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Saldova, R,Kilcoyne, M,Stockmann, H,Martin, SM,Lewis, AM,Tuite, CME,Gerlach, JQ,Le Berre, M,Borys, MC,Li, ZJ,Abu-Absi, NR,Leister, K,Joshi, L,Rudd, PM
2017
March
Methods
Advances in analytical methodologies to guide bioprocess engineering for bio-therapeutics
Published
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Bioprocess engineering Glycosylation CHO cells HILIC UPLC Lectin microarrays HAMSTER OVARY CELLS CHO-CELLS LIQUID-CHROMATOGRAPHY LECTIN MICROARRAYS MASS-SPECTROMETRY IMMUNOGLOBULIN-G GLYCOSYLATION PROTEIN GLYCANS BINDING
116
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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 alpha-(2,3)-linked, galactose beta-(1,4)-linked, with core fucose. Glycans attached to secreted glycoproteins were mostly complex with sialic acid alpha-(2,3)linked, galactose beta-(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. (C) 2016 Elsevier Inc. All rights reserved.
10.1016/j.ymeth.2016.11.002
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