title: Effect of production method and gene amplification on the glycosylation pattern of a secreted reporter protein in CHO cells.
abstract: We have investigated the independent effects of selective gene amplification (using the dhfr amplifiable selection marker) and culture operating strategy (batch vs repeated fed-batch vs semicontinuous perfusion) on the glycosylation of a recombinant reporter protein (secreted alkaline phosphatase, SEAP) produced in transfected Chinese hamster ovary (CHO) cells. HPLC analyses coupled with susceptibility to various exoglycosidases were used to determine the N-glycosylation profile of SEAP samples. The dhfr amplified cell line yielded an almost 10-fold increase in specific productivity as compared to that of the unamplified cell line. The glycosylation pattern of the reporter protein produced in batch bioreactor cultures of the amplified cell line showed only slight differences as compared to the glycosylation pattern of the protein from batch bioreactor cultures of the unamplified cell line. In contrast, analysis of SEAP glycosylation structures from the protein isolated from semicontinuous perfusion cultures indicated that both relative glycan content and extent of sialylation were increased as compared to samples isolated from repeated fed-batch cultures. These results suggest that the slow growing perfusion cultures produce more completely glycosylated proteins than the faster growing repeated fed-batch cultures.
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado, Boulder, CB 424, Boulder, Colorado 80309-0424, USA.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Lipscomb
authors.forename: Laura A
authors.initials: LA
authors.lastname: Palomares
authors.forename: Vanessa
authors.initials: V
authors.lastname: Hernández
authors.forename: Octavio T
authors.initials: OT
authors.lastname: Ramírez
authors.forename: Dhinakar S
authors.initials: DS
authors.lastname: Kompala
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003470:Culture Media; D046508:Culture Techniques / Q000379:methods; D005784:Gene Amplification; D006031:Glycosylation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase