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Development of Cell Lines for Controlled Proliferation and Apoptosis
Mohamed Al-Rubeai University College Dublin Cell Line Development and Engineering, 23 May, Zurich
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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
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Mammalian Cell Lines Used For Protein Production
Property CHO NS PER.C BHK Cell number ( cells/ml) <107 Productivity (pg/cell/day) 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
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Improvement of Product Expression
Expression Engineering - development of genetic tools comprising cloning and expression vectors Cell (Metabolic) Engineering - design or redirection of metabolic pathways
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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)
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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
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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
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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
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The Mammalian Cell Cycle preparation for mitosis
G0 reversible quiescent phase preparation for mitosis Apoptosis cyclin p21 P cdk DNA synthesis bcl-2 hTERT
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Growth of antibody-producing GS-CHO with and without anti-apoptosis gene- laboratory results
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Growth of antibody-producing GS-NS0: with and without anti-apoptosis gene- Industrially optimised condition
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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
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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
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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
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Effect of p21 expression on cell proliferation and cell productivity
Control IPTG p21- transfected CHO clones Increased productivity: cell cycle vs cell volume?
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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
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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)
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Adaptation of NS0 cells to serum free media using the p21 technology
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Adaptation of CHO cells to serum free media using the p21 technology
9 days 6 days 3 days arrest No arrest
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Adaptation of CHO cells to serum free media using the p21-Bcl2 technology
9 days 6 days 3 days No arrest
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Summary of time taken for adaptation to serum free in CHO cells using P21 and bcl2 technology
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Why arresting cells in G1 makes adaptation to serum-free easier?
DC DC G 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
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Adaptation of CHO cells to suspension using the p21 technology
3 days 6 days 9 days No arrest
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Adaptation of CHO cells to suspension using the p21-Bcl2 technology
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Why arresting cells in G1 makes adaptation to suspension easier?
DNA synthesis agitation rate agitation progression of cells from S to G2/M Disruption of cells by turbulent capillary Flow Mitotic index (%) Before After Mitotic index of remaining cells after capillary flow test
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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 Control cmyc
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Over-expression of hTERT in CHO cells
DNA Microarrays Acc No. Gene Ratio T/B Function AF296282 Icam4 2.2 Adhesion molecule that binds to LFA-1 adhesion protein. Binds to integrins (By similarity) M18933 Col3a1 26.5 Collagen type III occurs in most soft connective tissues along with type I collagen NM_015734 Col5a1 11.3 Type V collagen binds to DNA, heparan sulfate, thrombospondin, heparin, and insulin X65582 Col6a2 Collagen VI acts as a cell-binding protein NM_019759 Dpt 94.5 Mediate adhesion by cell surface integrin binding. Link between cell surface and its ECM NM_009242 Sparc 2.4 Regulate cell growth through interactions with the extracellular matrix and cytokines NM_010577 Itga5 2.6 Integrin alpha-5/beta-1 is a receptor for fibronectin and fibrinogen. Play a role in the survival of adult skeletal muscle M59912 C-kit ligand Mediates cell-cell adhesion NM_008608 Mmp14 3.0 Endopeptidase that degrades various components of the extracellular matrix, such as collagen NM_011777 Zyx Component of a signal pathway that mediates adhesion-stimulated changes in gene expression NM_011780 Adam23 May play a role in cell-cell and cell-matrix interactions NM_013798 Actg1 3.4 Actins are highly conserved proteins that are involved in various types of cell motility NM_021293 Cd33 9.0 Adhesion molecule that mediates sialic-acid dependent binding to cells NM_013681 Syn2 10.4 Phosphoprotein that binds to the cytoskeleton AB009674 Adam22 2.7 Probable ligand for integrin T: telomerase clones B: blank clones Telomerase cells Blank cells Cell Attachment and Survival in the Absence of Serum cDNA Microarray analysis of specific genes involved in cell attachment and formation of extra cellular matrix gene regulation in the CHO K1 cell lines
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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 Cells were transfected with the SEAP gene, passaged in culture for one year and activity of protein measured in the supernatants of batch cultures 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.
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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.
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Acknowledgments John Birch, Lonza Andy Racher, Lonza 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
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