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. 2000 Oct;34(1-2):59–70. doi: 10.1023/A:1008103730027

Relationship between cell size, cell cycle and specific recombinant protein productivity

David R Lloyd 1, Paul Holmes 1, Lee P Jackson 1, A Nicholas Emery 1, Mohamed Al-Rubeai 2,✉
PMCID: PMC3449736  PMID: 19003381

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.

Keywords: cell cycle, cell size, centrifugal elutriation, CHO, flow cytometry, NS0, productivity

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References

  1. Aggeler J, Kapp LN, Tseng SCG, Werb Z. Regulation of protein secretion in Chinese hamster ovary cells by cell cycle position and cell density. Exp Cell Res. 1982;139:275–283. doi: 10.1016/0014-4827(82)90252-x. [DOI] [PubMed] [Google Scholar]
  2. Al-Rubeai M, Emery AN. Mechanisms and kinetics of monoclonal antibody synthesis and secretion in synchronous and asynchronous hybridome cultures. J Biotechnol. 1990;16:67–86. doi: 10.1016/0168-1656(90)90066-k. [DOI] [PubMed] [Google Scholar]
  3. Al-Rubeai M, Emery AN, Chalder, Jan DC. Specific monoclonal antibody productivity and the cell cycle, comparisons of batch, continuous and perfusion cultures. Cytotechnology. 1992;9:85–97. doi: 10.1007/BF02521735. [DOI] [PubMed] [Google Scholar]
  4. Al-Rubeai M, Singh RP, Emery AN, Zhang Z. Cell cycle and cell size dependence of susceptibility to hydrodynamic forces. Biotechnol Bioeng. 1995;46:88–92. doi: 10.1002/bit.260460112. [DOI] [PubMed] [Google Scholar]
  5. Al-Rubeai M, Welzenbach K, Lloyd DR, Emery AN. A rapid method for evaluation of cell number and viability by flow cytometry. Cytotechnology. 1997;24:161–168. doi: 10.1023/A:1007910920355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Banik GG, Todd PW, Kompala DS. Foreign protein expression from S phase specific promoters in continuous cultures of recombinant CHO cells. Cytotechnology. 1996;22:179–184. doi: 10.1007/BF00353937. [DOI] [PubMed] [Google Scholar]
  7. Brandt J, Baird N, Lu L, Srour E, Hoffman R. Characterization of a human hematopoietic progenitor cell capable of forming blast cell containing coloniesin vitro. J Chin Invest. 1988;82:1017–1027. doi: 10.1172/JCI113658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Buell DN, Fahey JF. Limited periods of gene expression in immunoglobulin-synthesizing cells. Sciences. 1969;164:1524–1525. doi: 10.1126/science.164.3887.1524. [DOI] [PubMed] [Google Scholar]
  9. Chai H, Al-Rubeai M, Chua KL, Oh SKW, T Yap MGS. Insect cell line dependent gene expression of recombinant human tumor necrosis factor-β. Enzyme Microb Technol. 1996;18:126–132. [Google Scholar]
  10. Drying C, Hansen HA, Emborg C. Observations on the influence of glutamine, asparagine and peptone om growth and t-PA production of Chinmese hamster ovary (CHO) cells. Cytotechnology. 1994;16:37–42. doi: 10.1007/BF00761777. [DOI] [PubMed] [Google Scholar]
  11. Dulbecco R, Vogt M. Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med. 1954;99:167–182. doi: 10.1084/jem.99.2.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Feder JN, Assaraf YG, Seamer LC, Schimke RT. The pattern of dihydrofolate reductase expression through the cell cycle in rodent and human cultured cells. J Biol Chem. 1989;264:20583–20590. [PubMed] [Google Scholar]
  13. Fussenegger M, Mazur X, Bailey JE. A novel cytostatic process enhances the productivity of Chinese hamster overy cells. Biotechnol Bioeng. 1997;55:927–939. doi: 10.1002/(SICI)1097-0290(19970920)55:6<927::AID-BIT10>3.0.CO;2-4. [DOI] [PubMed] [Google Scholar]
  14. Fussenegger M, Shlatter S, Datwyler D, Mazur X, Bailey JE. Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster overy cells. Nature Biotechnol. 1998;16:468–472. doi: 10.1038/nbt0598-468. [DOI] [PubMed] [Google Scholar]
  15. Garatun-Tjeldstø O, Pryme IF, Weltman JK, Dowben RM. Synthesis and secretion of lightchain immunoglobulin in two successive cycles of synchronized cells. J Cell Biol. 1976;68:232–239. doi: 10.1083/jcb.68.2.232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ghosh R, Peng CH, Mitchell DL. Evidence for a novel DNA-damage binding-protein in human-cells. Proc Natl Acad Sci USA. 1996;93:6918–6923. doi: 10.1073/pnas.93.14.6918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gu MB, Tood P, Kompala DS. Cell cycle analysis of foreign gene (β-galactosidase) expression in recombinant mouse cells under control of mouse mammary tumor virus promoter. Biotechnol Bioeng. 1996;50:229–237. doi: 10.1002/(SICI)1097-0290(19960505)50:3<229::AID-BIT1>3.0.CO;2-I. [DOI] [PubMed] [Google Scholar]
  18. Gu MB, Todd P, Kompala DS. Metabolic burden in recombinant CHO cells: Effect ofdhfr gene amplification andlaxZ expression. Cytotechnology. 1996;18:159–166. doi: 10.1007/BF00767763. [DOI] [PubMed] [Google Scholar]
  19. Gu MB, Todd P, Kompala DS. Growth and induction kinetics of inducible and autoinducible expression of heterologous protein in suspension cultures of recombinant mouse L cell lines. Biotechnol Prog. 1996;12:226–237. doi: 10.1021/bp950074s. [DOI] [PubMed] [Google Scholar]
  20. Kromenaker SJ, Srienc F. Cell-cycle-dependent protein accumulation by producer and nonproducer murine hybridoma cell lines: A population analysis. Biotechnol Bioeng. 1991;28:665–677. doi: 10.1002/bit.260380612. [DOI] [PubMed] [Google Scholar]
  21. Kubbies M, Stockinger H. Cell cycle-dependent DHFR and t-PA production in cotransfected, MTX-amplified CHO cells revealed by dual-laser flow cytometry. Exp Cell Res. 1990;188:267–271. doi: 10.1016/0014-4827(90)90169-b. [DOI] [PubMed] [Google Scholar]
  22. Lee FYF, Flannery DJ, Siemann DW. Modulation of the cell cycle-dependent cytotoxicity of Adriamycin and 4-hydroperoxycyclophosphamide by Novobiocin, an inhibitor of mammalian topoisomerase-II. Cancer Res. 1992;52:3515–3520. [PubMed] [Google Scholar]
  23. Leelavatcharamas V, Emery AN, Al-Rubeai M. Use of flow cytometry for the monitoring of growth and productivity in batch culture of Chinese hamster ovary cells. In: Funatsu K, Shirai Y, Matsushita T, editors. Animal Cell Technology: Basic and Applied Aspects. Vol. 8. Dordrecht: Kluwer Academic Publishers; 1997. pp. 55–60. [Google Scholar]
  24. Lloyd DR, Al-Rubeai M. Cell cycle. In: Flickinger MC, Drew SW, editors. Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation. New York: John Wiley and Sons; 1999. pp. 465–476. [Google Scholar]
  25. Lloyd DR, Leelavatcharamas V, Emery AN, Al-Rubeai M. The role of the cell cycle in determining gene expression and productivity in CHO cells. Cytotechnology. 1999;30:49–57. doi: 10.1023/A:1008093404237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mariani BD, Slate DL, Schimke RT. S phase-specific synthesis of dihydrofolate reductase in Chinese hamster overy cells. Proc Natl Acad Sci USA. 1981;78:4985–4989. doi: 10.1073/pnas.78.8.4985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Matherley LH, Schuetz JD, Westin E, Goldman ID. 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. 1989;182:338–345. doi: 10.1016/0003-2697(89)90605-2. [DOI] [PubMed] [Google Scholar]
  28. McEwen CR, Stallard RW, Juhos ET. Separation of biological particles by centrifugation elutriation. Anal Biochem. 1968;23:369–377. doi: 10.1016/0003-2697(68)90228-5. [DOI] [PubMed] [Google Scholar]
  29. Scott FM, Sator de Serrano V, Castellino FJ. Appearance of plasminogen activator activity during a synchronous cycle of a rat adenocarcinoma cell line, PA-III. Exp Cell Res. 1987;169:39–46. doi: 10.1016/0014-4827(87)90222-9. [DOI] [PubMed] [Google Scholar]
  30. Suzuki N, Frapart M, Grdina DJ, Meistrich ML, Withers HR. Cell cycle dependency of metastatic lung colony formation. Cancer Res. 1977;37:3690–3693. [PubMed] [Google Scholar]
  31. van Es WL, Bont WS. An improved method for the fractionation of human blood cells by centrigugal elutriation. Anal Biochem. 1980;103:295–301. doi: 10.1016/0003-2697(80)90613-2. [DOI] [PubMed] [Google Scholar]
  32. Whittle N, Adair J, Lloyd C, Jenkins L, Schlom J, Raubitschek A, Colcher D, Bodmer M. Expression in COS cells of a mouse-human chimaeric B72.3 antibody. Protein Eng. 1987;1:499–505. doi: 10.1093/protein/1.6.499. [DOI] [PubMed] [Google Scholar]

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