Purification of Advanced Therapeutics by Flow‐Through Pseudo‐Affinity Chromatography: Bi‐Specific mABs, Fc Fusion Proteins, and Adeno‐Associated Viruses

ABSTRACT Modern biopharmaceutical manufacturing requires purification platforms capable of processing structurally and functionally diverse products while addressing the challenge of removing persistent and high‐risk host cell proteins (HCPs). Current purification processes utilize adsorbents that operate in bind‐and‐elute mode, which limits productivity, and often struggle to remove critical impurities. Addressing these limitations, our team developed a flow‐through pseudo‐affinity chromatography platform that utilizes size‐exclusion resins (Monomix Core 500) functionalized with mixed‐mode peptide ligands (AviGuard) to remove process‐related impurities while maximizing productivity and product integrity. This study evaluates this technology across three therapeutic modalities: two bi‐specific monoclonal antibodies (bsAbs) and an Fc‐fusion protein from Chinese hamster ovary (CHO) cell culture fluids, and adeno‐associated virus serotypes 2 and 9 (AAV2 and AAV9) from HEK293 cell lysates. For the CHO‐derived proteins, the platform comprised a pre‐capture step with LigaGuard‐Monomix Core 500 resin (LGMC500), a Protein A‐based capture step, and a polishing step using a single‐use size‐exclusion mixed‐mode (SEMM) resin. This configuration achieved global product yields exceeding 70%, final product pool concentration > 15 mg/mL, monomeric purity ~99%, and excellent cumulative HCP clearance (LRV > 4.5) to residual levels of 60–165 ng/mL (4–11 ppm). For AAV purification, the process utilized a mixed‐bed adsorbent comprising dextran‐coated charcoal and LGMC500 for removing HCPs, cell metabolites, and media components, followed by a capture step using either CaptureSelect AAVX resin or Natrix CH membranes. The optimization of the charcoal‐to‐resin volume ratio (1:3) and load volume (~ 10 14 vp per mL of resin) yielded ~50% AAV recovery with a residual HCP level of 350 ng/mL, representing < 100 ng per dose, in compliance with regulatory guidelines. Process productivity ranged from 19 to 23 g/L·hr for CHO‐derived proteins and ~7 × 10 14 vp/L·hr for AAV9, positioning this technology in the optimal operating space for modern bioprocessing.