Abstract
Two degradative activities were found in a recombinant Chinese hamster ovary cell culture. These activities became more dominant under high cell density and extended running time, as achieved in a semi-continous perfusion culture. The first, insulin degradative activity caused a growth upset in the 3rd cycle of the perfusion culture and shortened the length of the bioreactor process. The second activity, derived from the neutral pH stable sialidase, was found to affect the integrity of the carbohydrate structure of the recombinant protein, causing increase in heterogeneity in molecular weight and pI of the glycoforms. The most efficient way to overcome these problems may be the use of genetically altered ‘designer cells’ as the production cell line.
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Abbreviations
- IDA:
-
insulin degradative activity
- 4MU-NeuAc:
-
4-methylumbelliferyl acetyl neuraminic acid
- 4MU-Gal:
-
4-methylumbelliferyl-galactoside
- PVDF:
-
polyvinylidene difluoride
- qglucose :
-
specific glucose consumption rate
- μ :
-
specific growth rate
References
Ali M and Plas C (1989) Glucocorticoid regulation of chloroguine nonsensitive insulin degradation in cultured fetal rat hepatocytes. J. Biol. Chem. 264 (35): 20992–7.
Drapeau D, Luan Y-T, Popoloski J, Richards D, Cohen D, Sinacore M and Adamson S (1994) Extracellular insulin degrading activity creates instability in a CHO-based batch-refeed continuous process. Cytotechnology 15: 103–109.
Gramer M, Goochee C (1993) Glycosidase activities in Chinese hamster ovary cells lysate and cell culture supernatant. Biotechnol. Prog. 9: 366–373.
Gramer M, Goochee C, Chock V, Brousseau D and Sliwkowski M (1995) Removal of sialic acid from a glycoprotein in CHO cell culture supernatant by action of an extracellular CHO cell sialidase. Biotechnology 13: 692–698.
Kooistra T and Lloyd JB (1985) Degradation of insulin by human fibroblasts: effects of inhibitors of pinocytosis and lysosomal activity. Int. J. Biochem. 17 (7): 805–811.
Lund P (1985) L-glutamine and L-glutamate. In: Bergmeyer HU (ed.) Methods of enzymatic analysis. Vol. 8: 357–363. VCH Weinheim.
Minch SL, Kallio PT and Bailey JE (1995) Tissue plasminogen activator coexpressed in Chinese hamster ovary cells with α(2,6)-sialytransferase contains neuAc α(2,6)galβ(1,4)glc-N-AcR linkages. Biotechnol. Prog. 11: 348–351.
Omasa T, Higashiyama K, Shioya S and Suga K (1992) Effects of lactate concentration on hybridoma culture in lactate-controlled fed-batch operation. Biotech. Bioeng. 39: 556–564.
Renner WA, Lee KH, Hatzimanikatis V, Baily JE and Eppenberger HM (1995) Recombinant cyclin E expression activated proliferation and obviates surface attachment of Chinese hamster ovary (CHO) cells in protein-free medium. Biotech. Bioeng. 47: 476–482.
Schumpp B and Schlaeger E-J (1992) Growth study of lactate and ammonia double-resistant clones of HL-60 cells. In: Spier RE, Griffiths JB and MacDonald C (eds.) Animal cell technology: developments, processes and products (pp. 183–185). Butterworth-Heinemann.
Takahashi K, Tereda S, Ueda H, Makishima F and Suzuki E (1994) Growth rate suppression of cultured mammalian cells enhances protein productivity. Cytotechnology 15: 57–64.
Weidemann R, Ludwig A and Kretzmer G (1994) Low temperature cultivation-a step towards process optimisation. Cytotechnology 15: 111–116.
Xie L and Wang DIC (1994) Applications of improved stoichiometric model in medium design and fed-batch cultivation of animal cells in bioreactor. Cytotechnology 15: 17–29.
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Lao, MS., Toth, D., Danell, G. et al. Degradative activities in a recombinant chinese hamster ovary cell culture. Cytotechnology 22, 43–52 (1996). https://doi.org/10.1007/BF00353923
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DOI: https://doi.org/10.1007/BF00353923