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

• Published in: Analytical and Bioanalytical Chemistry 2024, Vol.416 (5), p.1281
• Main Authors: Gritti, Fabrice, Meyyappan, Sornanathan
• Format: Report
• Language: English
• Subjects: Analysis; Gel permeation chromatography; Methods; Monoclonal antibodies
• ISSN: 1618-2642
• DOI: 10.1007/s00216-023-05119-2

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 omitted] 150 mm coated hardware, 1.7 [Formula omitted]m 250 BEH [Formula omitted] Surface-modified Particles) and systems (ACQUITY [Formula omitted] UPLC [Formula omitted] I-class Bio Plus). The main issue still encountered is a persistent tail of the mAb peak. Here, the physical origin(s) of such peak tailing in size-exclusion chromatography (SEC) are investigated from both fundamental and practical approaches. Up to five relevant physical origins are analyzed: sample heterogeneity (glycoforms), UHPLC system dispersion, strong residual binding of the mAb to the SEC particles (via hydrophobic and/or electrostatic interactions) and to the stainless steel column/system hardware, slow escape kinetics of the mAb from the SEC particles, and flow heterogeneity caused by the non-ideal slurry packing of SEC columns. Experiments (testing sample heterogeneity, system dispersion, and strong residual interactions) and calculations (predicting the transient absorption/escape kinetics in a single SEC particle and the two-dimensional peak concentration profiles) altogether unambiguously demonstrate that the observed mAb peak tailing is caused primarily by the long-range velocity biases across the SEC column combined with the slow transverse dispersion of mAbs. Therefore, improvement in the resolution between mAb and sub-unit fragment impurities can only be achieved by increasing the column length, e.g., by applying recycling chromatography at acceptable pressures. Graphical abstract