Purification of Advanced Therapeutics by Flow-Through Pseudo-Affinity Chromatography: Bi-Specific mABs, Fc Fusion...

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 (~ 10¹⁴ 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 × 10¹⁴ vp/L·hr for AAV9, positioning this technology in the optimal operating space for modern bioprocessing.