Glycosylation characterization of recombinant human erythropoietin produced in glycoengineered Pichia pastoris by mass spectrometry.
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.
['Gong|Bing|B|', 'Burnina|Irina|I|', 'Stadheim|Terrance A|TA|', 'Li|Huijuan|H|']
D000818:Animals D016466:CHO Cells D006224:Cricetinae D003412:Cricetulus D004921:Erythropoietin D006801:Humans D060847:Metabolic Engineering D010843:Pichia D011994:Recombinant Proteins D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Pichia pastoris mass spectrometry rhEPO N-linked glycosylation O-linked glycosylation