Development of Cell Lines for Controlled Proliferation and Apoptosis Mohamed Al-Rubeai University College Dublin Cell Line Development and Engineering, 23 May, Zurich Criteria for Cell Line Selection Stability Product biological activity Product expression: level, duration and inducibility Growth and productivity in large scale culture Safety issues ease of selection of high producers Adaptation to protein free suspension culture apoptosis; proliferation rate; max cell number Mammalian Cell Lines Used For Protein Production Cell Line Property CHO Cell number ( cells/ml) Productivity (pg/cell/day) Product quality Impurities
++ + + <107 10 + ++ ++ ++ + + +++ Based partially on information from Robert D Kiss, Genetic Engineering News Improvement of Product Expression Expression Engineering - development of genetic tools comprising cloning and expression vectors Cell (Metabolic) Engineering - design or redirection of metabolic pathways 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) 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 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 Proliferation and Cell Death
Stimulated by cyclins, cdks, c-myc, signals from environment Proliferation Cell Population (numbers increase) Inhibited by cytostasis inducers, e.g. excess thymidine, hydroxyurea, nitrous oxide cdki p21Cip1, cdki p27Kip1 Stimulated by chemical compounds, NGF, Fas ligand Cell Death (numbers decrease) Inhibited by bcl-2, bclXL, p35, hTERT signal from environment The Mammalian Cell Cycle reversible quiescent phase preparation for mitosis G0 Apoptosis cyclin p21
DNA synthesis bcl-2 hTERT P cdk Growth of antibody-producing GS-CHO with and without anti-apoptosis gene- Viable cell count (cells/ml) E5 laboratory results 18 16 14 12 10 Parental CHO 8 CHO Bcl2 6 4 2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time (days) Growth of antibody-producing GS-NS0: with and without anti-apoptosis geneIndustrially optimised condition 12
parent bcl-2 transfectant Xv (106/mL) 10 8 6 4 2 0 0 100 200 Time (hr) 300 400 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
p21 bcl2 total cell count Batch culture Days Perfusion culture SPR (pg/cell/day) 15 5 Viable cell count /ml) 35 Cell density (10 Cell count (cell/ml) x The effect of ectopic p21CIP1 and Bcl-2 expression on IgG production in batch and perfusion culture Effect of p21 expression on cell proliferation and cell productivity 300 Col 15 Control Col IPTG18
MAb Productivity (pg/cell/day) Cell Density ( x 105 cells /ml) 12 10 8 6 4 2 0 250 Col 15 Col 18 200 150 100 50 0 3A1 1B5 2C12 2B7 2H3 3B2 3A1
1B5 2C12 2B7 p21- transfected CHO clones Increased productivity: cell cycle vs cell volume? 2H3 3B2 P21-dependent productivity: Cell size vs cell cycle P21 arrests cells in G1phase G1 is less productive than S and G2 6000 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 Cell volume (um3)
5000 4000 3000 2000 Control induced 1000 0 20 40 60 Time 80 100 120
cell dissociation over 10 minutes following exposure to cell disociation solution cell density x105 5 4 3 + IPTG 2 - IPTG 1 0 1 2 3 4 5 6 7 8 9 10
sampling time (mins) 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) Adaptation of NS0 cells to serum free media using the p21 technology 1.40E+06 12 NS0 p21 (4 Day Arrest) NS0 p21 Feotal Calf Serum (v/v) 10 10% 1.00E+06 8 8.00E+05 6 6.00E+05 5 % 4 4 % 4.00E+05 3%
IPTG Arrest Initiated 2 % 2.00E+05 2 IPTG Arrest Removed 1% 0.5% 0.00E+00 0 10 20 30 Passages (2 day) 0 40 50 [F e o ta l C a lf S e ru m ] v /v P eak C ell D en sity (cells/m l) 1.20E+06
V ia b le c e ll c o u n t (c e lls /m l) E 5 Adaptation of CHO cells to serum free media using the p21 technology 10 9 days 9 6 days 3 days arrest No arrest 8 7 6 5 4 3 0 20 40 Time (days) 60 80
100 V ia b le c e ll c o u n t ( c e lls /m l) E 5 Adaptation of CHO cells to serum free media using the p21-Bcl2 technology 12 9 days 6 days 3 days No arrest 10 8 6 4 2 0 10 20 30 Time (days) 40 50
60 Summary of time taken for adaptation to serum free in CHO cells using P21 and bcl2 technology 80 70 Time taken (days) 60 50 40 30 20 10 0 P21 arrested P21 BCL2 P21 arrested P21 BCL2 P21 arrested P21 BCL2 for 3 days arrested for 3 for 6 days arrested for 6 for 9 days arrested for 9 days days days P21 non arrested throughout P21 BCL2 non arrested throughout
Why arresting cells in G1 makes adaptation to serum-free easier? DC DC G1 S G2 M R Cells survive on minimum nutrition Highly variable duration, Less variable duration Dependent on soluble growth factors and Growth conditions to proceed to S DC V ia b le c e ll c o u n t (c e lls /m l) E 5 Adaptation of CHO cells to suspension using the p21 technology 8 7 9 days 3 days 6 days
No arrest 6 5 4 3 2 1 0 20 40 Time (Days) 60 80 V ia b le c e ll c o u n t (c e lls /m l) E 5 Adaptation of CHO cells to suspension using the p21-Bcl2 technology 7 6 5 4
3 2 1 0 10 20 30 Time (days) 40 50 agitation rate agitation progression of cells from S to G2/M Disruption of cells by turbulent capillary Flow Mitotic index (%) DNA synthesis Why arresting cells in G1 makes adaptation to suspension easier? Before
After Mitotic index of remaining cells after capillary flow test 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 % INCREASE IN CELL NUMBER / 3 DAYS (A) with serum 100 neo-cho cmyc-cho 80 60 40
20 0 control IGF-1 Transferrin IGF-1+Transferr Over-expression of hTERT in CHO cells DNA Microarrays Viable cell number (cells/ ml) 2.5e+6 T1F11 T2G7 T1G11 T1A8 B1E7 B3E12 B2F6 2.0e+6 1.5e+6 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 2.2 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 2.2 Mediates cell-cell adhesion NM_008608 Mmp14 3.0 Endopeptidase that degrades various components of the extracellular matrix, such as collagen NM_011777 Zyx
2.4 Component of a signal pathway that mediates adhesion-stimulated changes in gene expression NM_011780 Adam23 2.4 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 1.0e+6 5.0e+5 T: telomerase clones B: blank clones 0 5 10 15 20 25 Time (Days) 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
Over-expression of hTERT enhances chromosomal stability and possibly production stability CHO is not stable cell line! Aneuploidy Micronucleus Aneuploidy (loss of several chromosomes) 5 Activity (mU/ml) 4 Telomerase 3 2 Blank 1 0 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. Conclusions
Cell proliferation and apoptosis are coordinately linked processes. Genetic engineering of cellular and metabolic pathways can enhance cell robustness, adaptation and productivity. Genetic and chromosomal instability may affect production stability. 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
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