Glycosylation of a recombinant protein in the Tn5B1-4 insect cell line: Influence of ammonia, time of harvest, temperature, and dissolved oxygen

`Glycosylation is both cell line and protein dependent. Culture conditions can also influence the profile of glycoforms produced. To examine this poss ibility in the insect cell/baculovirus system, structures of N-linked oligo saccharides attached to SEAP (human secreted alkaline phosphatase), express ed under various culture conditions in BTI Tn5B1-4 cells, were characterize d using FACE (fluorescence-assisted carbohydrate electrophoresis). Paramete rs varied were time of harvest, ammonia added during infection, dissolved o xygen, and temperature. It was found that glycosylation in the insect cell/ baculovirus expression system is a robust, stable system that is less pertu rbed by variations in culture conditions than the lever of protein expressi on. Addition of ammonia and low oxygen conditions affected SEAP expression, but not the oligosaccharide profile of SEAP. Time of SEAP harvest increase d the amount of alpha-mannosidase resistant structures from 4.1% at 34 hour s postinfection (h pi), to 5.0% at 400 h pi, and to 7.5% at 120 h pi. These structures were primarily sensitive to N-acetylhexosaminidase digest, alth ough a small amount was insensitive to both mannosidase and N-acetylhexosam inidase digests. Lowering the temperature from 28 degrees C to 24 degrees C or even 20 degrees C, resulted in a twofold increase in oligosaccharides c ontaining terminal alpha(1,3)-mannose residues. This condition did not affe ct the amount of mannosidase-resistant structures. However, this could resu lt in more complete glycosylation of recombinant proteins in the BTI Tn5B1- 4 cell line, because more structures with the potential for further process ing would be produced. (C) 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 6 3: 255-262, 1999.