optimized clone screening ~5.6 g/l ~2 g/l Robust material supply DG44 BMSCO

13 Timeline Reduction with CE Months DG44 Selection MTX Master Well Development SCC Clone Development SCC GSKO Bulk Selection Clone Development 50-60% more efficient workflow Double CLD capacity to support portfolio L.Fan J. Biotechnology

14 Case study #3: CE Shorten CLD Timeline DNA QC/QA Transfection Bulk Selection SCC T Screening Top Clones ID Clone selection Banking releasing (Automated processes) (Accumulated weeks) ~36 Targeted integration technology More efficiently identify high producing clones Shortened CLD timeline

15 Current status of CO CLG process: Random integration of gene of interest (GOI) into CO genome Unpredictable clone behaviors due to genetic heterogeneity Need brute force screening (time + $) to identify high producing clones (several hundreds of clones/variant for mab) eterochromatin - where Targeted the DNA is Integration: more condensed, and usually there is not much transcriptional activity Targeted Integration in CO Cells Consistently deliver GOI into the same hot spot in CO genome Euchromatin - this is where the "active" genes are - usually this region is much less condensed Provide consistent micro environment for GOI uge potential impact on timeline and cost cbs.dtu.dk

16 TI Impact on CLG Timeline

17 Landing-pad Approaches of targeted Integration ID ot Spots Knock-in Landing pad New host cells

18 Benchmark Industrial TI Status Company Traditional CLD Timeline TI CLD Timeline FTE saving TI Expression (g/l) Stability A % Improved B % Improved C % Improved D NA Improved Application of the homogeneous bulk population to support Tox, and FI?

19 Summary Novel technologies will drive future evolving of CLD process Improve drug product quality to ensure safer and better drugs Improve cell line productivity to ensure robust drug supply Shorten timeline to provide patient new medicine sooner Regulatory guidance will be helpful for future direction

20 Acknowledgements