Multimodal chromatography solutions for the removal of light chain impurity from a fab therapeutic

Abstract

The production of Fab therapeutics is frequently challenged by an imbalanced expression of light chains (LC) and heavy chains (HC), often resulting in LC‐associated impurities. In this work, we evaluated multimodal chromatography as an alternative to affinity chromatography for the removal of an LC impurity. A panel of multimodal CEX and multimodal AEX resins was screened using linear salt and pH gradients to assess Fab monomer and LC impurity selectivities. Product and impurity characterization was carried out using analytical size‐exclusion chromatography. Screening data provided insight into the relative contributions of hydrophobic and electrostatic interactions in the Fab and LC impurity selectivities across different multimodal ligands. A previously developed separability scoring metric (Fractional Peak Overlap) was employed to identify Eshmuno HCX and CMM HyperCel as the top resin candidates from the screening data. Finally, bench‐scale refinement was carried out to develop an Eshmuno HCX bind‐elute step with a 95%–100% recovery and 95%–98% purity of Fab monomer and a CMM HyperCel bind‐elute step with a 98%–99% recovery and 98% purity of Fab monomer. This work demonstrates the efficacy of this PD workflow and the utility of multimodal chromatography for removal of LC impurities from Fabs.