Potential Use of Supercharging Agents for Improved Mass Spectrometric Analysis of Monoclonal Antibodies and Antibody-Drug Conjugates.

The addition of supercharging (SC) reagents in electrospray ionization coupled mass spectrometry (ESI-MS) has demonstrated several advantages for protein analysis such as reduced required mass range of the instrument, narrowed charge-state distribution, increased sensitivity, and adduct suppression. The potential use of SC reagents to improve analyses of larger and complex protein molecules such as monoclonal antibodies and antibody-drug conjugates (ADCs) has not been previously reported. In this study, the effect of seven SC reagents (-nitrobenzyl alcohol (-NBA), dimethyl sulfoxide (DMSO), -nitroanisole (-NA), propane sultone (PS), ethylene carbonate (EC), propylene carbonate (PC), and sulfolane) on ESI-MS acquired spectra of deglycosylated, intact, and reduced trastuzumab and a vcMMAE-trastuzumab ADC was investigated under denaturing conditions.

The Wittig bioconjugation of maleimide derived, water soluble phosphonium ylides to aldehyde-tagged proteins.

Herein we disclose the transformation of maleimides into water-soluble tris(2-carboxyethyl)phosphonium ylides and their subsequent application in the bioconjugation of protein- and peptide-linked aldehydes. The new entry into Wittig bioconjugate chemistry proceeds under mild conditions and relies on highly water soluble reagents, which are likely already part of most biochemists' inventory.

Application of triple quadrupole mass spectrometry for the characterization of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are an inherently heterogeneous class of biotherapeutics, the development of which requires extensive characterization throughout. During the earliest phases of preclinical development, when synthetic routes towards the desired conjugate are being assessed, the main interest lies in the determination of the average drug-to-antibody ratio (DAR) of a given batch as well as information about different conjugation species. There has been a trend in mass spectrometry (MS)-based characterization of ADCs towards the use of high-resolving mass spectrometry for many of these analyses.

Particle-based N-linked glycan analysis of selected proteins from biological samples using nonglycosylated binders.

Glycosylation is one of the most common and important post-translational modifications, influencing both the chemical and the biological properties of proteins. Studying the glycosylation of the entire protein population of a sample can be challenging because variations in the concentrations of certain proteins can enhance or obscure changes in glycosylation. Furthermore, alterations in the glycosylation pattern of individual proteins, exhibiting larger variability in disease states, have been suggested as biomarkers for different types of cancer, as well as inflammatory and neurodegenerative diseases.