Brian Rivera | Authors


Glycosylation Analysis Through Released N-Glycan Workflows

Post-translational modifications are potential critical quality attributes (pCQAs) routinely assessed in biotherapeutic development. Glycosylation is one of the most important attributes to assess because it affects protein function as well as antigen receptor binding. N-glycosylation of asparagine residues is the most common pCQA assessed during monoclonal antibody (mAb) therapeutic development. There are a few protocols to assess and quantitate N-glycans, but the most common approach is through an enzymatic release and labelling procedure, followed by separation and detection. This article demonstrates the method development considerations for sample preparation and chromatographic analysis of N-glycans of therapeutic mAbs.

High-Flow Weak Cation Exchange for Charge Variant Analysis

Within the broad scope of analytical techniques required to characterize a protein, chromatographic methods have shifted towards high-flow analyses that can drop development time significantly. However, fast analytical methods for charge heterogeneity have lagged in development because current column technologies are ultrahigh-pressure liquid chromatography (UHPLC)-incompatible. This article will demonstrate the development of a high-flow method for charge variant analysis made possible through a bioinert titanium column flow path.

Addressing the Challenges of Drug-to-Antibody Measurement

Antibody–drug conjugates (ADC) are an emerging pharmaceutical technology, with the potential of being the true “magic bullet”. However, these inherently complex biomolecules have unique analytical challenges. One is the determination of the drug-to-antibody ratio (DAR). The average DAR and the drug distribution need to be monitored and could determine the success of an ADC. This article discusses the chromatographic methods used to determine DAR, including hydrophobic interaction chromatography (HIC), reversed phase, and liquid chromatography–mass spectrometry (LC–MS).