MONITORING OF IGG ANTIBODY THERMAL-STABILITY BY MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY AND MATRIX-ASSISTED LASER DESORPTION IONIZATION MASS-SPECTROMETRY/

Monitoring the stability of immunoglobulin G (IgG) type antibodies is a crucial analytical issue spanning a wide variety of immunological/bi otechnological studies, which includes the analysis of conjugated IgG' s for drug delivery. Capillary electrophoresis (CE) has proven valuabl e for the analysis of proteins and has the potential to separate and d etect native antibody components. An ideal complement to CE, which is capable of providing the desired detection specificity to provide spec ies identification information, is matrix-assisted laser desorption/io nization 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 anti body (BR96). Electropherograms of the antibody after up to 166 h of th ermal stress are presented; MALDI mass spectra of the stressed antibod y were acquired at the same time points. At 166 h, the percentage of i onization carried by the intact antibody molecular ions M(+), M(2+), e tc., had clearly decreased, while that due to additional ion species h ad significantly increased. Ions corresponding in mass to loss of one light chain, loss of an F-ab arm to yield an F-ab/c type fragment, and formation of separated heavy-chain and light-chain moieties were obse rved. Several of these fragments result from simple disulfide linkage disruption, In addition, species less in mass than common antibody sub units were also observed, demonstrating peptide as well as disulfide b ond cleavage. The observation that a small number of well-defined spec ies were formed during the study suggests that the cleavage induced by thermal stress is very site-specific within the IgG.