Rapid High Performance Liquid Chromatography methodologies for analytical characterization of biotherapeutic products

• Major developments over last 6 years (2019-2025) in rapid HPLC analysis of biotherapeutics reviewed • Topics addressed include chromatography equipment, column innovations, and advanced methodologies • Data analytics and software-driven method development have also been addressed • Case studies showcase rapid analysis of charge variants, size-variants, glycans, and virus particles • Challenges in implementing rapid HPLC for routine biotherapeutic analysis are discussed The last two decades have witnessed an increasing interest in rapid analytical methods amongst the biopharmaceutical industry. This interest has, in turn, resulted in advancements in analytical hardware, data analytics, and practices. In this article, we review major developments that have occurred over the last 6 years (2019-2025) in rapid chromatographic analysis of proteins, including monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), and other therapeutic proteins. Conventional high-performance liquid chromatography (HPLC) is vital for characterizing critical quality attributes (CQAs), but has limitations like long run times, manual handling, and low throughput. Recent developments in rapid HPLC have given a new direction to biopharmaceutical analysis by reducing analysis times from hours to minutes, while maintaining resolution and sensitivity. Key aspects of improvements that have been addressed in this review include chromatography equipment, column innovations, and advanced methodologies that have contributed towards the implementation of rapid HPLC methods in the biopharmaceutical industry. Furthermore, integrating process analytical technology (PAT) with rapid HPLC has made it possible to monitor CQAs in real-time, either inline or at-line, which is crucial for manufacturers engaged in continuous processing. The review also discusses data analytics and software-driven method development, which have significantly lowered experimental efforts and strengthened method reliability. The article focuses mainly on rapid HPLC-based analysis and does not include the mass spectrometry (MS) oriented faster analytical methods.