title: Monitoring of IgG antibody thermal stability by micellar electrokinetic capillary chromatography and matrix-assisted laser desorption/ionization mass spectrometry.
abstract: Monitoring the stability of immunoglobulin G (IgG) type antibodies is a crucial analytical issue spanning a wide variety of immunological/biotechnological studies, which includes the analysis of conjugated IgG's for drug delivery. Capillary electrophoresis (CE) has proven valuable for the analysis of proteins and has the potential to separate and detect native antibody components. An ideal complement to CE, which is capable of providing the desired detection specificity to provide species identification information, is matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Utilizing these two techniques we have developed an antibody examination procedure and monitored the degradation of an internalizing chimeric (human/mouse) monoclonal antibody (BR96). Electropherograms of the antibody after up to 166 h of thermal stress are presented; MALDI mass spectra of the stressed antibody were acquired at the same time points. At 166 h, the percentage of ionization carried by the intact antibody molecular ions M+, M2+, etc., had clearly decreased, while that due to additional ion species had significantly increased. Ions corresponding in mass to loss of one light chain, loss of an Fab arm to yield an Fab/c type fragment, and formation of separated heavy-chain and light-chain moieties were observed. Several of these fragments result from simple disulfide linkage disruption. In addition, species less in mass than common antibody subunits were also observed, demonstrating peptide as well as disulfide bond cleavage. The observation that a small number of well-defined species were formed during the study suggests that the cleavage induced by thermal stress is very site-specific within the IgG.
authors.affiliation: Bristol-Myers Squibb, Analytical Research & Development, Pharmaceutical Research Institute, Syracuse, New York 13221-4755, USA.
authors.forename: A J
authors.initials: AJ
authors.lastname: Alexander
authors.forename: D E
authors.initials: DE
authors.lastname: Hughes
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D004355:Drug Stability; D019075:Electrophoresis, Capillary; D006168:Guinea Pigs; D006358:Hot Temperature; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins