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Flocculation of CHO cells for primary separation of recombinant glycoproteins: Effect on glycosylation profiles

Gómez, Itzcóatl A., Hernández, Vanessa, Palomares, Laura A., Ramírez, Octavio T.
Biochemical engineering journal 2018 v.132 pp. 244-254
DNA, acids, amino acids, cell culture, centrifugation, composite polymers, erythropoietin, flocculants, flocculation, glycosylation, harvest date, microfiltration, models, monoclonal antibodies, polyacrylamide, polyamines, polyethyleneimine, product quality, sedimentation rate
Centrifugation and microfiltration are the most commonly used steps for initial recovery of recombinant glycoproteins. However, such operations have significant drawbacks at large-scales. Accordingly, the use of flocculants was studied here as an alternative to traditional processes for the clarification of CHO cell cultures producing two model glycoproteins: erythropoietin (EPO) and a monoclonal antibody (MAb), selected for their distinctive glycosylation profiles. Ten polymers, including poly-l-amino acids, polyacrylamide co-polymers and polyamines, were evaluated at concentrations between 60 and 100 ppm at different harvest times. The sedimentation rate, clarification efficiency, and DNA and host cell protein (HCP) removal were evaluated. Poly-l-amino acids were the most effective for removing cells, HCP, and DNA. Whereas increasing harvest time decreased cell removal efficiency for most flocculants, it increased HCP and DNA removal. Polyethylenimine was among the best flocculants tested. Its effect on the glycosylation profile of recovered glycoproteins was determined. No effect on the quality (glycosylation profile and charge variant distribution) and interaction with flocculant of the MAb was observed. In contrast, polyethylenimine affected EPO glycosylation, as it removed highly sialylated glycoforms, reducing product quality. These results illustrate cases where flocculation can be successfully used as initial clarification step for recovery of glycoproteins without affecting their glycosylation. To our knowledge, this is the first report on how flocculants affect glycosylation profiles of recovered recombinant glycoproteins.