Many protein modifications of monoclonal antibody (mAb) drugs have profound impact on their structural heterogeneity, function, efficacy, pharmacokinetic properties and safety. Such modifications are known as critical quality attributes (CQAs) of the mAb. Quality-by-Design (QbD) principles supported by the US Food and Drug Administration mandates a thorough understanding of these CQAs in the early stages of manufacturing. Implementation of QbD requires deployment of monitoring methods to assess the levels of these critical quality attributes at pre and post-development in addition to the QC environment.
Several biopharmaceutical companies have developed mass spectrometry-based methods that confirm and relatively quantify multiple CQAs of mAbs. This approach is commonly referred to as multi-attribute monitoring method (MAM). While some methods use a peptide mapping approach, most recently, mAb subunit level methods have made its way from biopharmaceutical characterization to process development and quality control as a viable approach that benefit from simple sample preparation and reduced data complexity. Subunit level analysis of mAb modifications has been successfully deployed for process development and optimization as well as QC release testing, and was initially developed on a QTof mass spectrometer platforms for rapid LC-MS analysis and monitoring CQAs such as glycosylation and methionine oxidation.
Fucosylation of N-glycan core GlcNAcs affects the potency of the mAb through antibodydependent cell-mediated cytotoxicity (ADCC), and is an important CQA to monitor during process development, optimization, product manufacturing and quality control. Here, we demonstrate the use of a streamlined LC-MS platform under compliant-ready informatics (UNIFI Scientific Information System) for Fc-core fucosylation and aglycosylation confirmation and monitoring their relative levels. In addition, we also performed instrument-to-instrument and day-to-day repeatability assessment to demonstrate that this method has the potential to be validated according to ICH guideline Q2R1.