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A platform for high-throughput molecular characterization of recombinant monoclonal antibodies

被引：32
作者：
Bailey, MJ
Hooker, AD
Adams, CS
Zhang, SH
James, DC [1 ]
机构：
[1] Univ Queensland, Sch Engn, St Lucia, Qld 4072, Australia
[2] Pfizer Analyt Res & Dev, Sittingbourne ME9 8AG, Kent, England
[3] Pfizer Global Res & Dev, St Louis, MO USA
来源：
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES | 2005年 / 826卷 / 1-2期
关键词：
IgG2; recombinant monoclonal antibody; glycosylation; glycopeptide; high-throughput characterization; normal phase liquid chromatography; MALDI-TOF MS;
D O I：
10.1016/j.jchromb.2005.08.021
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
We describe quantitative characterization of a sample preparation platform for rapid and high-throughput analysis of recombinant monoclonal antibodies (MAbs) and their post-translational modifications. MAb capture, desalting and in situ reduction/alkylation were accomplished by sequential adsorption of analyte to solid phase beads (protein A, reverse-phase) suspended in microtiter plate wells. Following elution and rapid tryptic digestion in the presence of acid-labile surfactant (RapiGest (TM)), peptides were fractionated by stepwise elution from reverse-phase pipet tips and the fraction containing Fc N-glycopeptides isolated. Direct quantitative analysis of the relative abundance of peptide glycoforms by MALDI-TOF MS in linear mode closely correlated with normal phase HPLC analysis of fluorophore labeled N-glycans released by PNGaseF. (c) 2005 Elsevier B.V. All rights reserved.
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页码：177 / 187
页数：11
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