Rapid selection of high yielding GS-CHO cell lines using the GS expression system in a protein-free, chemically defined, animal component-free process

Transcription

1 Rapid selection of high yielding GS-CHO cell lines using the GS expression system in a protein-free, chemically defined, animal component-free process David Mainwaring, Lonza Biologics.

2 Introduction Chemically defined animal component-free processes Cell line selection Fermentation optimisation Case study Summary Slide 2

3 Chemically defined animal component-free processes Cell line selection Fermentation optimisation Case study Summary Slide 3

4 Chemically defined, animal component-free media (CDACF) Why animal component-free? Increasing emphasis from regulatory authorities on removal of animal-derived raw materials from antibody production processes Potential source of adventitious agents and product contaminants Sourcing of animal derived proteins, such as New Zealand bovine serum albumin, becoming more difficult as demand increases due to expanding worldwide production capacity Potentially a similar issue with pharmaceutical grade components used as protein replacements Slide 4

5 Chemically defined, animal component-free media (CDACF) Simplification of downstream processing Reduced protein contaminant levels Increased harvest material purity Why chemically defined? Lot to lot variability of raw materials reduces manufacturing process robustness and consistency Issue associated with both hydrolysates and peptones May necessitate extensive raw material testing to identify good raw material lots Chemical definition alleviates these issues Slide 5

6 Chemically defined animal component-free processes Cell line selection Fermentation optimisation Case study Summary Slide 6

7 Cell line selection Creation of highly expressing cell lines critical to improving process productivity Cell line creation is on the critical path to the clinic GS expression system facilitates rapid creation of highly productive cell lines High specific production rate coupled with good growth characteristics Ease of selection using glutamine-free medium No requirement for amplification Slide 7

8 Cell line selection By definition, the transfectants with potentially the highest specific productivities are rare To find these rare events, it is necessary to have: A transfection method that generates large numbers of stable transfectants Selection method that eliminates the vast majority of low producers from the transfectant pool Slide 8

9 Cell line selection CHO cell lines widely used within the industry Suspension variant of CHO-K1 which grows in chemically defined medium without need for adaptation Efficiency and stringency of transfection conditions increased to improve selection of highly productive clones Methionine sulfoxamine (MSX) Slide 9

10 Cell line selection Influence of selection conditions for GS-CHO cell lines with cb72.3 antibody Antibody (mg/l) Cell lines have not been amplified µm 50 µm Selection conditions - MSX concentration Slide 10

11 Cell line selection Antibody production by non-amplified GS-CHO cell lines in a shake-flask model of a fed-batch production process Cell line ID C6 C7 C11 C12 C01 C18 C23 LB01 cb72.3 antibody concentration at harvest (mg/l) Slide 11

12 Timeline reduction Use of suspension variant of CHO K1 pre-adapted to growth in CDACF media substantially reduces time taken to generate cell lines Serum-Containing Process Vector construction Transfection Selection and Expansion Adaptation to Suspension Growth CDACF Process Vector construction Transfection Selection and Expansion Adaptation to Suspension Growth 20 weeks Time (weeks) Slide 12

13 Cell line selection summary Increasing the stringency of the selection conditions substantially increases the median antibody productivity GS-CHO cell lines producing up to 1150 mg/l in a protein-free, chemically defined medium can be constructed Amplification was not needed Slide 13

14 Chemically defined animal component-free processes Cell line selection Fermentation optimisation Case study Summary Slide 14

15 Fermentation process optimisation Purpose of process optimisation Increase manufacturing-scale productivity Minimize cost of goods Considerations Fermentation process productivity Maintenance of product quality Downstream process yields Maintenance of product quality Facility throughput Balance increased yield and process duration Slide 15

16 Fermentation process optimisation Physicochemical environment Culture ph in particular can have dramatic effect on cell growth and productivity Responses are cell line specific Nutritional environment Processes typically operated in fed-batch mode Higher productivity than batch processes Development of improved basal medium, feed formulations and feed addition strategies Slide 16

17 Generic processes Basis for process optimisation is efficient generic processes Well defined with proven performance Use allows rapid cell line evaluation and early phase product supply Provide some of the information required for subsequent process optimisation Updated as technology advances Slide 17

18 Generic process Generic processes need to be based on a sound platform Effective, robust and operable at manufacturingscale Unconstrained by raw material supply and regulatory issues Fermentation process optimisation example LB01, GS-CHO producing cb72.3 IgG Slide 18

19 Process optimisation for a model GS-CHO Cell line Process Time integral of viable cell concentration (10 9 cell h/l) Antibody (mg/l) Q p pg/cell/h LB01 Iteration LB01 Iteration LB01 Iteration LB01 Iteration Iteration 3 feed modification Iteration 4 medium, ph control and feed modifications Iteration 5 feed modification and extended culture duration Product quality analysed throughout Slide 19

20 GS-CHO growth characteristics 1000 Viable Cell Concentration (10 5 /ml) Time (h) Iteration 2 Iteration 3 Iteration 4 Iteration 5 Slide 20

21 GS-CHO product accumulation Product Concentration (mg/l) Time Integral of Viable Cell Concentration (10 9 cell h/l) Iteration 2 Iteration 3 Iteration 4 Iteration 5 Slide 21

22 Fermentation process optimisation summary Generic processes form the basis of process optimisation strategies Fermentation optimization can result in significant increases in process productivity Additional increases can be achieved through downstream process optimization The goal is increased productivity from the manufacturing-scale facility Compromise between process productivity and process duration Slide 22

23 Chemically defined animal component-free processes Cell line selection Fermentation optimisation Case study Summary Slide 23

24 Cell line construction case study 350 cell lines assayed by assembly ELISA for antibody expression 150 cell lines selected and expanded from 96 well plates to 24 well plates Productivity assessed Top 60 adapted to CDACF medium Productivity assessed Top ten cell lines selected for further evaluation in fed-batch culture Lead cell line selected based on growth, productivity kinetics and product quality Time from transfection to cgmp manufacture <12 months Slide 24

25 Antibody production data from candidate cell lines Shake-flask cultures operated in fed-batch mode 2000 Harvest Product Concentration (mg/l) Candidate Cell Lines Slide 25

26 Bioreactor comparison Bioreactor Laboratory-scale (10 L) Pilot-scale (130 L) Manufacturing (200 L) Maximum Viable Cell Concentration (10 6 cells/ml) Product Concentration (g/l) Specific Production Rate (pg/cell/h) Harvest day Slide 26

27 Chemically defined animal component-free processes Cell line selection Fermentation optimisation Case study Summary Slide 27

28 Summary High yielding GS-CHO cell lines can be created rapidly Product concentrations of > 4 g/l have been achieved Chemically-defined protein-free processes are the preferred route Regulatory compliance Improved process robustness Simplification of downstream processing Slide 28

29 Acknowledgements Cell culture process development Assay development group for support Rinat neuroscience Slide 29