Development of Cell Lines for Controlled Proliferation and Apoptosis

1. Development of Cell Lines for Controlled Proliferation and Apoptosis Mohamed Al-Rubeai University College Dublin Cell Line Development and Engineering, 23 May, Zurich

2. Criteria for Cell Line Selection • Stability • Product biological activity • Product expression: level, duration and inducibility • Growth and productivity in large scale culture • ease of selection of high producers • Adaptation to protein free suspension culture • apoptosis; proliferation rate; max cell number • Safety issues

3. Mammalian Cell Lines Used For Protein Production Cell Line Property CHO NS0 PER.C6 BHK Cell number ( cells/ml) 107 107 107 <107 Productivity (pg/cell/day) 15-65 15-65 10-20 10 Product quality ++ + ++ + Impurities ++ + ++ ++ Ease of manufacture +++ +++ ++ ++ Economics ++ ++ ++ ++ Time to clinic + + + + Intellectual property + + - + Regulatory issues +++ ++ + +++ Based partially on information from Robert D Kiss, Genetic Engineering News

4. Improvement of Product Expression • Expression Engineering - development of genetic tools comprising cloning and expression vectors • Cell (Metabolic) Engineering - design or redirection of metabolic pathways

5. Selection of cell lines with high-level, regulated gene expression • Rational cell engineering • Multicistronic expression: Coordinated, constitutive or adjustable high expression level of several genes • Suppressing gene expression (siRNA technology) • Selection of high producers and monitoring of stability • Selection markers and reporter genes • DHFR and GS • Flow cytometric based methods • DHFR/fluorescent MTX • Cell encapsulation (Gel Microdrops) and Affinity matrix based secretion assay (Carroll and Al-Rubeai, 2004, Expert Opin. Biol. Ther. 4, 1821)

6. Approaches for Cell line Engineering • Identification of genes/proteins that are specifically up-regulated in bioprocessing conditions (-omics approach) • Engineering of cells and selection of a new cell line • Engineering of cells to over-express the gene(s) of interest (historical approach) • Examine the effect and select new cell line • Further understanding of genes/pathways directly regulated by the gene of interest

7. Key Genes in Proliferation and Apoptosis • bcl-2 suppresses cell death • p21 arrests the cell proliferation and enhance specific productivity • c-myc enhances proliferation rate, reduces serum dependency and induces anchorage independence • hTERT reduces apoptosis, enhances proliferation and increases attachment tendency in the absence of serum

8. Proliferation and Cell Death Stimulated by cyclins, cdk’s, c-myc, signals from environment Stimulated by chemical compounds, NGF, Fas ligand Proliferation Cell Population Cell Death (numbers increase) (numbers decrease) Inhibited by cytostasis inducers, e.g. excess thymidine, hydroxyurea, nitrous oxide cdki p21Cip1, cdki p27Kip1 Inhibited by bcl-2, bclXL, p35, hTERT signal from environment

9. G0 cyclin p21 P cdk The Mammalian Cell Cycle reversible quiescent phase preparation for mitosis Apoptosis DNA synthesis bcl-2 hTERT

10. Growth of antibody-producing GS-CHO with and without anti-apoptosis gene- laboratory results

11. Growth of antibody-producing GS-NS0: with and without anti-apoptosis gene- Industrially optimised condition

12. Bcl-2 over-expression: The Advantages • Increases cell viability • Prolongs culture duration • Reduces serum dependency • Improves nutrient metabolism • Protects cells in stressful conditions • Enhances adaptation in serum free media

13. Bcl-2 over-expression: Productivity? • Culture dependant • Increased productivity • laboratory scale • stressed conditions • serum supplemented culture • Decreased productivity • optimised culture conditions • Fed batch industrial scale • Cell line dependant

14. The effect of ectopic p21CIP1 and Bcl-2 expression on IgG production in batch and perfusion culture Total cell count Viable cell count Arrested total & viable cell count Time (days) Batch culture Perfusion culture

15. Control IPTG p21- transfected CHO clones Increased productivity: cell cycle vs cell volume? Effect of p21 expression on cell proliferation and cell productivity

16. P21-dependent productivity: Cell size vs cell cycle • P21 arrests cells in G1phase • G1 is less productive than S and G2 • P21 leads to increased: • mitochondrial activity • oxygen uptake rate • cell volume • total cellular protein • dry cell weight • ribosomal biogenesis • intracellular IgG • H:L chain ratio • Larger cells are more productive than smaller cells

17. The effect of cell cycle arrest on cell adhesion cells grown in static for 48 hours with or without IPTG. Cells dissociated using 50% dilution cell dissociation solution (non enzymatic)

18. Adaptation of NS0 cells to serum free media using the p21 technology

19. Adaptation of CHO cells to serum free media using the p21 technology 9 days 6 days 3 days arrest No arrest

20. Adaptation of CHO cells to serum free media using the p21-Bcl2 technology 9 days 6 days 3 days No arrest

21. Summary of time taken for adaptation to serum free in CHO cells using P21 and bcl2 technology

22. Why arresting cells in G1 makes adaptation to serum-free easier? DC DC G1 S G2 M R Cells survive on minimum nutrition DC Highly variable duration, Less variable duration Dependent on soluble growth factors and Growth conditions to proceed to S

23. Adaptation of CHO cells to suspension using the p21 technology 3 days 6 days No arrest 9 days

24. Adaptation of CHO cells to suspension using the p21-Bcl2 technology

25. DNA synthesis agitation rate agitation progression of cells from S to G2/M Why arresting cells in G1 makes adaptation to suspension easier? Disruption of cells by turbulent capillary Flow Mitotic index (%) Before After Mitotic index of remaining cells after capillary flow test

26. Control cmyc The effect of c-myc expression on growth of CHO cells • stimulates cell proliferation • decreases attachment dependency • improves adaptation to suspension • enhances response to feeding • works synergistically with growth factors to promote proliferation

27. Over-expression of hTERT in CHO cells DNA Microarrays T: telomerase clones B: blank clones Telomerase cells Blank cells cDNA Microarray analysis of specific genes involved in cell attachment and formation of extra cellular matrix gene regulation in the CHO K1 cell lines Cell Attachment and Survival in the Absence of Serum

28. Over-expression of hTERT enhances chromosomal stability and possibly production stability CHO is not stable cell line! Aneuploidy Micronucleus Aneuploidy (loss of several chromosomes) Telomerase Blank The distribution of chromosomal number at different times. A: one day, B: 4 months and C: one year. The x axis represents the number of chromosomes in the cell line and the y axis represents the percentage of cells in population. Cells were transfected with the SEAP gene, passaged in culture for one year and activity of protein measured in the supernatants of batch cultures

29. Conclusions • Cell proliferation and apoptosis are co-ordinately linked processes. • Genetic engineering of cellular and metabolic pathways can enhance cell robustness, adaptation and productivity. • Genetic and chromosomal instability may affect production stability.

30. Kelly Astley Paul Clee Gary Khoo Darrin Kuystermans Amelia Petch Jenny Bi Funding: Lonza Biologics (p21 work), BBSRC, EU Framework (Bcl-2 work), SFI (Ireland), Cambrex Biosciences John Birch, Lonza Andy Racher, Lonza Acknowledgments