Abstract
Centrifugal elutriation was used to produce cell cycle enrichedfractions of four commercially relevant recombinant cell lines,chosen to allow for variation in properties due to construct,expression system and parent cell type, from normally growingheterogeneous batch cultures. As these fractions had identicalculture histories and had not been subjected to any insult orstress which was likely to have adversely affected cellularmetabolism, they were ideal for further study of cellularproperties. Specific productivity, cell size and cell cyclestate of replicate elutriated fractions were measured for eachcell line. Results showed that cell size was the major cellulardeterminant of productivity for all cell lines examined. Productformation was not restricted to any particular cell cycle phaseand in all cases, production occurred irrespective of cell cyclephase. Specific productivity was lowest when the majority ofcells in the fraction were G1, intermediate when themajority of cells in the fraction were S phase and greater whenthe majority of cells in the fraction were in G2/M. However, the evidence suggests that size is the major cellulardeterminant of productivity; the apparent relationship betweencell cycle and productivity is secondary and can simply beascribed to the increasing size of cells as they progress thoughthe cell cycle. Thus, in addition to cell density and viabilitycell size is the cellular parameter which should be incorporatednot only into mathematical models of recombinant mammalian cellproduction processes but also into process monitoring andcontrol strategies.
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Aggeler J, Kapp LN, Tseng SCG and Werb Z (1982) Regulation of protein secretion in Chinese hamster ovary cells by cell cycle position and cell density. Exp Cell Res 139: 275–283.
Al-Rubeai M and Emery AN (1990) Mechanisms and kinetics of monoclonal antibody synthesis and secretion in synchronous and asynchronous hybridome cultures. J Biotechnol 16: 67–86.
Al-Rubeai M, Emery AN, Chalder and Jan DC (1992) Specific monoclonal antibody productivity and the cell cycle, comparisons of batch, continuous and perfusion cultures. Cytotechnology 9: 85–97.
Al-Rubeai M, Singh RP, Emery AN and Zhang Z (1995) Cell cycle and cell size dependence of susceptibility to hydrodynamic forces. Biotechnol Bioeng 46: 88–92.
Al-Rubeai M, Welzenbach K, Lloyd DR and Emery AN (1997) A rapid method for evaluation of cell number and viability by flow cytometry. Cytotechnology 24: 161–168.
Banik GG, Todd PW and Kompala DS (1996) Foreign protein expression from S phase specific promoters in continuous cultures of recombinant CHO cells. Cytotechnology 22: 179–184.
Brandt J, Baird N, Lu L, Srour E and Hoffman R (1988) Characterization of a human hematopoietic progenitor cell capable of forming blast cell containing coloniesin vitro. J Chin Invest 82: 1017–1027.
Buell DN and Fahey JF (1969) Limited periods of gene expression in immunoglobulin-synthesizing cells. Sciences 164: 1524–1525.
Chai H, Al-Rubeai M, Chua KL, Oh SKW and T Yap MGS (1996) Insect cell line dependent gene expression of recombinant human tumor necrosis factor-β. Enzyme Microb Technol 18: 126–132.
Drying C, Hansen HA and Emborg C (1994) Observations on the influence of glutamine, asparagine and peptone om growth and t-PA production of Chinmese hamster ovary (CHO) cells. Cytotechnology 16: 37–42.
Dulbecco R and Vogt M (1954) Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med 99: 167–182.
Feder JN, Assaraf YG, Seamer LC and Schimke RT (1989) The pattern of dihydrofolate reductase expression through the cell cycle in rodent and human cultured cells. J Biol Chem 264: 20583–20590.
Fussenegger M, Mazur X and Bailey JE (1997) A novel cytostatic process enhances the productivity of Chinese hamster overy cells. Biotechnol Bioeng 55: 927–939.
Fussenegger M, Shlatter S, Datwyler D, Mazur X and Bailey JE (1998b) Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster overy cells. Nature Biotechnol 16: 468–472.
Garatun-Tjeldstø O, Pryme IF, Weltman JK and Dowben RM (1976) Synthesis and secretion of lightchain immunoglobulin in two successive cycles of synchronized cells. J Cell Biol 68: 232–239.
Ghosh R, Peng CH and Mitchell DL (1996) Evidence for a novel DNA-damage binding-protein in human-cells. Proc Natl Acad Sci USA 93: 6918–6923.
Gu MB, Tood P and Kompala DS (1996a) Cell cycle analysis of foreign gene (β-galactosidase) expression in recombinant mouse cells under control of mouse mammary tumor virus promoter. Biotechnol Bioeng 50: 229–237.
Gu MB, Todd P and Kompala DS (1996b) Metabolic burden in recombinant CHO cells: Effect ofdhfr gene amplification andlaxZ expression. Cytotechnology 18: 159–166.
Gu MB, Todd P and Kompala DS (1996c) Growth and induction kinetics of inducible and autoinducible expression of heterologous protein in suspension cultures of recombinant mouse L cell lines. Biotechnol Prog 12: 226–237.
Kromenaker SJ and Srienc F (1991) Cell-cycle-dependent protein accumulation by producer and nonproducer murine hybridoma cell lines: A population analysis. Biotechnol Bioeng 28: 665–677.
Kubbies M and Stockinger H (1990) Cell cycle-dependent DHFR and t-PA production in cotransfected, MTX-amplified CHO cells revealed by dual-laser flow cytometry. Exp Cell Res 188: 267–271.
Lee FYF, Flannery DJ and Siemann DW (1992) Modulation of the cell cycle-dependent cytotoxicity of Adriamycin and 4-hydroperoxycyclophosphamide by Novobiocin, an inhibitor of mammalian topoisomerase-II. Cancer Res 52: 3515–3520.
Leelavatcharamas V, Emery AN and Al-Rubeai M (1997) Use of flow cytometry for the monitoring of growth and productivity in batch culture of Chinese hamster ovary cells. In: Funatsu K, Shirai Y and Matsushita T (eds) Animal Cell Technology: Basic and Applied Aspects. Vol. 8 (pp. 55–60) Kluwer Academic Publishers, Dordrecht.
Lloyd DR and Al-Rubeai M (1999) Cell cycle. In: Flickinger MC and Drew SW (eds) Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation (pp. 465–476) John Wiley and Sons, New York.
Lloyd DR, Leelavatcharamas V, Emery AN and Al-Rubeai M (1999) The role of the cell cycle in determining gene expression and productivity in CHO cells. Cytotechnology 30: 49–57.
Mariani BD, Slate DL and Schimke RT (1981) S phase-specific synthesis of dihydrofolate reductase in Chinese hamster overy cells. Proc Natl Acad Sci USA 78: 4985–4989.
Matherley LH, Schuetz JD, Westin E and Goldman ID (1989) A method for the synchronization of cultured cells with aphidicolin: Application to the large-scale synchronization of L1210 cells and the study of the cell cycle regulation of thymidylate synthetase and dihydrofolate reductase. Anal Biochem 182: 338–345.
McEwen CR, Stallard RW and Juhos ET (1968) Separation of biological particles by centrifugation elutriation. Anal Biochem 23: 369–377.
Scott FM, Sator de Serrano V and Castellino FJ (1987) Appearance of plasminogen activator activity during a synchronous cycle of a rat adenocarcinoma cell line, PA-III. Exp Cell Res 169: 39–46.
Suzuki N, Frapart M, Grdina DJ, Meistrich ML and Withers HR (1977) Cell cycle dependency of metastatic lung colony formation. Cancer Res 37: 3690–3693.
van Es WL and Bont WS (1980) An improved method for the fractionation of human blood cells by centrigugal elutriation. Anal Biochem 103: 295–301.
Whittle N, Adair J, Lloyd C, Jenkins L, Schlom J, Raubitschek A, Colcher D and Bodmer M (1987) Expression in COS cells of a mouse-human chimaeric B72.3 antibody. Protein Eng 1: 499–505.
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Lloyd, D.R., Holmes, P., Jackson, L.P. et al. Relationship between cell size, cell cycle and specific recombinant protein productivity. Cytotechnology 34, 59–70 (2000). https://doi.org/10.1023/A:1008103730027
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DOI: https://doi.org/10.1023/A:1008103730027