Alain Beck

The impact of ionic strength, buffer capacity, and pH-response on the retention behavior and peak shape of mAb species characterization is evaluated for IEX-MS. The aim of the present study was to understand the impact of ionic strength, buffer capacity, and pH-response on the retention behavior and peak shape of mAb species.

Characterization of mAbs and related products requires the identification of chromatographic peaks with MS. However, the conventional salt- and pH-gradient elution techniques used in IEX are inherently incompatible with MS. Ammonium acetate- and ammonium carbonate-based mobile phase systems have been recently applied in IEX-MS, but the influence of the eluent composition on peak shape and retention has not been discussed nor studied systematically until now. The aim of the present study was to understand the impact of ionic strength, buffer capacity, and pH-response on the retention behaviour and peak shape of mAb species.

With this method, a single injection was sufficient to characterize the amino acid sequence with complete sequence coverage. In addition, glycosylation and drug-loaded peptides could be identified from MS/MS spectra. A drug-loaded peptide fragmentation mass spectra study yielded drug-specific fragments, which reinforced the confidence about the identifications. The results reveal the ability of the sheathless CZE–MS/MS method to characterize an ADC’s primary structure in a single experiment.

This is the final instalment in a series of four articles exploring topics that will be addressed at the HPLC 2016 conference in San Francisco, USA, from 19–24 June 2016.

As a result of advances in multilevel state-of-the-art mass spectrometry (MS) methods, combined with chromatographic and electrophoretic techniques, very precise characterization of biotherapeutics such as monoclonal antibodies (mAbs) and antibody drug conjugates (ADCs) is now possible. Until recently, however, these techniques were considered suitable only for research use. With the advent of robust and user-friendly solutions, these techniques are now amenable for routine use, as illustrated by examples of applications of the characterization of mAbs and ADCs. This is the fourth in a series of four articles exploring topics that will be addressed at the HPLC 2016 conference in San Francisco, from June 19 to 24.

Recent progress in high-resolution mass spectrometry (HRMS) and liquid chromatography–mass spectrometry (LC–MS) methods for the structural characterization of brentuximab vedotin and trastuzumab emtansine are presented.

An analytical methodology for the characterization of the primary structure of biotherapeutic proteins using sheathless CE–ESI-MS-MS instrumentation is presented. For the first time, complete sequence coverage can be achieved using a bottom-up proteomic approach from a single analysis of a tryptic digest. In a biosimilarity assessment, a single amino acid substitution was detected.