Size-Exclusion Ultra Performance Liquid Chromatography Method Development for the Analysis of the Degradation Products of the Trastuzumab Antibody

■ ■ Robust method development to facilitate a successful SE-UPLC method transfer IN T RO DU C T IO N Size-exclusion chromatography (SEC) has been the predominant technique for the analysis of biotherapeutic protein aggregation, or high molecular weight forms (HMW), an important degradation pathway that has been found to correlate with undesired immunogenic effects and/or decreased efficacy.1,2 An additional degradation pathway for monoclonal antibodies (mAb) that can be observed by SEC is non-enzymatic peptide bond hydrolysis in the hinge region of these proteins resulting in antibodies that are missing one or both FAb arms.3 Identification of these low molecular weight degradants (LMW) are provided in a previous Waters Application Note 720004254EN: “Analysis of Proteins by Size-Exclusion Chromatography Coupled with Mass Spectrometry Under NonDenaturing Conditions”.