Problems with serum-free production of antithrombin III regarding proteolytic activity and product quality

Human antithrombin III (AT-III) was produced using a recombinant BHK-21 cell line with a microcarrier culture in spinner flasks. Cells were cultivated for the first 4 days in a medium containing 10% fetal calf serum (FCS). Afterwards, the medium was exchanged and production of AT-III occurred at high cell numbers in a serum-free medium. The product was determined by an immunoassay and further analysed after isolation from the culture medium. During cultivation, high proteolytic activity was detected which caused a considerable product decomposition. Furthermore, a higher level of non-glycosylated AT-III was found after serum-free production.