Charge variant analysis of intact monoclonal antibody reference standards using microfluidic capillary zone...

Monoclonal antibodies (mAbs) are among the most common biotherapeutics, and their clinical applications and global markets have demonstrated increased growth during the last decade, which are expected to grow further due to the shift of therapeutic treatments from general to personalized approaches. As the number and complexity of mAb-based therapeutics grow, there is an urgent need for highly efficient and high-throughput analytical techniques to characterize them and ensure their safety, potency, stability, and efficacy both from a development and regulatory standpoints. A key component in ensuring the methods used for the characterization of antibody-based therapeutics can provide accurate information, with the potential to influence important pipeline decisions, is the availability of diverse commercially available reference standards with well-known and catalogued properties that can serve as benchmarks and validation for the quality assurance of biotherapeutics in development. In this study, we developed and optimized a microfluidic capillary zone electrophoresis electrospray ionization-mass spectrometry (CZE-ESI-MS) method, utilizing the high-mass range MS data acquisition, to characterize three intact mAb reference standards and their proteoforms under near-native conditions using short analysis times (<15 min), low sample amounts (500 picograms), and minimal sample preparation. The developed CZE-MS method identified a number of modifications at the intact mAb level, including relatively high abundance C-terminal lysine clipping (>95 %) and N-linked glycosylation (>99 %), as well as lower abundance modifications, such as deamidation (∼3-7 %) and N-glycan sialylation (<1.5 %). The developed technique is uniquely suited for thorough and reproducible characterization of mAb-based biotherapeutics in industrial settings.