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).
Sinha
Sandipan
S
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas, USA.
Pipes
Gary
G
Topp
Elizabeth M
EM
Bondarenko
Pavel V
PV
Treuheit
Michael J
MJ
Gadgil
Himanshu S
HS
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000032:analysis* / Q000378:metabolism; D007074:Immunoglobulin G / Q000032:analysis* / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011134:Polysaccharides; D011994:Recombinant Proteins; D014357:Trypsin