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

Author:
Saldova, Radka, Kilcoyne, Michelle, Stöckmann, Henning, Millán Martín, Silvia, Lewis, Amanda M., Tuite, Catherine M.E., Gerlach, Jared Q., Le Berre, Marie, Borys, Michael C., Li, Zheng Jian, Abu-Absi, Nicholas R., Leister, Kirk, Joshi, Lokesh, Rudd, Pauline M.
Source:
Methods 2017 v.116 pp. 63-83
ISSN:
1046-2023
Subject:
biopharmaceuticals, bioprocess engineering, bioreactors, cell culture, fucose, galactose, glycoproteins, glycosylation, lectins, mannose, microarray technology, monitoring, pellets, polysaccharides, recombinant antibodies, sialic acids
Abstract:
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.
Agid:
5586577