Peptide mapping of therapeutic monoclonal antibodies: Improvements for increased speed and fewer artifacts

Dick, Lawrence W.; Mahon, David; Qiu, Difei; Cheng, Kuang-Chuan

doi:10.1016/j.jchromb.2008.12.009

Cited by 69 publications

(45 citation statements)

References 30 publications

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“…32,33 The peptide map method could also be used as an alternative or be combined with an oligosaccharide mapping method to acquire site-specific information for proteins with multiple glycosylation sites. For complicated sample preparation, we might treat them with trypsin combined with other proteolytic enzymes to digest the protein into peptide fragments that were amenable to analysis by LC-MS.…”

Section: Resultsmentioning

confidence: 99%

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

Zhu

Guo

Guo³

et al. 2014

mAbs

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These authors contributed equally to this work.Keywords: characterization, CTLA4-Ig fusion protein, glycan, glycosylation modification, intact protein, mass spectrometry, peptide mapping, similarity CTLA4-Ig is a highly glycosylated therapeutic fusion protein that contains multiple N-and O-glycosylation sites. Glycosylation plays a vital role in protein solubility, stability, serum half-life, activity, and immunogenicity. For a CTLA4-Ig biosimilar development program, comparative analytical data, especially the glycosylation data, can influence decisions about the type and amount of animal and clinical data needed to establish biosimilarity. Because of the limited clinical experience with biosimilars before approval, a comprehensive level of knowledge about the biosimilar candidates is needed to achieve subsequent development. Liquid chromatography-mass spectrometry (LC-MS) is a versatile technique for characterizing N-and O-glycosylation modification of recombinant therapeutic proteins, including 3 levels: intact protein analysis, peptide mapping analysis, and released glycans analysis. In this report, an in-depth characterization of glycosylation of a candidate biosimilar was carried out using a systematic approach: N-and O-linked glycans were identified and electron-transfer dissociation was then used to pinpoint the 4 occupied O-glycosylation sites for the first time. As the results show, the approach provides a set of routine tools that combine accurate intact mass measurement, peptide mapping, and released glycan profiling. This approach can be used to comprehensively research a candidate biosimilar Fc-fusion protein and provides a basis for future studies addressing the similarity of CTLA4-Ig biosimilars.

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Section: Resultsmentioning

confidence: 99%

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

Zhu

Guo

Guo³

et al. 2014

mAbs

View full text Add to dashboard Cite

show abstract

“…For the purpose of product characterization, results from peptide mapping need to be interpreted with caution as artifacts during lengthy digestion could occur and lead to incorrect conclusions. [7][8][9][10][11] Another important issue to consider is that any correlation between modifications of different sites can be lost after the protein is cleaved into small peptides.…”

Section: Introductionmentioning

confidence: 99%

A new tool for monoclonal antibody analysis

Zhang

Mueller

et al. 2014

mAbs

131

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“…Deamidation is, in general, accelerated at high temperatures and high pH values. 6 One way to minimize the degree of induced deamidation is to lower the pH of the digestion buffer. SMART Digest is performed at elevated temperatures but at a pH of 7.2, which is much lower than the pH of classical in-solutiondigestion methods.…”

Section: Resultsmentioning

confidence: 99%