A size-exclusion chromatography fingerprinting workflow for the development of flow-through polishing operations for mAbs derived from continuous precipitation processes

Abstract

In this work we present a workflow for developing two-step, flow-through polishing processes for monoclonal antibodies (mAbs). The approach is demonstrated using redissolved precipitates from three CHO-derived mAbs generated by a continuous, PEG/ZnCl₂-mediated precipitation capture process. Size Exclusion Chromatography (SEC) fingerprinting and a percent SEC clearance (PSC) metric are developed to enable simultaneous quantification of monomer yield and impurity removal during high-throughput screening and scale-down column studies. Batch slurry plate screens are used to evaluate multimodal anion exchange (MMA) resins and an activated carbon composite adsorber under varying pH and ionic strengths, assessing partition coefficients and PSC values against both low-molecular-weight (LMW) and high-molecular-weight (HMW) impurities. Top candidates were then assessed in single-column, higher-loading flow-through experiments using the redissolved precipitates as feeds. Activated carbon emerged as a highly effective first polishing step for LMW impurity removal under acidic, low-conductivity conditions, while MMA resins provided complementary LMW and HMW clearances in a subsequent flow-through step. The two-step processes achieved overall mAb recoveries of 80%–87%, reduced HMW species from >1.7% down to 1.1%, and decreased host-cell protein levels from >10,000 to <40 ppm for all three mAbs. SEC fingerprints showed the ability to identify orthogonal impurity removal opportunities between the two polishing materials, validating the screening methodology for a process devoid of bind-elute processing steps. This work demonstrates that SEC-based impurity profiling and PSC metrics can guide the development of flow-through polishing processes and offer a useful intensification strategy to alleviate DSP bottlenecks and reduce reliance on affinity capture.