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.

Trap for MAbs: Characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry

“…However, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) [7,8], ESI orthogonal-TOF [9,10] and ESI quadrupole [2,[11][12][13] mass spectrometers have been the instruments of choice for MAbs and macromolecules, largely due to the large m/z range of the TOF and high ion transmission efficiency of the quadrupole mass analyzers. Unfortunately, on-line RP-HPLC has not accompanied these previous studies of intact monoclonal antibodies.…”

The impact of mass spectrometry on the study of intact antibodies: from post-translational modifications to structural analysis

“…Early examples used MALDI-TOF MS or triple quadrupole instruments for such analyses; [16][17][18] however, the high m/z requirements make such experimental approaches challenging. Currently, analysis of intact proteins is generally in the realm of ESI-TOF-like instruments due to the accessibility of a suitable m/z range, i.e., high m/z, whereby the maximum m/z required is typically 5000 for denatured proteins ( Fig.…”

Indium Tin Oxide-Polyaniline Biosensor: Fabrication and Characterization

“…Temperature levels at this region, however, are too high for any biological element, such as antibodies, in the biosensor design. An antibody thermal stability study concluded that a heat treatment at 60ºC resulted in the cleavage of the antibody heavy and light chains and promoted the denaturation of the protein (18). For this reason, most biosensor operations are conducted in room temperature (~ 25ºC) (19,20).…”