Enhancing Biotherapeutic Characterization With MALDI Mass Spectrometry

From Discovery to Development: Enhancing Biotherapeutic Characterization With MALDI-MS Enhancing Biotherapeutic Characterization With Advanced Mass Spectrometry Biotherapeutics, including engineered proteins and therapeutic antibodies, are vital in treating diseases such as cancer and immune disorders. With advancements in recombinant protein engineering, multi-specific therapeutic candidates have emerged, necessitating rigorous quality characterization to optimize development workflows. Traditional analytical methods, such as Size Exclusion Chromatography (SEC) and Hydrophobic Interaction Chromatography (HIC), often present challenges for mass spectrometry (MS) compatibility, particularly with Electrospray Ionization (ESI), which can impede rapid impurity identification. To overcome these hurdles, a streamlined LC-MALDI-MS offline approach that leverages Matrix-Assisted Laser Desorption Ionization (MALDI) for high-throughput screening was developed. This method facilitates efficient peak identification using an automated liquid chromatography fraction collector and MALDI-TOF-MS, allowing comprehensive analysis of various biotherapeutics, including monoclonal and bispecific antibodies, fusion proteins, peptides, and cytokines. Further, many of these cytokines are engineered to serve as promising immuno-oncology therapies, yet their structural complexity poses analytical challenges. A recently developed MALDI-MS platform effectively addresses these challenges, integrating seamlessly with existing risk assessment workflows for biopharmaceutical development. This orthogonal technique enables rapid characterization of molecular degradation in engineered PEGylated cytokine formulations, streamlining the pathway to clinical application. To expedite peptide variant selection regarding cell membrane permeability, we developed a rapid high-throughput label-free methodology, Cell-based Approach Membrane Permeability Assay (CAMPA) based on the MALDI-hydrogen-deuterium exchange mass spectrometry (MALDI-HDX-MS) approach to rank-order peptide cell membrane permeability using live cancer cell cultures (THP1, AsPc1 and CACO2). A differential hydrogen-deuterium exchange approach was used to distinguish the peptides outside of the cells from those inside. This new workflow has allowed us to assess target-agnostic cell-membrane permeability of peptides in a fast, robust, high-throughput and label-free manner. The systematic exploration of automated workflows demonstrates the capability of this platform to meet the evolving demands of the pharmaceutical industry in characterizing complex biotherapeutics efficiently and effectively. Biotherapeutics such as monoclonal antibodies, fusion proteins, peptides and cytokines are increasingly complex, creating challenges for rapid and accurate quality characterization. Traditional analytical methods often struggle with mass spectrometry (MS) compatibility, slowing impurity identification and structural analysis. This webinar introduces a refined matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS workflow that streamlines analysis, enables high-throughput screening and integrates with existing development pipelines. Attendees will gain insights into approaches that address structural complexity, enhance analytical efficiency and support faster decision-making in biotherapeutic development.