Domain contributions to antibody retention in multimodal chromatography systems.

Although a platform process has been established for purification of antibodies, a deep, fundamental understanding of how these molecules interact with chromatography resins has yet to be developed. The increasing prevalence of antibody-related therapeutics and associated purification challenges further motivate research into these molecular level interactions. The objective of this work is to understand the nature (i.e. size and properties) of preferred protein-ligand binding regions for large, multi-domain molecules such as antibodies. In this work, three antibodies with pI 7.5-8.3 and varying hydrophobicity were enzymatically digested to create (Fab), Fab, and F fragments. Linear salt gradient chromatography experiments from 0 to 1M NaCl were carried out with the full mAbs and the fragments in several multimodal chromatography systems at pH 6. The retention of the constituent fragments was then compared to that of the mAb to gain insight into the relative importance of these different domains and the contribution of each domain to the binding of the full mAb in these systems. While some mAbs were dominated by contribution from the F constant region, others were primarily driven by the (Fab) interactions. The domain contributions for each mAb were connected to the unique distribution of surface charge and hydrophobicity using protein surface property maps. This work lays the foundation for identifying the key surface patches on large, multi-domain molecules that are important interaction sites in various multimodal systems. Finally, this work has important implications for the separation of product related variants as well as the design of complex therapeutics for biomanufacturability.