ABSTRACT Monoclonal antibodies and other biotherapeutics are subject to a variety of modifications that can impact activity and stability and therefore must be analyzed as part of QC and comparability. Mass spectrometry (MS) has become a workhorse for biopharmaceutical analytical laboratories due to its ability to detect protein modifications at a molecular level. Over the past few years, the Multi-Attribute Method (MAM) has gained traction throughout pharmaceutical development and QC labs, with several developers implementing some form of MAM in characterization or release. While replacing multiple QC tests provides an opportunity to streamline lab work and decrease development time and post-approval costs, several challenges remain. While some large biopharma companies are implementing MAM in QC, MAM is not as commonly used in biosimilar and small biopharma companies. This proposal addresses one of the key areas of consideration for implementation of MAM in QC as outlined in a 2019 publication from FDA staff: the performance of MAM vs conventional methods. Collecting data to support transitioning from conventional techniques to MAM is a significant investment that can prevent or delay development of biosimilars. The objective of this work is to assess the performance of the MS-based MAM versus conventional QC methods to identify changes in product quality attributes (PQAs) upon forced degradation and to correlate changes in those PQAs with bioactivity, binding affinity, and structure. Results of this study will help support transitioning from conventional techniques to MAM by creating a knowledge base that can lower the barrier to adoption of MAM and enable wider use of MAM by biosimilar manufacturers. The work proposed here will assess and compare PQAs of a monoclonal antibody (adalimumab) and Fc fusion protein (etanercept) acquired from three different sources using both conventional QC methods and MAM-based approaches. Sensitivity of each technique to changes during forced degradation will be evaluated and compared to functional and structural changes detected using cell-based assays, binding assays and circular dichroism. We propose to first conduct forced degradation studies of adalimumab and etanercept from multiple sources (Specific Aim 1). PQAs will be evaluated using conventional methods to assess charge variants, glycosylation, and size variants, (Specific Aim 2) and compared to the specific molecular modifications detected using an MS-based MAM workflow (Specific Aim 3) to assess comparability and sensitivity to changes in PQAs. Lastly, under Specific Aim 4, we will assess the bioactivity and structure of biotherapeutic products exhibiting significant differences in PQAs to correlate molecular changes to changes in structure and function.