In silico mediated workflow for rapid development of downstream processing: Orthogonal product-related impurity removal for a Fc-containing therapeutic

Abstract

Therapeutic formats derived from the monoclonal antibody structure have been gaining significant traction in the biopharmaceutical market. Being structurally similar to mAbs, most Fc-containing therapeutics exhibit product-related impurities in the form of aggregates, charge variants, fragments, and glycoforms, which are inherently challenging to remove. In this work, we developed a workflow that employed rapid resin screening in conjunction with an in silico tool to identify and rank orthogonally selective processes for the removal of product-related impurities from a Fc-containing therapeutic product. Linear salt gradient screens were performed at various pH conditions on a set of ion-exchange, multimodal ion-exchange, and hydrophobic interaction resins. Select fractions from the screening experiments were analyzed by three different analytical techniques to characterize aggregates, charge variants, fragments, and glycoforms. The retention database generated by the resin screens and subsequent impurity characterization were then processed by an in silico tool that generated and ranked all possible two-step resin sequences for the removal of product-related impurities. A highly-ranked process was then evaluated and refined at the bench-scale to develop a completely flowthrough two-step polishing process which resulted in complete removal of the Man5 glycoform and aggregate impurities with a 73% overall yield. The successful implementation of the in silico mediated workflow suggests the possibility of a platformable workflow that could facilitate polishing process development for a wide variety of mAb-based therapeutics.