Flow-through pseudo-affinity chromatography platform purifies bi-specific antibodies, Fc-fusion proteins, and AAVs with high yield and impurity clearance
Background
Efficient purification of advanced biotherapeutics, including bi-specific monoclonal antibodies (bsAbs), Fc-fusion proteins, and adeno-associated viruses (AAVs), is critical for manufacturing. Current bind-and-elute chromatography methods often limit productivity and struggle to adequately remove host cell proteins (HCPs) and other process-related impurities, posing significant challenges for regulatory compliance and product safety. A need exists for purification platforms that can handle diverse product types while maximizing throughput and product integrity.
Study Design
Researchers developed a flow-through pseudo-affinity chromatography platform using Monomix Core 500 resins functionalized with mixed-mode peptide ligands (AviGuard). This technology was evaluated across three therapeutic modalities: two bi-specific monoclonal antibodies 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 CHO-derived proteins, the platform included a pre-capture step with LigaGuard-Monomix Core 500 resin (LGMC500), a Protein A capture, and a polishing step with a single-use SEMM resin. AAV purification utilized a mixed-bed adsorbent of dextran-coated charcoal and LGMC500, followed by CaptureSelect AAVX resin or Natrix CH membranes for capture. The charcoal-to-resin volume ratio was optimized to 1:3, with a load volume of ~10^14 vp per mL of resin.
Results
The flow-through pseudo-affinity chromatography platform demonstrated robust performance across diverse biotherapeutics. For CHO-derived proteins (bsAbs and Fc-fusion), the process achieved global product yields exceeding 70%, with final product pool concentrations greater than 15 mg/mL. Monomeric purity reached approximately 99%, and cumulative HCP clearance (LRV) was greater than 4.5, reducing residual HCP levels to 60-165 ng/mL (4-11 ppm).
Key Findings
- Global product yields exceeded 70% for CHO-derived proteins (bsAbs, Fc-fusion).
- Monomeric purity reached ~99% for CHO-derived proteins.
- Cumulative
HCPclearance (LRV) was >4.5, reducing residualHCPto 60-165 ng/mL. - AAV recovery was ~50% with residual
HCPlevels of 350 ng/mL (<100 ng per dose). - Optimized charcoal-to-resin ratio of 1:3 and load volume of ~10^14 vp per mL for AAV purification.
Why It Matters
This novel flow-through purification platform represents a significant advancement for biopharmaceutical manufacturing, particularly for complex modalities like bi-specific antibodies, Fc-fusion proteins, and AAVs. The high productivity and impurity clearance achieved could streamline manufacturing processes, reduce costs, and accelerate the availability of advanced therapeutics. By moving away from traditional bind-and-elute methods, this technology offers a more efficient and scalable approach, potentially enabling higher throughput and lower residual HCP levels that meet stringent regulatory guidelines. This could lead to more robust and cost-effective production of next-generation biologics and gene therapies.
biopharmaceutical-purification
flow-through-chromatography
monoclonal-antibodies
fc-fusion-proteins
adeno-associated-virus
host-cell-proteins