Improving GS-CHO Cell Line Selection: Reducing Time to Clinic

Cell Line Development and Engineering 2-6 March 2009, Berlin Improving GS-CHO Cell Line Selection: Reducing Time to Clinic Adrian Haines, 2009 Lonza Biologics plc, Slough, UK

Disclaimer Certain matters discussed in this presentation may constitute forward-looking statements. These statements are based on current expectations and estimates of Lonza Group Ltd, although Lonza Group Ltd can give no assurance that these expectations and estimates will be achieved. The actual results may differ materially in the future from the forward-looking statements included in this presentation due to various factors. Furthermore, Lonza Group Ltd has no obligation to update the statements contained in this presentation. Note: All slides are incomplete without verbal comments. slide 2

Overview Historical view of cell line constructions: Traditional 95 week process (what processes looked like 10+ years ago) Classic 40 week process Developing a shorter process: Rapid 23 week process Considerations in developing a 23 week process How faster processes fit with cgmp material supply Summary slide 3

Considerations for cgmp Cell Line Development High productivity >2 g/l monoclonal antibody Product characteristics e.g. glycosylation, de-amidation, aggregates, bioactivity, other PTMs Clonality demonstrate monoclonality e.g. formal cloning round(s) No animal components e.g. chemically defined, animal component free Scaleability consistent growth and productivity as scale increases Stability consistent expression and product characteristics SPEED fast cell line construction slide 4

High Productivity But Rapidly! Use of platform process Media, feeds, fermentation conditions, purification (especially for antibodies) Rapid generation of cell lines Cell line creation is on critical path Carry out as rapidly as possible without compromising productivity, clonality or product characteristics Predictive scale-up systems slide 5

Manufacture of a Therapeutic Protein: The Process from Cell to Clinic Cell Line Construction cgmp Cell banking Process development Scale-up & pilot prodn. cgmp manufacture slide 6

Historical 95 Week Cell Line Construction Process Total Process Transfection & selection Amplification & selection (1 round) Suspension evaluation (low serum) Limiting diln. cloning (2 rounds) Suspension evaluation (serum-free) Select lead cell lines 0 10 20 30 40 50 60 70 80 90 100 Weeks slide 7

Key Contributors to Historical 95 Week Timeline Gene amplification (~10 weeks per round) Adaptation of cells (CHO) to single cell suspension culture in serum-free medium (~ 30 weeks) Cloning (~ 20 weeks for 2 rounds of limiting dilution cloning) slide 8

How have Timelines for Cell Line Development been Reduced? Avoid amplification Use of single cell suspension adapted host cells Avoid multiple cloning steps Direct cloning from transfectant pools Screening process which is relevant to platform production process slide 9

Avoid Amplification Choice of expression technology the GS System Highly stringent selection Use weak promoter on GS gene selects for integration into highly transcriptionally active sites in genome increase stringency of selection by use of GS inhibitor methionine sulphoximine (MSX) Linked product gene driven by strong promoter (hcmv) to give high expression Time saving ~ 10 weeks per round of amplification omitted slide 10

Number of Cell Lines Product Concentration Distribution from Cell Lines Grown in Fed-Batch Shake-Flasks 175 cell lines were isolated without screening from a single cell line construction 80 70 assessed in fed-batch shake-flask 60 50 40 30 20 10 0 0-500 501-1000 1001-1500 1501-2000 2001-2500 Product Concentration (mg/l) slide 11

How can Timelines for Cell Line Creation be Reduced? Avoid amplification Use of single cell suspension adapted host cells Time saving ~ 30 weeks Avoid multiple cloning steps Direct cloning from transfectant pools Screening process which is relevant to platform production process slide 12

Contribution of CHOK1SV Cell Line CHOK1SV is a variant of CHOK1 Grows spontaneously in single cell suspension culture in protein free, chemically defined medium, free of animal derived components Improved growth characteristics compared with parent line - benefits productivity Fast suspension re-adaptation after transfection/cloning Switch very quickly between static and suspension culture slide 13

Comparison: CHOK1 vs. CHOK1SV cell lines Viable cell concentration (10 6 /ml) 12 8 4 0 0 100 200 300 400 Time (h) LB01 22H11 Host cell line IVC 10 6 cells /ml.h Qp pg/cell.h mab mg/l CHOK1SV 2266 0.89 1917 CHO-K1 1041 0.53 585 GS cell lines making the same recombinant antibody Chemically defined protein free medium Fed-batch culture slide 14

How can Timelines for Cell Line Creation be Reduced? Avoid amplification Use of single cell suspension adapted host cells Avoid multiple cloning steps Direct cloning from transfectant pools Screening process which is relevant to platform production process slide 15

Avoiding Multiple Cloning Steps Traditional: limiting dilution methods takes ~ 20 weeks for 2 rounds Classic: use of Capillary Aided Cell Cloning Direct visualisation to confirm monoclonality Saves 10 weeks With other improvements resulted in cell line creation from transfection to lead candidates in ~ 40 weeks slide 16

Clonal GS-CHO Cell Lines: Classic Two Step Process 40 Weeks from Transfection to Lead Cell Lines Transfer high-ranked cell lines Transfect CHOK1SV host cells with vector Bioreactor studies etc. cells in 96-well plates Week 40: choose lead cell lines capillary aided cell cloning cells Static in 24-well flasks plates High-ranked cell lines progressed GMP cell banking Stability study Screen and grow cells in shaking flasks: evaluate productivity and growth characteristics for manufacturability slide 17

Number of Cell Lines (Each Making a Different Product) Antibody Concentrations from GS-CHO Cell Lines 8 7 6 5 4 3 2 1 0 0-1 1-2 2-3 3-4 >4 Product Concentration (g/l) cgmp manufacturing bioreactors at scale (200 to 10 000 L) Harvested no later than 15 days after inoculation slide 18

Improved Cell Line Construction Process Total CLC Transfection & selection Amplification & selection (1 round) Suspension evaluation (low serum) Limiting diln. cloning (2 rounds) Suspension evaluation (serum-free) Select lead cell lines Total CLC Transfection & selection Suspension evaluation (protein-free) Capillary-aided cell cloning (1 round) Suspension evaluation (protein-free) Select lead cell lines Traditional: 95 week process Classic: 40 week process 0 20 40 60 80 100 Weeks slide 19

How can Timelines for Cell Line Creation be Reduced Further? Avoid amplification Use of suspension adapted host cells Avoid multiple cloning steps Direct cloning from transfectant pools slide 20

Direct Cloning From Transfectant Pools Collapse two rounds of screening to one round Transfectant pools are mixtures of high and low producing cell lines Manual capilliary aided cell cloning, without identifying high expressors, not suitable as number of clones to be screened is substantially higher Can use methods such as antibody capture and flow cytometry analysis to identify putative high producers at the point of cloning, but technically complex often with poor robustness frequently requires animal derived materials Automated single cell cloning by use of cell sorter without identifying high producers is suitable slide 21

Clonal GS-CHO Cell Lines: Rapid One Step Process 23 Weeks from Transfection to Lead Cell Lines Generate pools of transfectants Automated cloning Identify monoclonal cell lines Productivity assessment Transfer highranked cell lines Cell sorter Automated imaging Transfect CHOK1SV host cells with vector Productivity assessment Week 23: choose lead cell lines High-ranked cell lines progressed Bioreactor studies, etc. High ranked cell lines progressed GMP cell banking Stability study Fed-batch assessment CSI of automated growth, productivity imaging platform and product quality Productivity assessment 5 10 cell lines (abridged fed-batch) slide 22

Considerations Pool generation and selection Pools contain cells with a range of expression levels Generate a variety of pools and pre-screen for expression to identify the best pools Is the proportion of non- or low expressing cell lines derived from sorting a transfectant pool greater than when derived by a classic method? Verification likelihood of monoclonality slide 23

Proportion of non or low expressing cell lines: Classic v sorting from pool Classic: Productivity assessment Transfect CHOK1SV host cells with vector Sorting: Generate pools of transfectants Automated cloning Cell sorter Productivity assessment Transfect CHOK1SV host cells with vector slide 24

Proportion of non- or low expressing cell lines: Classic v sorting from pool Classic: 208 colonies were recovered after transfection Colonies were assessed for secreted production of antibody in static multi-well plates (un-fed culture) mean productivity = 40 mg/l proportion producing less than 25 mg/l = 46% Sorting: 275 colonies were recovered after cell sorting 156 colonies were assessed for production of antibody in static multi-well plates (un-fed culture) mean productivity = 91 mg/l proportion producing less than 25 mg/l = 5% slide 25

Proportion of cell lines (%) Productivity distribution of colonies: Classic v sorting from pool 100 80 60 40 20 0 0-1 1-24 25-49 50-99 100-149 150-200 > 200 Productivity distribution (mg/l) Classic (n=208) Sorting (n=156) slide 26

Summary of cell growth and productivity: Classic v sorting from pool Method used No. of multiwell plates No. of colonies recovered Colonies per plate No. of colonies screened Antibody product conc. Classic 80 208 2.6 208 1-179 Sorting 113 275 2.4 156 13-192 Cloning from transfectant pools without screening at the cloning stage for higher producers, does not result in an increased proportion of non- or low expressers slide 27

Considerations Pool generation and selection Pools contain cells with a range of expression levels Generate a variety of pools and pre-screen for expression to identify the best pools Is the proportion of non- or low expressing cell lines derived from sorting a transfectant pool greater than when derived by a classic method? Verification likelihood of monoclonality slide 28

Sorting: Verify Likelihood of Monoclonality Three arms to verify the likelihood of monoclonality: Correct instrument set-up and operational checks Historical data set and statistical model for analysing data Visual monitoring of plating, pre- and post-sort slide 29

Sorting: Why do we Need to Assess Clonality? Surely a fluorescent activated cell sorter operating in single cell mode will only deposit a single cell per well? slide 30

Sorting: Historical Data Analysis Sorted cells into 67 96-well plates 5982 wells were analysed for presence or absence of cells 1626 wells were empty (no cells) 4330 wells contained one cell 26 wells contained 2 or more cells Overall population containing 2 or more cells = 0.43% Probability that a well contains 1 particle = 0.72 If sort into 240 96-well plates 240 x 96 x 0.43% = 99 @ >1 cell / well Need to ensure optimal set up slide 31

Sorting: Optimal Set Up At each sort session: Check single particle deposition on subset of plates - visual inspection of one particle per well Perform the sort Repeat single particle deposition on subset of plates - visual inspection of one particle per well Statistical analysis slide 32

Data Analysis Historical data Data obtained from pre + post sort checks on single cell deposition Calculated probability for the sort Start, p(x=0) probability of 0 cells/well Start, p(x=1) probability of 1 cell/well Bayes theorem used to combine historical data on single particle deposition (prior) and data from the experiment itself (likelihood) to estimate probabilities that wells contain different numbers of particles (posterior) slide 33

P(monoclonality P(monoclonality) Once have probabilities for a well containing n cells, can calculate p(monoclonality) Using probabilities from start and end of session, can estimate range of likely values for p(monoclonality) 1.002 1.000 0.998 0.996 0.994 0.992 0.990 0.988 Session slide 34

Sorting: Further Assurance of Clonality Screen and select colonies using a Clone Select Imager Automated imaging of 96-well plates Identifies single colonies Repeat imaging allows colony growth to be tracked slide 35

Assurance of monoclonality 3 orthogonal approaches: Use of cell sorter Confirmation of correct instrument function Pre- and post-sort checks Statistical analysis Use of image analysis to track colony formation slide 36

Effect of Shortening CLC: Typical Examples DNA Sequence to Clinical supply Traditional 95 week CLC GMP material Classic two step (Capillary aided cell cloning) 40 week CLC GMP material 35 weeks Rapid one step (Cell Sorting) 23 week CLC GMP material 0 6 12 18 24 30 Months DNA seq. optimization and prep. Cell line stability studies MCB Process development and pilot cgmp manufacture slide 37

Summary 1 Traditional 95 week process can be shortened to 40 weeks by Removing amplification stages Avoiding adapting cells to suspension culture in chemically defined medium by using a suspension variant (e.g.chok1sv) Using one round of cloning, such as capillary aided cloning Process can be further shortened to 23 weeks by cloning directly from transfected pools using a cell sorter Eliminates a round of screening No increase in proportion of low or non-expressers compared to classic method Demonstrate likelihood of monoclonality slide 38

Summary 2 Reduce historical timeline of >18 months to < 6 months (from transfection to selection of clonally derived lead candidates) GMP material can be prepared within ~14 months from DNA sequence slide 39

Acknowledgments Andy Racher Hilary Metcalfe James Rance Tabitha Bullock Frances Kenny Kasia Szymaniec Deepa Pillai Marc Smith Matthieu Stettler Sarah Turner Alison Porter John Birch Other members of CCPD past and present Analytical Development Dept. slide 40