Mass spectrometric glycoform profiling of the innovator and biosimilar erythropoietin and darbepoetin by LC/ESI-MS

Harazono, Akira; Hashii, Noritaka; Kuribayashi, Ryosuke; Nakazawa, Shiori; Kawasaki, Nana

doi:10.1016/j.jpba.2013.04.031

Cited by 48 publications

(43 citation statements)

References 33 publications

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“…N38 and N83 sites of EPO seem more exposed than N24 sites, permitting selective introduction of two N- glycans at these two sites, and a different N- glycan at the N24 site. Previous site-specific glycosylation profiling of recombinant EPO indicated that the glycans attached at the N38 and N83 sites were mainly tetra-antennary glycans while that at the N24 site was a mixture of bi-, tri-, and tetra-antennary glycans 38 . The data also imply that the N38 and N83 sites might be more accessible for enzyme processing to give the fully processed and highly branched tetra-antennary N-glycans as the major glycoforms, while the N24 site might be less accessible for enzymatic processing to yield the less branched glycoforms.…”

Section: Resultsmentioning

confidence: 99%

Glycan Remodeling of Human Erythropoietin (EPO) Through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation

Yang

Zhu

et al. 2017

ACS Chem. Biol.

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The tremendous structural heterogeneity of N-glycosylation of glycoproteins poses a great challenge for deciphering the biological functions of specific glycoforms and for developing protein-based therapeutics. We have previously reported a chemoenzymatic glycan remodeling method for producing homogeneous glycoforms of N-glycoproteins including intact antibodies, which consist of endoglycosidase-catalyzed deglycosylation and novel glycosynthase-catalyzed transglycosylation, but its application to complex glycoproteins carrying multiple N-glycans remains to be examined. We report here site-selective chemoenzymatic glycosylation remodeling of recombinant human erythropoietin (EPO) that contains three N-glycans. We found that the generation of a HEK293S GnT I knockout FUT8 overexpressing cell line enabled the production of an unusual Man5GlcNAc2Fuc glycoform, which could be converted to the core-fucosylated GlcNAc-EPO intermediate acceptor for enzymatic transglycosylation. With this acceptor, homogeneous sialylated glycoform or azide-tagged glycoform were produced using the glycosynthase (EndoF3-D165A) catalyzed transglycosylation. Interestingly, a remarkable site-selectivity was observed in the transglycosylation reactions, leading to the introduction of two N-glycans selectively at the Asn-38 and Asn-83 sites, which was confirmed by a detailed MS/MS analysis of the transglycosylation product. Finally, a different N-glycan was attached at the third (Asn-24) site by pushing the enzymatic transglycosylation with a distinct glycan oxazoline, achieving the site-selective glycosylation modification of the protein. This study represents the first example of site-selective chemoenzymatic glycan engineering of complex glycoproteins carrying multiple N-glycans.

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Section: Resultsmentioning

confidence: 99%

Glycan Remodeling of Human Erythropoietin (EPO) Through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation

Yang

Zhu

et al. 2017

ACS Chem. Biol.

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“…However, a more thorough evaluation of biopharmaceutical glycosylation at all stages of drug development and production becomes obligatory, since glyco-engineering is emerging. This is demonstrated by the targeted insertion of additional glycosylation sites in darbepoetin alpha or alpha 1-antitrypsin [9][10][11], concurrent with increasing availability of cost-effective glyco-analytical methods.…”

Section: Current Methods In Glycosylation Analysis Of Biopharmaceuticalsmentioning

confidence: 99%

“…Starting with intact or reduced protein, LC-MS is a straightforward technique for profiling glycoforms in biopharmaceuticals, as applied in biosimilar research [10,44,45], clone selection [38], batch-to-batch comparison [46,47], or as routine check for molecular weight and heterogeneity in biopharma industry. LC-MS is commonly applied in reversed-phase (RP) or size-exclusion chromatography modes with ESI-TOF-MS detection [43,48].…”

Section: Lc-esi-ms and Lc-ms/msmentioning

confidence: 99%

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Mass spectrometry for glycosylation analysis of biopharmaceuticals

Dotz

Haselberg

Shubhakar

et al. 2015

TrAC Trends in Analytical Chemistry

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“…In retrospect, however, the inability to obtain a supportive mass spectrum of this material raises concerns with respect to the nature of the folded product. Related sialic acid-containing recombinantly derived EPO mixtures of unspecified structure gave rise to useful mass spectra in the context of glycoform profiling studies ([105, 106] and references therein). The corresponding author of reference [101] assumes full responsibility for not taking cognizance of these precedents.…”

Section: Chemical Synthesis Of Glycoproteinsmentioning

confidence: 99%

Total Synthesis of Glycosylated Proteins

Fernández‐Tejada

Brailsford

Zhang

et al. 2014

Topics in Current Chemistry

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Glycoproteins are an important class of naturally occurring biomolecules which play a pivotal role in many biological processes. They are biosynthesized as complex mixtures of glycoforms through post-translational protein glycosylation. This fact, together with the challenges associated with producing them in homogeneous form, has hampered detailed structure-function studies of glycoproteins as well as their full exploitation as potential therapeutic agents. By contrast, chemical synthesis offers the unique opportunity to gain access to homogeneous glycoprotein samples for rigorous biological evaluation. Herein, we review recent methods for the assembly of complex glycopeptides and glycoproteins and present several examples from our laboratory towards the total chemical synthesis of clinically relevant glycosylated proteins that have enabled synthetic access to full-length homogeneous glycoproteins.

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Mass spectrometric glycoform profiling of the innovator and biosimilar erythropoietin and darbepoetin by LC/ESI-MS

Cited by 48 publications

References 33 publications

Glycan Remodeling of Human Erythropoietin (EPO) Through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation

Glycan Remodeling of Human Erythropoietin (EPO) Through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation

Mass spectrometry for glycosylation analysis of biopharmaceuticals

Total Synthesis of Glycosylated Proteins

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