Comparison of LC and LC/MS Methods for Quantifying N-Glycosylation in Recombinant IgGs

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).

Graphical AbstractDeconvoluted mass spectra of IgG-Fc/2 generated from two different lots, (Lot a and Lot b) of the same recombinant monoclonal antibody. The peak labels refer to the various glycoforms identified based on the deconvoluted mass. MS quantitation based on the analysis of Fc/2 is accurate and gives results that are comparable to normal phase HPLC analysis of N-glycans.Figure optionsDownload full-size imageDownload high-quality image (111 K)Download as PowerPoint slide