Inactivation of viruses using novel protein A wash buffers

Area of Science:

• Biotechnology
• Biopharmaceutical Manufacturing
• Viral Inactivation

Background:

• Low pH viral inactivation is standard post-Protein A capture for monoclonal antibodies (mAbs) and Fc-fusion proteins.
• Low pH exposure can negatively impact protein quality during manufacturing.
• Alternative methods are needed to inactivate viruses without compromising protein integrity.

Purpose of the Study:

• To develop novel wash buffers for viral inactivation while mAbs or Fc-fusion proteins are bound to affinity or mixed-mode resins.
• To assess the efficacy of these buffers in inactivating XMuLV virus.
• To evaluate the impact of these buffers on protein binding and elution characteristics.

Main Methods:

• Developed high salt (2 M ammonium sulfate, 3 M sodium chloride) and detergent (LDAO) wash buffers.
• Equilibrated Protein A columns in high salt buffers to enhance hydrophobic interactions and prevent premature elution at pH 3.
• Tested viral inactivation of XMuLV using 2 M ammonium sulfate (pH 3), 1 M arginine, and LDAO buffers with a 1-hour contact time.
• Quantified viral removal using PCR and infectivity assays.

Main Results:

• High salt buffers (2 M ammonium sulfate, 3 M sodium chloride) at pH 3 enhanced hydrophobic interactions, preventing antibody elution from Protein A resins.
• Ammonium sulfate facilitated antibody binding to mixed-mode resins at pH values typically causing elution.
• Wash buffers containing 2 M ammonium sulfate (pH 3) or LDAO achieved approximately five logs of viral removal (PCR) and complete inactivation (>6 logs) by infectivity assay.
• Novel Protein A washes demonstrated potential for rapid, automated viral inactivation with reduced pool conductivity.

Conclusions:

• Novel wash buffers offer an effective strategy for viral inactivation during biopharmaceutical manufacturing.
• These methods can be integrated into Protein A capture steps, improving process efficiency and protein quality.
• The developed buffers provide a viable alternative to traditional low pH inactivation, enabling faster, automated viral clearance with lower conductivity.