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 Property Cell Line CHO NS0 PER.C6 BHK Cell number ( cells/ml) 10 7 10 7 10 7 <10 7 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 1.bcl-2 suppresses cell death 2.p21 arrests the cell proliferation and enhance specific productivity 3.c-myc enhances proliferation rate, reduces serum dependency and induces anchorage independence 4.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 ProliferationCell PopulationCell Death (numbers increase) (numbers decrease) Inhibited by cytostasis inducers, e.g. excess thymidine, hydroxyurea, nitrous oxide cdki p21 Cip1, cdki p27 Kip1 Inhibited by bcl-2, bcl XL, p35, hTERT signal from environment Stimulated by chemical compounds, NGF, Fas ligand

9. The Mammalian Cell Cycle G0G0 DNA synthesis preparation for mitosis reversible quiescent phase Apoptosis bcl-2 hTERT cyclin p21 P cdk

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 p21 CIP1 and Bcl-2 expression on IgG production in batch and perfusion culture Total cell count Viable cell count Arrested total & viable cell count Batch culture Perfusion culture Time (days)

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

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 No arrest 3 days arrest 6 days 9 days

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

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 G1 S G2 M DC Highly variable duration, Less variable duration Dependent on soluble growth factors and Growth conditions to proceed to S R Cells survive on minimum nutrition

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

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

25. Why arresting cells in G1 makes adaptation to suspension easier? DNA synthesis agitation rate agitation progression of cells from S to G 2 /M Mitotic index (%) Before After Mitotic index of remaining cells after capillary flow test Disruption of cells by turbulent capillary Flow

26. zstimulates cell proliferation zdecreases attachment dependency zimproves adaptation to suspension zenhances response to feeding zworks synergistically with growth factors to promote proliferation Control cmyc The effect of c-myc expression on growth of CHO cells

27. Over-expression of hTERT in CHO cells Cell Attachment and Survival in the Absence of Serum Acc No.GeneRatio T/B Function AF296282Icam42.2Adhesion molecule that binds to LFA-1 adhesion protein. Binds to integrins (By similarity) M18933Col3a126.5Collagen type III occurs in most soft connective tissues along with type I collagen NM_015734Col5a111.3Type V collagen binds to DNA, heparan sulfate, thrombospondin, heparin, and insulin X65582Col6a22.2Collagen VI acts as a cell-binding protein NM_019759Dpt94.5Mediate adhesion by cell surface integrin binding. Link between cell surface and its ECM NM_009242Sparc2.4Regulate cell growth through interactions with the extracellular matrix and cytokines NM_010577Itga52.6Integrin alpha-5/beta-1 is a receptor for fibronectin and fibrinogen. Play a role in the survival of adult skeletal muscle M59912C-kit ligand2.2Mediates cell-cell adhesion NM_008608Mmp143.0Endopeptidase that degrades various components of the extracellular matrix, such as collagen NM_011777Zyx2.4Component of a signal pathway that mediates adhesion-stimulated changes in gene expression NM_011780Adam232.4May play a role in cell-cell and cell-matrix interactions NM_013798Actg13.4Actins are highly conserved proteins that are involved in various types of cell motility NM_021293Cd339.0Adhesion molecule that mediates sialic-acid dependent binding to cells NM_013681Syn210.4Phosphoprotein that binds to the cytoskeleton AB009674Adam222.7Probable ligand for integrin Telomerase cells Blank cells T: telomerase clones B: blank clones cDNA Microarray analysis of specific genes involved in cell attachment and formation of extra cellular matrix gene regulation in the CHO K1 cell lines DNA Microarrays

28. Over-expression of hTERT enhances chromosomal stability and possibly production stability Micronucleus Aneuploidy (loss of several chromosomes) Aneuploidy 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. CHO is not stable cell line! Cells were transfected with the SEAP gene, passaged in culture for one year and activity of protein measured in the supernatants of batch cultures Telomerase Blank

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. Acknowledgments 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