Ken Cook

Trypsin is one of the most commonly used proteases in peptide mapping protocols because of its high level of specificity. However, trypsin alone is not always sufficient for full sequence coverage. In this article, the authors detail how trypsin was combined with chymotrypsin to overcome this, and the benefits of an automated platform.

Pepsin digestion and guanidine hydrochloride post-digestion can improve sequence coverage in antibody peptide mapping compared with trypsin digestion.

This instalment of “Perspectives in Modern HPLC” provides an overview of antibody–drug conjugates (ADCs) as a new class of biotherapeutics and describes their analytical characterization for quality assessment with examples from extensive applications libraries.

Comprehensive characterization of ADCs requires increasingly powerful approaches consisting of small- and large-molecule techniques.

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are heterogeneous and exist as variant mixtures of structurally similar molecules. The heterogeneity of monoclonal antibodies is revealed by charge-sensitive methods, such as ion exchange chromatography (IEX). Changes in charge profile can significantly impact the structure, stability, binding affinity, and efficacy of the biotherapeutic drug. It is therefore necessary to understand the profile of the drug so that variants are identified and controlled. This article describes advances in ion exchange column chemistries, elution buffers, and ultrahigh-pressure liquid chromatography (UHPLC) instruments to meet the needs for modern, robust analysis of charge variants in monoclonal antibodies and therapeutic proteins.