Abstract
High-performance liquid chromatography (HPLC) remains the cornerstone of analytical characterization in the biopharmaceutical industry, despite the proliferation of newer orthogonal and hyphenated techniques. The rapid expansion of biologics including monoclonal antibodies, peptides, oligonucleotides, viral vectors, and antibody–drug conjugates has necessitated substantial advances in chromatographic science. Innovations in column chemistry, encompassing superficially porous particles, hybrid organic–silica materials, and monolithic supports, have enhanced the resolution, sensitivity, and speed of biomolecule separations. Parallel to these material advancements, the integration of automation, artificial intelligence (AI), and advanced data analytics has transformed chromatographic workflows into highly intelligent, self-optimizing systems. Modern HPLC instruments are now embedded within digital ecosystems that comply with regulatory expectations, promote sustainability, and align with the principles of Quality-by-Design (QbD) and Process Analytical Technology (PAT). This review provides an in-depth analysis of the evolution of HPLC in the biopharmaceutical era, focusing on developments in column design, mobile-phase optimization, detection systems, and digital transformation. Furthermore, it discusses how emerging paradigms such as green chromatography, AI-driven decision support, and continuous analytics are redefining the future of pharmaceutical quality assurance. By integrating scientific innovation with data-driven intelligence, HPLC continues to serve as an indispensable analytical platform in biopharmaceutical discovery, process development, and regulatory compliance.