Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2008-11

AUTHORS

Sandipan Sinha, Gary Pipes, Elizabeth M. Topp, Pavel V. Bondarenko, Michael J. Treuheit, Himanshu S. Gadgil

ABSTRACT

High-performance liquid chromatography (LC) and liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-MS) methods with various sample preparation schemes were compared for their ability to identify and quantify glycoforms in two different production lots of a recombinant monoclonal IgG1 antibody. IgG1s contain a conserved N-glycosylation site in the fragment crystallizable (Fc) subunit. Six methods were compared: (1) LC/ESI-MS analysis of intact IgG, (2) LC/ESI-MS analysis of the Fc fragment produced by limited proteolysis with Lys-C, (3) LC/ESI-MS analysis of the IgG heavy chain produced by reduction, (4) LC/ESI-MS analysis of Fc/2 fragment produced by limited proteolysis and reduction, (5) LC/MS analysis of the glycosylated tryptic fragment (293EEQYNSTYR301) using extracted ion chromatograms, and (6) normal phase HPLC analysis of N-glycans cleaved from the IgG using PNGase F. The results suggest that MS quantitation based on the analysis of Fc/2 (4) is accurate and gives results that are comparable to normal phase HPLC analysis of N-glycans (6). More... »

PAGES

1643-1654

References to SciGraph publications

Identifiers

URI

http://scigraph.springernature.com/pub.10.1016/j.jasms.2008.07.004

DOI

http://dx.doi.org/10.1016/j.jasms.2008.07.004

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1020303650

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/18707900


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