ISSN:
1573-0778
Keywords:
CD40
;
COS
;
low protein serum-free medium
;
scale up
;
stransfection
;
transient expression
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Medicine
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
Abstract We demonstrate here that transient expression with COS cells can be performed at the one litre scale for a period of more than 10 days. Cells grown in T225 flasks were transfected by electroporation, transferred into spinners, and then grown either in suspension or on microcarriers. A daily medium change significantly extented culture life and production time, compared with standard protocols. Concentrations of the product, the secreted fusion protein CD40-Fc, were comparable in microcarrier and suspension culture. Cultures were started in fetal calf serum containing medium and the subsequent production process was performed in a low protein serum free medium which allowed easy downstream processing. 10 litres of supernatant, collected from one transfected batch of cells, yielded 30 mg of purified and biologically active protein. In addition to developing a simplified protocol for generation of cells we also reduced the material (DNA, cuvettes) required for electroporation. Our results show that scale up of transient expression to the litre scale can be successfully acieved. This provides a new tool to generate milligram quantities of protein within weeks of gene cloning.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00767766
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