Physical origin of the peak tailing of monoclonal antibodies in size-exclusion chromatography using bio-compatible systems and columns.

This study focuses on physical origin of the peak tailing of monoclonal antibodies in size-exclusion chromatography using bio-compatible systems and columns.. The research employs high-performance liquid chromatography (HPLC) techniques to address analytical challenges in the biopharmaceutical field. The analysis of mixtures containing monoclonal antibody (mAb) (approximately 150 kDa molecular weight) and sub-unit impurities (approximately 100 kDa) is challenging, even when adopting the latest ultra-high-pressure liquid chromatography (UHPLC) columns (4.6 mm [Formula: see text] 150 mm coated hardware, 1.7 [Formula: see text]m 250 BEH[Formula: see text] Surface-modified Particles)... Research Background and Significance Monoclonal antibodies (mAbs) are a cornerstone of modern biopharmaceutical therapeutics, necessitating robust analytical techniques to ensure product quality, safety, and efficacy. Size-exclusion chromatography (SEC) is widely employed in the characterization of mAbs to separate monomeric forms from aggregates and sub-unit impurities based on molecular size. However, peak tailing—a phenomenon where chromatographic peaks exhibit asymmetry with elongated trailing edges—presents significant challenges in accurate quantification and resolution. This study by Gritti and Meyyappan addresses the physical origins of peak tailing in mAb analysis using bio-compatible SEC systems and columns, emphasizing the impact on analytical performance. Understanding and mitigating peak tailing is critical for reliable biopharmaceutical characterization, especially when working with large proteins (~150 kDa) and closely related impurities (~100 kDa). Experimental Design and Methodology The investigation employed state-of-the-art ultra-high-pressure liquid chromatography (UHPLC)