Behaviour of Cell Lines in a Selection Strategy

Cell Line Development and Engineering 27 Behaviour of Cell Lines in a Selection Strategy Alison Porter, Lonza Biologics plc, Slough, GB, 6 March 27

Scope of Talk Cell line construction Issues and Requirements Current cell line construction strategy Challenging the current cell line selection strategy slide 2

Cell Line Construction Issues and Requirements

Cell Line Construction Issues and Requirements Issue: Transfection results in heterogeneous population Measurements made early in construction of a cell line may not reflect how it behaves in the final production process Requirement: Selection of cell lines with appropriate specific production rates (Q P ) and growth characteristics slide 4

Cell Line Construction Issues and Requirements A possible strategy Take large numbers (possibly thousands) of cell lines through to the final production process Scientifically sound Lengthy and resource intensive Developing other strategies Designed with reference to the final production process Requires prediction of manufacturing behaviour of cell lines at very early stage Economic Compatible with resources slide 5

Cell Line Construction Objectives Objective of any cell line selection strategy is to isolate cell line(s) that: Are high volumetric producers Show acceptable growth in final inoculum processes Show desired product quality Including bioactivity slide 6

Current Cell Line Construction Strategy

Current Cell Line Construction Strategy Vectors designed to promote expression of the gene(s) of interest (GOIs) Strong promoter (hcmv) drives expression of the GOIs Weak promoter (SV4) on GS gene is coupled with selection in high (stringent) levels of MSX selects for integration at transcriptionally active loci GOIs and GS gene are on the same plasmid and tightly linked Integration of GS gene into transcriptionally active loci results in co-integration of GOIs into same loci slide 8

Current GS-CHO Cell Line Construction Method Transfect CHOK1SV host cells with vector 96-well plates, single colonies per well Static culture - CDACF medium 2 3 cell lines Semi-quantitative productivity assessment 1-15 cell lines Quantitative productivity assessment 3 6 cell lines Suspension (shake-flask) culture - CDACF medium Select cell lines for stability assessment 5-1 cell lines Adapt to suspension culture 3 6 cell lines Preliminary quantitative assessment Select cell lines to clone Fed-batch assessment of growth, productivity and product quality slide 9

Fed-Batch Shake-Flask Screen of GS-CHO Cell Lines 4 2.5 Product Concentration (mg/l) 35 3 25 2 15 1 5 Q P (pg/cell/hour) 2 1.5 1.5 35 Antibody 2 Antibody IVC (1 6 cells.h/ml) 3 25 2 15 1 5 Max X V (1 6 cells/ml) 18 16 14 12 1 8 6 4 2 Antibody Antibody slide 1

Batch and Fed-Batch Culture: Comparison of Ranking Positions Examples from two GS-CHO construction programmes 2 2 4 Rank Position 4 6 8 1 Rank Position 6 8 1 12 14 16 18 12 Preliminary Productivity Assessment Scale-down Model of the Final Production Process Individual cell lines respond differently to feed Change in rank position 2 Preliminary Productivity Assessment Scale-down Model of the Final Production Process slide 11

Fed-Batch Shake-Flask vs. Fed-Batch Bioreactor 3 Value in Bioreactor Relative to Shake-Flask 2.5 2 1.5 1.5 Product Concentration Parameter Relationship between productivity characteristics of the lead GS-CHO cell lines making ten different antibodies Individual cell lines respond differently to culture in bioreactors Q P slide 12

Current Cell Line Construction Strategy - Summary Individual cell lines respond differently to environment Feed Bioreactor Suggests requirement for more powerful strategies for selecting best cell lines A better model of the bioreactor likely to be a key requirement slide 13

Challenging The Current Cell Line Selection Strategy

Challenging The Current Cell Line Construction Strategy Objective: to increase the frequency of isolating the best 5% of cell lines With no change in resource requirement or elapsed time Potential issue High producing cell lines are isolated using the current cell line construction strategy Are these good cell lines rather than the best cell lines? Lowest producers discarded at each stage and response of cells to environment differs. Limitations with assessment of available data Initial work to fully understand what happens in current selection strategy may therefore help fulfil the objective slide 15

Challenging The Current Cell Line Construction Strategy Hypothesis: A screen stage fully predicts the behaviour of the cell lines in the subsequent screens Is any screen truly predictive? At each stage cell line(s) are discarded/selected which do/don t do well in a particular system How? Progression of 175 randomly selected cell lines from transfection through all selection stages prior to fermentation Assessment of a subset of these in bioreactors slide 16

Challenging The Current Cell Line Selection Strategy - Method Transfect CHOK1SV host cells with vector 96-well plates, single colonies per well 175 cell lines Semi-quantitative productivity assessment spot Static culture - CDACF medium 175 cell lines Quantitative productivity assessment 24-well plate 175 cell lines Suspension (shake-flask) culture - CDACF medium Adapt to suspension culture Bioreactor assessment 175 cell lines 175 cell lines Preliminary quantitative assessment batch Pseudo-random selection of 29 cell lines for bioreactors Fed-batch assessment slide 17

Challenging The Current Cell Line Selection Strategy Productivity Results Specific Activity Spot Product Concentration (mg/l) 35 3 25 2 15 1 5 24-well plate Product Concentration (mg/l) 9 8 7 6 5 4 3 2 1 Batch Cell Line Product Concentration (mg/l) 3 25 2 15 1 5 Fed-batch Cell Line Cell Line Cell Line slide 18

Challenging The Current Cell Line Selection Strategy Location of Selected Top 1 Current selection strategy used to identify top 1 cell lines from the 175 cell lines Spot : 175 12 cell lines 24-well plate : 12 6 cell line Batch : 6 1 cell lines These cell lines designated the selected top 1 Would progress to assessment in fed-batch screen Where are these ten cell lines at each screen stage? slide 19

Specific Activity Challenging The Current Cell Line Selection Strategy Location of Selected Top 1 Spot Product Concentration (mg/l) 35 3 25 2 15 1 5 24-well plate Product Concentration (mg/l) 9 8 7 6 5 4 3 2 1 Batch Cell Line Product Concentration (mg/l) 3 25 2 15 1 5 Fed-batch Cell Line Cell Line Cell Line slide 2

Challenging The Current Cell Line Selection Strategy Location of selected Top 1 Use of the selection strategy has not resulted in the selection of the top ten highest producers at the fed-batch stage Where are the real top ten cell lines at each stage? Location of the ten highest producers identified in the fed-batch screen What happens if screen stages are missed out? slide 21

Specific Activity Challenging The Current Cell Line Selection Strategy Location of the Real Top 1 Spot Product Concentration (mg/l) 35 3 25 2 15 1 5 24-well plate Product Concentration (mg/l) 9 8 7 6 5 4 3 2 1 Batch Cell Line Product Concentration (mg/l) 3 25 2 15 1 5 Fed-batch Cell Line Cell Line Cell Line slide 22

Challenging The Current Cell Line Selection Strategy Removal of Screen Stages Product Concentration (mg/l) 3 25 2 15 1 5 Selected top ten Product Concentration (mg/l) 3 25 2 15 1 5 Assessing spot data only Product Concentration (mg/l) 3 25 2 15 1 5 Cell Line Removing the 24-well plate screen Product Concentration (mg/l) 3 25 2 15 1 5 Cell Line Removing the batch screen Cell Line Cell Line slide 23

Challenging The Current Cell Line Selection Strategy Cell Line Locations Summary Use of the current selection strategy does result in the selection of highly productive cell lines Use of the current selection strategy does not result in the selection of all ten cell lines which are the highest producers in the fed-batch screen It is possible to discard cell lines which would be high producers Use of the spot screen alone is not sufficient to identify a high producing cell line May be possible to have just the 24-well plate or batch screen stage rather than both? Could then refocus resource on the fed-batch screen Further statistical analysis is in progress to investigate this slide 24

Challenging The Current Cell Line Selection Strategy Ranking Progress to examine whether ranking position of cell line changes between each screen Are the screens truly predictive? slide 25

Challenging The Current Cell Line Selection Strategy Ranking -2 High ranking Rank Position 2 4 6 8 1 12 14 16 18 2 'spot' '24-well plate' 'batch' 'fed-batch' Low ranking slide 26

Challenging The Current Cell Line Selection Strategy Ranking -2 High ranking Rank Position 2 4 6 8 1 12 14 16 18 2 'spot' '24-well plate' 'batch' 'fed-batch' Low ranking slide 27

Challenging The Current Cell Line Selection Strategy Ranking Ranking position of a cell line can change as it moves through the different screens Movement both up and down can be observed Those cell lines which are initially the lowest producers tend to remain as low producers throughout each screen Is the ranking position in one screen predictive of the ranking position in the next screen? slide 28

Challenging The Current Cell Line Selection Strategy Contingency Tables 4 4 Number of Cell Lines 3 2 1 1 2 3 Quartile in 'spot' 4 1 2 3 4 Quartile in '24- Well Plate' Number of Cell Lines 3 2 1 1 Quartile in '24-Well Plate' 2 3 4 1 2 3 4 Quartile in 'Batch' Initial statistical analysis suggests there is a relationship between variables Number of Cell Lines 4 3 2 1 1 2 3 Quartile in 'Batch' 4 1 2 3 4 Quartile in 'Fed- Batch' Propose that this relationship primarily due to those which are low producers tending to remain low producers through each screen slide 29

Challenging The Current Cell Line Selection Strategy Bioreactor Results A subset of the 175 cell lines were assessed in 1 L bioreactors 29 cell lines assessed How do results in shake-flasks compare to results in bioreactors for these cell lines? slide 3

Bioreactor: Product Concentration (mg/l) Bioreactor: IVC (1 6 cells/ml.h) Challenging The Current Cell Line Selection Strategy Bioreactor Results 35 3 25 2 15 1 5 7 6 5 4 3 2 1 5 1 15 2 25 3 35 Shake-flask: Product Concentration (mg/l) 1 2 3 4 5 6 7 Shake-Flask: IVC (1 6 cells/ml.h) Bioreactor: Q P (pch) 1.4 1.2 1..8.6.4.2...2.4.6.8 1. 1.2 1.4 Shake-Flask: Q P (pch) Positive correlation between shake-flask and bioreactor results for: Product concentration (r =.86, p =.5) Q P (r =.94, p =.5) IVC (r =.46, p =.5) slide 31

Challenging The Current Cell Line Selection Strategy Bioreactor Results Selected top ten highlighted: Bioreactor: Product Concentration (mg/l) 35 3 25 2 15 1 5 Changes in rank position observed 5 1 15 2 25 3 35 Shake-flask: Product Concentration (mg/l) A cell line which is highly productive in the final production process can be selected It may not be the highest producer slide 32

Challenging The Current Cell Line Selection Strategy Bioreactor Results There is positive correlation between results obtained in shake-flasks and bioreactors for productivity characteristics and for IVC Seen for a range of productivities Rank positions may change between shake-flasks and bioreactors Although prediction of the highest producer in the bioreactor did not occur, a high producing cell line was still identified slide 33

Challenging The Current Cell Line Selection Strategy Summary Variation in measured parameters observed at each assessment stage Ranking positions not consistent as cell lines progress through the different assessment stages Heterogeneous response of cell lines to environment Cell lines which would exhibit good characteristics in the final production process can be discarded during the selection strategy Fed-batch shake-flask screen not truly predictive of behaviour in bioreactor Reject the hypothesis that any screen fully predicts the behaviour in subsequent screens However, use of the current selection strategy still enables identification of highly productive cell lines slide 34

Challenging The Current Cell Line Selection Strategy Summary Initial statistical analysis does suggest a relationship between the screens Low producers tend to remain low producers slide 35

Challenging The Current Cell Line Selection Strategy Discussion Possible reasons for why each assessment stage is not truly predictive of the next: Loss of GOI A cell line which is high producing at one screen and then low producing at the rest Pseudo-random integration of the integrated genes throughout the genome Susceptibility of loci to epigenetic affects Affect of loci on mrna transport Affect of loci on functioning of other genes in adjacent areas slide 36

Challenging The Current Cell Line Selection Strategy The future As current selection strategy is not truly predictive of behaviour in final selection strategy, there is an opportunity to improve the selection strategy Statistical analysis to determine how many cell lines to screen at each stage to increase frequency of identification of best 5% cell lines Remove a screen stage and refocus resource onto one of the other screen stages? Screen more cell lines at the later stages? Develop more predictive early assessments? Although not necessarily the best cell lines, very good cell lines can be isolated in the current selection strategy slide 37

Acknowledgements Lonza Biologics plc, Slough, GB Andy Racher Juanita Porter Atul Mohindra Emma Allen Tracy Root Eric Le Guern Richard Aldcroft Cell Culture Process Development Group Analytical Development Group University of Manchester Alan Dickson Louise Barnes slide 38