Enhanced erythropoietin heterogeneity in a CHO culture is caused by proteolytic degradation and can be eliminated by a high glutamine level.
The molecular heterogeneity of recombinant humanerythropoietin (EPO) increased during the course of abatch culture of transfected Chinese hamster ovary(CHO) cells grown in serum-free medium. This wasshown by both an increased molecular weight and pIrange of the isolated EPO at the end of the culture. However, analysis of the N-glycan structures of themolecule by fluorophore-assisted carbohydrateelectrophoresis (FACE) and HPLC anion exchangechromatography indicated a consistent pattern ofglycosylation. Seven glycoforms were identified, thepredominant structure being a fully sialylatedtetra-antennary glycan. The degree of sialylationwas maintained throughout the culture. Analysis ofthe secreted EPO indicated a time-dependent increasein the molecular weight band width of the peptideconsistent with proteolytic degradation. A highglutamine concentration (16-20 mM) in the culturedecreased the apparent degradation of the EPO.
Yang
M
M
Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada.
Butler
M
M