Open AccessComparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs
Author(s) -
Sandipan Sinha,
Gary D. Pipes,
Elizabeth M. Topp,
Pavel V. Bondarenko,
Michael J. Treuheit,
Himanshu S. Gadgil
Publication year - 2008
Publication title -
journal of the american society for mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.961
H-Index - 127
eISSN - 1879-1123
pISSN - 1044-0305
DOI - 10.1016/j.jasms.2008.07.004
Subject(s) - chemistry , chromatography , glycosylation , pngase f , electrospray , electrospray ionization , mass spectrometry , high performance liquid chromatography , glycan , reversed phase chromatography , liquid chromatography–mass spectrometry , fragment crystallizable region , recombinant dna , biochemistry , glycoprotein , receptor , gene
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).