Accelerating Biotherapeutics Discovery: Blueprint for a Universal LC-MALDI-MS Offline Approach

Biotherapeutics are a prominent class of molecules used to treat a wide range of diseases, including cancer, infections, inflammatory and immune disorders. Advances in recombinant protein engineering have led to the development of therapeutic antibodies with multi-specific attributes and increasingly complex structural and functional formats. The discovery workflow involves high-throughput screening of critical quality attributes using liquid chromatography (LC) to optimize cell line development and facilitate drug candidate selection. However, several LC techniques, such as Size Exclusion Chromatography (SEC) and Hydrophobic Interaction Chromatography (HIC), are not directly compatible with Electrospray Ionization (ESI), hindering mass spectrometry (MS)-based identification of impurity peaks. Consequently, significant time and effort are often required to adapt these method conditions to ESI-MS, which slows down discovery efforts. Matrix-Assisted Laser Desorption Ionization (MALDI) is a surface-based ionization technique that offers an attractive alternative to ESI for high-throughput screening due to its rapid analysis times and resistance to buffer/matrix interferences. MALDI-MS is highly compatible with automated liquid handling tools for sample transfer and requires minimal sample volume. To address the increasingly complex needs and challenging timelines of next-generation biologics development, we have developed an LC-MALDI-MS offline approach for rapid peak identification using an LC-fraction collector and MALDI-TOF-MS. This streamlined approach is adaptable to multiple chromatographic techniques, regardless of their inherent MS compatibility, and is suitable for automation. The workflow is versatile and can be applied universally in combination with various chromatographic techniques and analytes, including monoclonal and bispecific antibodies, fusion proteins, and peptides. Importantly, fraction collection and MS data acquisition can be completed within hours without the need for additional method development or sample preparation for MS-based measurements. Herein, we systematically demonstrate the use of an automated workflow involving SEC, HIC, and Reversed Phase for proteins and peptides, as well as a use case with a bispecific antibody and fusion protein using SEC.