Abstract
A recombinant Chinese hamster ovary cell line, producing human erythropoietin, was cultivated in a continuous mode in a stirred tank reactor applying different dilution rates. In order to monitor the stability of this expression system, product and non-product proteins of the cell culture supernatant were analyzed by two-dimensional electrophoresis. The consistency of the isoforms of the recombinant product was determined by western blot combined with specific staining.
The same cell line was propagated in a high cell density cultivation system based on macro-cell-aggregates. The patterns of secreted proteins of the cell line cultivated in the different systems were compared in order to detect modifications in protein expression of the product and of non product proteins relevant for cell culture supernatant.
Hardly any alterations in two-dimensional pattern were detectable. The isoforms of erythropoietin, as well as the overall pattern of secreted proteins, detectable with the two-dimensional electrophoresis method were remarkably stable under different cultivation conditions.
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Abbreviations
- 2-DE:
-
two-dimensional electrophoresis
- rCHO:
-
recombinant Chinese hamster ovary cell
- EPO:
-
erythropoietin
- FBR:
-
fluidized bed reactor
- IEF:
-
isoelectric focusing
- IPG:
-
immobilized pH gradient
- pI:
-
isoelectric point
- SDS:
-
sodium dodecylsulfate
- STR:
-
stirred tank reactor
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Wimmer, K., Harant, H., Reiter, M. et al. Two-dimensional gel electrophoresis for controlling and comparing culture supernatants of mammalian cell culture productions systems. Cytotechnology 16, 137–146 (1994). https://doi.org/10.1007/BF00749900
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DOI: https://doi.org/10.1007/BF00749900