Abstract
UCOE vectors contain non-tissue specific chromatin-opening-elements that permit rapid expression of a protein in anintegration independent manner. Efficient expression can bederived from a single copy of an integrated gene site resulting ina higher percentage of cells expressing the marker gene in theselected pool in comparison to standard non-UCOE containingvectors. This, in combination with the utilization of a serum-free, suspension adapted parent cell line allows for rapidproduction of large quantities of protein in a short period oftime. Utilizing this system more than 300 mg of a recombinantantibody has been produced in less than 1 month from transfectionpools in shake flask. Selected subclones have been scaled intosmall bioreactors in less than 2 months, producing significantquantities of monoclonal antibody using a protocol generic for theparent cell line. The increased efficiency obtained with the UCOEvector reduces the number of transfectants which need to bescreened in order to obtain high productivity subclones.Transfection of a standard host cell line, preadapted to grow in alarge-scale setting, allows for rapid cell line developmentdecreasing the transition time from research into development andmanufacturing. Alternatively, the traditional approach of using aparent cell line which requires serum-free and suspensionadaptation after transfection further increases the need forscreening a large number of subclones, because many of thesubclones will not be able to grow under conditions that allowlarge-scale protein production. The use of a preadapted cell linecan reduce the time required to develop a cell line from months toweeks.
Keywords: preadapted serum-free cell culture, rapid proteinproduction, UCOE
Full Text
The Full Text of this article is available as a PDF (80.5 KB).
References
- Borrebeack CM, Ohlin M. Does endogenous glycosylation prevent the use of mouse monoclonal antibodies as cancer therapeutics? Immunol Today. 1993;14:477–479. doi: 10.1016/0167-5699(93)90259-n. [DOI] [PubMed] [Google Scholar]
- Cho SK, Cummings RD. A soluble form of α1,3-galactosyltransferase functions within cells to glycosylate proteins. J Biol Chem. 1997;272:13622–13628. doi: 10.1074/jbc.272.21.13622. [DOI] [PubMed] [Google Scholar]
- Crombie R & Antoniou M (2000) A Polynucleotide Comprising a Ubiquitous Chromatin Opening Element (UCOE). Patent Number WO0005393, 2000-02-03.
- D'Anna JA, Valdez JG, Habbersett RC, Crissman HA. Association of G1/S-Phase and Late S-Phase Checkpoints with Regulation of Cyclin-Dependent Kinases in Chinese Hamster Ovary Cells. Rad Res. 1997;148:260–271. [PubMed] [Google Scholar]
- D'Anna JA, Crissman HA, Valdez JG, Gurley LR, Tobey RA. Syncronization of mammalian cells in S-phase by sequential use of isoleucine-deprivation G1-or serum-withdrawl G0-arrest and aphidicolin block. Meth Cell Sci. 1996;18:115–125. [Google Scholar]
- Deaven LL, Petersen DF. The Chromosomes of CHO, an Aneuploid Chinese Hamster Cell Line: G-Band, C-Band, and Autoradiographic Analysis. Chromosoma. 1973;41:129–144. doi: 10.1007/BF00319690. [DOI] [PubMed] [Google Scholar]
- Gorelik EL, Anaraki DF, Galili U. Alterations of cell surface carbohydrate and inhibition of metastatic property of murine melanomas by α1,3 galactosyltransferase gene transfection. Cancer Res. 1995;55:4185–4173. [PubMed] [Google Scholar]
- Gorfien SF, Dzimian JL, Tilkins ML, Godwin GP, Fike R. Recombinant Protein Production by CHO Cells Cultures in a Chemically Defined Medium. In: Kitagawa Y, Matsuda T, Iijima S, editors. Animal Cell Technology: Basic & Applied Aspects. (Netherlands): Kluwer Academic Publishers; 1998. pp. 247–252. [Google Scholar]
- Kagawa YT, Utsumi S, Hosoi K, Shimizu H, Kochibe N, Kobata A. Comparative study of the asparagines-linked sugar chains of natural human interferon-1 and recombinant human interferon produced by three different mammalian cell lines. J Biol Chem. 1988;263:7508–17515. [PubMed] [Google Scholar]
- Puck TT, Cieciura SJ, Robinson A. Genetics of somatic mammalian cells: Long term cultivation of euploid cells from human and animal subjects. J Exp Med. 1958;108:945–955. doi: 10.1084/jem.108.6.945. [DOI] [PMC free article] [PubMed] [Google Scholar]
