Abstract
Therapeutic proteins require proper folding and posttranslational modifications to be effective and biologically active. Chinese hamster ovary (CHO) cells are by far the most frequently used host for commercial production of therapeutic proteins. However, an unpredictable decrease in protein productivity during the time required for scale up impairs process yields, time, finance, and regulatory approval for the desired product. Therefore, it is important to assess cell lines at stages throughout the period of long-term culture in terms of productivity and various molecular parameters including plasmid and mRNA copy numbers and location of the plasmid on the host cell chromosome. Here, we describe methods, which are frequently used to analyze stability of the recombinant CHO cells over long-term culture. These procedures include the following; western blotting, ELISA to evaluate protein production, real-time PCR to analyze plasmid and mRNA copy numbers, and fluorescent in situ hybridization (FISH) to assess the location of the inserted plasmid on host cell chromosomes.
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References
Bailey LA, Hatton D, Field R, Dickson AJ (2012) Determination of Chinese hamster ovary cell line stability and recombinant antibody expression during long-term culture. Biotechnol Bioeng 109:2093–2103. doi:10.1002/bit.24485/abstract
Barnes LM, Bentley CM, Dickson AJ (2003) Stability of protein production from recombinant mammalian cells. Biotechnol Bioeng 81:631–639. doi:10.1002/bit.10517
Betts Z, Croxford AS, Dickson AJ (2015) Evaluating the interaction between UCOE and DHFR-linked amplification and stability of recombinant protein expression. Biotechnol Prog 31:1014–1025. doi:10.1002/btpr.2083
Kim SJ, Lee GM (1999) Cytogenetic analysis of chimeric antibody-producing CHO cells inthe course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure. Biotechnol Bioeng 64:741–749
Derouazi M, Martinet D, Besuchet Schmutz N et al (2006) Genetic characterization of CHO production host DG44 and derivative recombinant cell lines. Biochem Biophys Res Commun 340:1069–1077. doi:10.1016/j.bbrc.2005.12.111
Betts Z, Dickson AJ (2016) Ubiquitous chromatin opening elements (UCOEs) effect on transgene position and expression stability in CHO cells following methotrexate (MTX) amplification. Biotechnol J 11:554–564. doi:10.1002/biot.201500159
Kim M, O'Callaghan PM, Droms KA, James DC (2011) A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies. Biotechnol Bioeng 108:2434–2446. doi:10.1002/bit.23189
Osterlehner A, Simmeth S, Göpfert U (2011) Promoter methylation and transgene copy numbers predict unstable protein production in recombinant chinese hamster ovary cell lines. Biotechnol Bioeng 108:2670–2681. doi:10.1002/bit.23216
Kim NS, Kim SJ, Lee GM (1998) Clonal variability within dihydrofolate reductase-mediated gene amplified Chinese hamster ovary cells: stability in the absence of selective pressure. Biotechnol Bioeng 60:679–688
Chusainow J, Yang YS, Yeo JHM et al (2009) A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng 102:1182–1196. doi:10.1002/bit.22158
Barnes LM, Bentley CM, Dickson AJ (2001) Characterization of the stability of recombinant protein production in the GS-NS0 expression system. Biotechnol Bioeng 73:261–270
Mahmood T, Yang P-C (2012) Western blot: technique, theory, and trouble shooting. N Am J Med Sci 4:429–434. doi:10.4103/1947-2714.100998
Feit C, Bartal AH, Tauber G et al (1983) An enzyme-linked immunosorbent assay (ELISA) for the detection of monoclonal antibodies recognizing surface antigens expressed on viable cells. J Immunol Methods 58:301–308
Hornbeck P, Winston SE, Fuller SA (2001) Enzyme-linked immunosorbent assays (ELISA). Curr Protoc Mol Biol 11:112
Blin N, Stafford DW (1976) A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res 3:2303–2308
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159. doi:10.1006/abio.1987.9999
Chomczynski P, Sacchi N (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on. Nat Protoc 1:581–585. doi:10.1038/nprot.2006.83
Gall JG, Pardue ML (1969) Formation and detection of Rna-Dna hybrid molecules in cytological preparations. Proc Natl Acad Sci U S A 63:378
Lichter P, Cremer T, Borden J et al (1988) Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum Genet 80:224–234
Rudkin GT, Stollar BD (1977) High resolution detection of DNA-RNA hybrids in situ by indirect immunofluorescence. Nature 265:472–473
Trask BJ (2002) Human cytogenetics: 46 chromosomes, 46 years and counting. Nat Rev Genet 3:769–778. doi:10.1038/nrg905
Speicher MR, Carter NP (2005) The new cytogenetics: blurring the boundaries with molecular biology. Nat Rev Genet 6:782–792. doi:10.1038/nrg1692
Rens W, Fu B, O'Brien PCM, Ferguson-Smith M (2006) Cross-species chromosome painting. Nat Protoc 1:783–790. doi:10.1038/nprot.2006.91
Rigby PW, Dieckmann M, Rhodes C, Berg P (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237–251
Cox WG, Singer VL (2004) Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling. Biotechniques 36:114–122
O'connor C (2008) Fluorescence in situ hybridization (FISH). Nat Educ 1:171
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Betts, Z., Dickson, A.J. (2017). Improved CHO Cell Line Stability and Recombinant Protein Expression During Long-Term Culture. In: Meleady, P. (eds) Heterologous Protein Production in CHO Cells. Methods in Molecular Biology, vol 1603. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6972-2_8
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DOI: https://doi.org/10.1007/978-1-4939-6972-2_8
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