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Vol. 116, 2017, Pages: 63-83

Advances in analytical methodologies to guide bioprocess engineering for bio-therapeutics

RadkaSaldova, MichelleKilcoyne, HenningStöckmann, SilviaMillán Martín, Amanda M.Lewis, Catherine M.E.Tuite, Jared Q.Gerlach, MarieLe Berre, Michael C.Borys, Zheng

NIBRT GlycoScience Group, The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Dublin 4, Ireland.


This study was performed to monitor the glycoform distribution of a recombinant antibody fusion protein expressed in CHO cells over the course of fed-batch bioreactor runs using high-throughput methods to accurately determine the glycosylation status of the cell culture and its product. Three different bioreactors running similar conditions were analysed at the same five time-points using the advanced methods described here. N-glycans from cell and secreted glycoproteins from CHO cells were analysed by HILIC-UPLC and MS, and the total glycosylation (both N- and O-linked glycans) secreted from the CHO cells were analysed by lectin microarrays. Cell glycoproteins contained mostly high mannose type N-linked glycans with some complex glycans; sialic acid was α-(2,3)-linked, galactose β-(1,4)-linked, with core fucose. Glycans attached to secreted glycoproteins were mostly complex with sialic acid α-(2,3)-linked, galactose β-(1,4)-linked, with mostly core fucose.

There were no significant differences noted among the bioreactors in either the cell pellets or supernatants using the HILIC-UPLC method and only minor differences at the early time-points of days 1 and 3 by the lectin microarray method. In comparing different time-points, significant decreases in sialylation and branching with time were observed for glycans attached to both cell and secreted glycoproteins. Additionally, there was a significant decrease over time in high mannose type N-glycans from the cell glycoproteins.

A combination of the complementary methods HILIC-UPLC and lectin microarrays could provide a powerful and rapid HTP profiling tool capable of yielding qualitative and quantitative data for a defined biopharmaceutical process, which would allow valuable near ‘real-time’ monitoring of the biopharmaceutical product.

Graphical abstract

Keywords: Bioprocess engineering, Glycosylation, CHO cells, HILIC, UPL, CLectin microarrays.

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