Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs.

Journal of the American Society for Mass Spectrometry
Sandipan SinhaHimanshu 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).

References

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Citations

Jul 20, 2010·Analytical and Bioanalytical Chemistry·Michael MelmerAndreas Premstaller
Apr 15, 2010·Journal of Clinical Immunology·Johannes StadlmannFriedrich Altmann
Mar 28, 2013·Chemical Reviews·William R AlleyMilos V Novotny
Mar 26, 2014·Journal of the American Society for Mass Spectrometry·Bhavana ShahZhongqi Zhang
Feb 3, 2011·Journal of Pharmaceutical Sciences·Amanda K MillerHimanshu S Gadgil
Jul 26, 2011·Journal of Pharmaceutical Sciences·Steven D StroopScott D Leigh
Oct 30, 2015·Journal of Proteome Research·Qiuting HongCarlito B Lebrilla
Nov 10, 2015·Proteomics. Clinical Applications·Yuting CongJingkai Gu
Jul 15, 2011·Journal of Pharmaceutical and Biomedical Analysis·Song C KlapoetkeSteven Becht
May 15, 2012·Journal of Pharmaceutical and Biomedical Analysis·Ryosuke KuribayashiNana Kawasaki
Aug 21, 2016·Analytica Chimica Acta·Giorgio Gianini MorbioliEmanuel Carrilho
Jul 28, 2016·Clinica Chimica Acta; International Journal of Clinical Chemistry·Joannes F M JacobsMonique van Scherpenzeel
Jun 22, 2011·British Journal of Haematology·Roberto F MachadoMark T Gladwin
Jan 18, 2018·Biotechnology Journal·Tianhua WangPauline M Rudd
Mar 6, 2019·Protein Science : a Publication of the Protein Society·Seiji SaitoShigeru Iida
Mar 31, 2019·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·María L CeruttiMauricio Seigelchifer
Nov 2, 2019·Journal of Pharmaceutical and Biomedical Analysis·Julien CamperiNathalie Delaunay
May 8, 2018·Journal of the American Society for Mass Spectrometry·Vera B IvlevaQ Paula Lei
Nov 10, 2010·Analytical Chemistry·Yang Shen, Hongcheng Liu

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