A typical protein purification process involves a chromatography capture step, several filtration steps, and one to two polishing chromatography steps. The polishing steps in downstream biologics purification are critical for achieving final product quality and ensuring robustness in manufacturing. Its integration must balance impurity clearance, process stability, and scalability—particularly when rapid development timelines and complex molecules are involved. In this study, a polishing step was redesigned during commercial process development to address stability, manufacturing, and cell line-specific challenges associated with a highly potent, multi-specific molecule. This molecule exhibits dynamic, reversible aggregation behavior dependent on pH, conductivity, and concentration. The initial purification processes required impractical 5–7× fold product dilutions and offered limited intermediate hold times, creating barriers to manufacturing scale-up. Evaluations of the cation exchange (CEX) step of the first generation purification process showed significant product loss and aggregation due to the molecule’s sensitivity to the high pH and conductivity conditions required. Additionally, changing cell lines resulted in an increased level of residual host cell proteins (rHCP) and lipase which leads to a polysorbate 20 (PS-20) degradation liability. The new polishing step was designed to achieve three core objectives: (1) to clear lipase and residual host cell proteins, (2) to enable operation within a stable range (pH 5–6, conductivity <5 mS/cm) thus reducing reversible aggregation, and (3) to eliminate excessive product dilutions to support scalable clinical and commercial manufacturing. Process development of this polishing step considered two hydrophobic interaction chromatography (HIC) resins and an anion exchange (AEX) based chromatographic depth filter. The selected HIC resin enabled the most effective separation via frontal chromatography, driven by stronger hydrophobic interactions between impurities and resin matrix allowing the product to flow through with minimal impact on dimerization. Neutralized Protein A Pool (NPAP) was used to simulate worst-case impurity profiles and evaluate step performance under high-stress conditions. The optimized polishing column process was robust across all-studied range of loadings, achieved a 35.9% reduction in rHCP, a 74.3% reduction in lipase activity, and did not induce additional reversible aggregation. These results demonstrate that the redesigned polishing step significantly improves product quality, enables operational feasibility, and aligns with internal platform standards—supporting successful tech transfer and clinical manufacturing across two facilities on an accelerated timeline.
