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

J Am Soc Mass Spectrom. 2008 Nov;19(11):1643-54. doi: 10.1016/j.jasms.2008.07.004. Epub 2008 Jul 16.

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).

Publication types

  • Comparative Study
  • Evaluation Study

MeSH terms

  • Chromatography, High Pressure Liquid / methods*
  • Glycosylation
  • Humans
  • Immunoglobulin Fc Fragments / analysis*
  • Immunoglobulin Fc Fragments / metabolism
  • Immunoglobulin G / analysis*
  • Immunoglobulin G / metabolism
  • Polysaccharides / metabolism
  • Recombinant Proteins / analysis
  • Recombinant Proteins / metabolism
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Trypsin / metabolism

Substances

  • Immunoglobulin Fc Fragments
  • Immunoglobulin G
  • Polysaccharides
  • Recombinant Proteins
  • Trypsin