A novel in vitro serum stability assay for antibody therapeutics incorporating internal standards.

Abstract

Antibody-based therapeutics have demonstrated remarkable therapeutic benefit, but their susceptibility to biotransformation and degradation in the body can affect their safety, efficacy, and pharmacokinetic/pharmacodynamic (PK/PD) profiles. In vitro stability assessments play a pivotal role in proactively identifying potential liabilities of antibody therapeutics prior to animal studies. Liquid chromatography-mass spectrometry (LC-MS)-based in vitro stability assays has been developed and adopted in the biopharmaceutical industry for the characterization of antibody-based therapeutics. However, these methodologies often overlook operational error and random variation during sample preparation and analysis, leading to inaccurate stability estimation. To address this limitation, we have developed an LC-MS-based in vitro serum stability assessment that incorporates two internal standards (ISs), National Institute of Standards and Technology monoclonal antibody (NISTmAb) and its crystallizable fragment (Fc), to improve assay performance. Our method involves three steps: incubation of antibody therapeutics along with an IS in biological matrices, affinity purification, and LC-MS analysis. The stability of 21 monoclonal or bispecific antibodies was assessed in serums of preclinical species using this method. Our results showed improved accuracy and precision of recovery calculations with the incorporation of ISs, enabling a more confident stability assessment even in the absence of biotransformation or aggregation. In vitro stability correlated with in vivo exposure, suggesting that this in vitro assay could serve as a routine screening tool to select and advance stable antibody therapeutic candidates for subsequent in vivo studies.