Fuente: Biomolecules - Revista científica (MDPI) Biomolecules, Vol. 16, Pages 785: Multimodal Anion-Exchange Resins for Chromatographic Separation of Antibody Aggregates
Biomolecules doi: 10.3390/biom16060785
Authors:
Simona Kotuličová
Tomáš Molnár
Milan Polakovič

Efficient protein aggregate removal remains a major challenge in downstream bioprocessing because high aggregate clearance must be achieved without compromising monomer yield. Mixed-mode chromatography (MMC) has emerged as a promising approach, offering enhanced selectivity through combined ionic and hydrophobic interactions and salt-tolerant behavior. However, the relative roles of matrix pore accessibility and ligand density remain insufficiently understood. In this study, MMC adsorbents based on 4% and 6% agarose matrices were functionalized with a BMEA ligand. Inverse size-exclusion chromatography revealed that functionalization caused matrix syneresis, increasing dry matter content to 23% and enhancing mechanical rigidity. MMC-Ag4, with a larger mean pore radius (19.1 nm), exhibited a selectivity factor of 2 toward aggregates in static binding experiments, whereas the denser MMC-Ag6 (15.7 nm) showed no selectivity. In column studies using a feed containing 10% aggregates, MMC-Ag4 outperformed the commercial benchmark Capto Adhere, achieving monomer yields of 80–90% at 97–98% purity with salt tolerance up to 300 mM NaCl. These findings indicate that while MMC-Ag6 is limited by pore blockage, the optimized pore accessibility of MMC-Ag4 enables effective aggregate recognition. In conclusion, multimodal adsorbent design must balance ligand density with matrix porosity to ensure high resolution and yield in aggregate removal.