Analysis of human antibody IgG2 domains by reversed-phase liquid chromatography and mass spectrometry.
It has been well documented that papain cleaves an IgG1 molecule to release Fab and Fc domains; however, papain was found unable to release such domains from an IgG2. Here we present a new combinatory strategy to analyze the heterogeneity of the light chain (LC), single chain Fc (sFc), and Fab portion of the heavy chain (Fd) of an IgG2 molecule released by papain cleavage under mild reducing conditions. These domains were well separated on reversed-phase high performance liquid chromatography (RP-HPLC) and analyzed by in-line liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS). In addition, some modifications of these domains were revealed by in-line mass spectrometry, and confirmed by the peptide mapping on LC-MS/MS analysis. This same strategy was proven suitable for IgG1 molecules as well. This procedure provides a simplified approach for the characterization of antibody biomolecules by facilitating the detection of low-level modifications in a domain. In addition, the technique offers a new strategy as an identification assay to distinguish IgG2 molecules on RP-HPLC, by which highly conserved Fc domains remain at a constant retention time (RT) unique to its subisotype, while varying RTs of the light chain and the Fd distinguish the monoclonal antibody from other molecules of the same isotype based on the underlying characteristics of each antibody.
Yan
Boxu
B
Department of Analytical and Formulation Science, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, USA. byan@amgen.com
Eris
Tamer
T
Yates
Zac
Z
Hong
Robert W
RW
Steen
Sean
S
Kleemann
Gerd
G
Wang
Weichun
W
Liu
Jennifer L
JL
D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D053719:Tandem Mass Spectrometry / Q000379:methods*
D007074:Immunoglobulin G