Effect of culture conditions on the degree of sialylation of a recombinant glycoprotein expressed in insect cells.
Secreted human placental alkaline phosphatase (SEAP) was produced in a nonengineered Trichoplusia ni insect cell line, Tn-4s, using a recombinant Autographa californica baculovirus expression vector. The effect of culture conditions on SEAP specific yield and glycosylation was studied. When cultured in the high aspect ratio vessel (HARV) or in tissue culture flasks (T-flasks), baculovirus-infected Tn-4s cells produced high levels of SEAP (13 and 23 U/10(6) cells, respectively; 4 days postinfection), but in those conditions SEAP possessed only high mannose, paucimannosidic, and hybrid structures. In spinner flasks, lower SEAP yields were obtained (<4 U/10(6) cells, 3 days postinfection), but in such cultures, sialylation of SEAP could be achieved. Several spinner-flask culture conditions were tested and resulted in different SEAP specific yields and levels of sialylation. The highest level of sialylation (9%) was obtained in the culture with the lowest agitation rate and lowest yield (1.2 U/10(6) cells), suggesting a limiting capacity of the Tn-4s cells to process glycoproteins to sialylation. High specific yield, low passage number Tn5B1-4 cells did not produce SEAP with complex glycosylation when cultured in a low agitation rate spinner-flask. On the basis of these results, we propose that the Golgi apparatus has a limited capacity for processing proteins to complex glycosylation and sialylation and that this capacity is easily overwhelmed by high levels of foreign protein productivity. Selected media additives such as Pluronic F-68, dextran sulfate (MW 12 500) and a lipids premix did not allow improvement of the specific yield of sialylated SEAP when supplemented to spinner-flask cultures.
Joosten
Christoph E
CE
School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, New York 14853-5201, USA. cej6@cornell.edu
Shuler
Michael L
ML
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000502:physiology; D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival / Q000502:physiology; D003470:Culture Media / Q000378:metabolism; D005822:Genetic Vectors; D006019:Glycolysis / Q000502:physiology*; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007915:Lepidoptera / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development* / Q000821:virology; D010920:Placenta / Q000201:enzymology; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection / Q000379:methods*
D003470:Culture Media; D006023:Glycoproteins; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase