title: Glycosylation characterization of recombinant human erythropoietin produced in glycoengineered Pichia pastoris by mass spectrometry.
abstract: 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.
authors: GlycoFi, Biologics Discovery, Merck & Co., Inc., 16 Cavendish Court, Lebanon, NH, 03766, USA.
authors: Bing
authors: B
authors: Gong
authors: Irina
authors: I
authors: Burnina
authors: Terrance A
authors: TA
authors: Stadheim
authors: Huijuan
authors: H
authors: Li
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D060847:Metabolic Engineering; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011994:Recombinant Proteins; D004921:Erythropoietin
keywords: N-linked glycosylation; O-linked glycosylation; Pichia pastoris; mass spectrometry; rhEPO