Comparison of LC and LC/MS methods for quantifying <Emphasis Type="Italic">N</Emphasis>-glycosylation in recombinant IgGs |
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Authors: | Sandipan Sinha Gary Pipes Elizabeth M Topp Pavel V Bondarenko Michael J Treuheit Himanshu S Gadgil |
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Institution: | Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas, USA. |
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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). |
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