Complementary MS Methods Assist Conformational Characterization of Antibodies with Altered S–S Bonding Networks

Abstract

As therapeutic monoclonal antibodies (mAbs) become a major focus in biotechnology and a source of the next-generation drugs, new analytical methods or combination methods are needed for monitoring changes in higher order structure and effects of post-translational modifications. The complexity of these molecules and their vulnerability to structural change provide a serious challenge. We describe here the use of complementary mass spectrometry methods that not only characterize mutant mAbs but also may provide a general framework for characterizing higher order structure of other protein therapeutics and biosimilars. To frame the challenge, we selected members of the IgG2 subclass that have distinct disulfide isomeric structures as a model to evaluate an overall approach that uses ion mobility, top-down MS sequencing, and protein footprinting in the form of fast photochemical oxidation of proteins (FPOP). These three methods are rapid, sensitive, respond to subtle changes in conformation of Cys→Ser mutants of an IgG2, each representing a single disulfide isoform, and may be used in series to probe higher order structure. The outcome suggests that this approach of using various methods in combination can assist the development and quality control of protein therapeutics.

Native MS and ECD Characterization of a Fab–Antigen Complex May Facilitate Crystallization for X-ray Diffraction

“…This approach was previously used for mAbs [13, 37, 38], where the goal was to provide high sequence coverage. Our strategy is to keep together the complex by native MS to provide binding stoichiometry, flexible-region location, and even partial sequence, which cannot be obtained by denaturing ESI.…”

Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting

“…Hydrogen peroxide (H 2 O 2 ) (30%), formic acid, PBS, L-glutamine, and L-methionine were obtained from Sigma-Aldrich. The FPOP labeling protocol was modified from that previously described (52,53,(66)(67)(68)(69). The FPOP method uses a KrF excimer laser to generate 248-nm wavelength laser pulses to photolyze molecular H 2 O 2 into •OH radicals that covalently modify solvent-exposed side chains of amino acids (described in refs.…”

Integrated Microfluidic Capillary Electrophoresis-Electrospray Ionization Devices with Online MS Detection for the Separation and Characterization of Intact Monoclonal Antibody Variants

“…Individually, these modifications can introduce a significant amount of heterogeneity in the final product, and it is likely that mixtures of multiple modifications will coexist in finished products that may ultimately increase upon storage or degradation. 1,4,7,8 Consequently, it is critical to characterize such molecules from the amino acid sequence to structural conformation to ensure clinically significant bioactivity, drug effectiveness, and quality. Common techniques used to analyze mAbs include chromatographic methods such as size exclusion chromatography, ion-exchange chromatography, reversed-phase liquid chromatography, and electrophoresis based methods such as isoelectric focusing, gel electrophoresis, and capillary zone electrophoresis.…”