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Glycosylation characterization of recombinant human erythropoietin produced in glycoengineered Pichia pastoris by mass spectrometry
Author(s) -
Gong Bing,
Burnina Irina,
Stadheim Terrance A.,
Li Huijuan
Publication year - 2013
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.3291
Subject(s) - pichia pastoris , glycosylation , chemistry , chinese hamster ovary cell , glycan , recombinant dna , pichia , n linked glycosylation , biochemistry , erythropoietin , glycoprotein , biology , receptor , gene , endocrinology
Glycosylation plays a critical role in the in vivo efficacy of both endogenous and recombinant erythropoietin (EPO). Using mass spectrometry, we characterized the N ‐/ O ‐linked glycosylation of recombinant human EPO (rhEPO) produced in glycoengineered Pichia pastoris and compared with the glycosylation of Chinese hamster ovary (CHO) cell‐derived rhEPO. While the three predicted N ‐linked glycosylation sites (Asn24, Asn38 and Asn83) showed complete site occupancy, Pichia ‐ and CHO‐derived rhEPO showed distinct differences in the glycan structures with the former containing sialylated bi‐antennary glycoforms and the latter containing a mixture of sialylated bi‐, tri‐ and tetra‐antennary structures. Additionally, the N ‐linked glycans from Pichia ‐produced rhEPO were similar across all three sites. A low level of O ‐linked mannosylation was detected on Pichia ‐produced rhEPO at position Ser126, which is also the O ‐linked glycosylation site for endogenous human EPO and CHO‐derived rhEPO. In summary, the mass spectrometric analyses revealed that rhEPO derived from glycoengineered Pichia has a highly uniform bi‐antennary N ‐linked glycan composition and preserves the orthogonal O ‐linked glycosylation site present on endogenous human EPO and CHO‐derived rhEPO. Copyright © 2013 John Wiley & Sons, Ltd.