Characterization of lower molecular weight artifact bands of recombinant monoclonal IgG1 antibodies on non-reducing SDS-PAGE.
SDS-PAGE under non-reducing conditions is one of the most commonly used techniques for recombinant monoclonal antibody purity and stability indicating assay. On non-reducing SDS-PAGE, bands with a lower molecular weight than the intact antibody are routinely observed and is a common feature of IgG molecules. These fragments were analyzed by in-gel digestion followed by matrix-assisted-laser-desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry, Western blot and by comparing the banding pattern of sample prepared in the presence of a reducing reagent. The fragments bands were identified as antibody lacking one light chain, two heavy chains, one light chain and one heavy chain, free heavy chain and free light chain. Sensitivity of fragmentation to sample buffer pH, incubation time, reducing reagent and alkylation reagents indicated that fragments were formed during sample preparation, but not present in the samples analyzed. Disulfide bond scrambling and beta-elimination are the two major mechanisms of the formation antibody fragments. Mass spectrometry analysis suggested that disulfide bond scrambling can be prevented by specifically modifying free sulhydryl using alkylation and thus reduced the amount of artifacts on non-reducing SDS-PAGE. Breakage of disulfide bonds by beta-elimination was evidenced by the detection of dehydroalanine using mass spectrometry.
Liu
Hongcheng
H
Protein Analytics, Process Sciences Department, Abbott Bioresearch Center, Worcester, MA, 01605, USA. hongcheng.liu@abbott.com
Gaza-Bulseco
Georgeen
G
Chumsae
Chris
C
Newby-Kew
Abigail
A
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification*; D007147:Immunoglobulin Light Chains / Q000302:isolation & purification; D007142:Immunoglobulin gamma-Chains / Q000302:isolation & purification; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007147:Immunoglobulin Light Chains; D007142:Immunoglobulin gamma-Chains; D011994:Recombinant Proteins