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J Mass Spectrom. 2013 Dec;48(12):1308-17. doi: 10.1002/jms.3291.

Glycosylation characterization of recombinant human erythropoietin produced in glycoengineered Pichia pastoris by mass spectrometry.

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GlycoFi, Biologics Discovery, Merck & Co., Inc., 16 Cavendish Court, Lebanon, NH, 03766, USA.


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.


N-linked glycosylation; O-linked glycosylation; Pichia pastoris; mass spectrometry; rhEPO

[Indexed for MEDLINE]

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