Developing a process of lentivirus purification from recombinant fluids using peptide affinity ligands

Area of Science:

• Biotechnology
• Gene Therapy
• Process Development

Background:

• Lentiviral vectors (LVVs) are crucial for gene and cell therapies, but scalable purification methods are limited.
• Existing LVV purification relies on affinity ligands, particularly for Vesicular Stomatitis Virus G (VSV-G) protein-pseudotyped vectors.
• Camelid antibody ligands were previously used, but peptide-based ligands offer a novel approach.

Purpose of the Study:

• To develop and evaluate novel affinity resins and membranes using VSV-G-targeting peptides for LVV purification.
• To optimize binding capacity, productivity, and host cell contaminant removal in LVV isolation.
• To establish a multi-step LVV purification process for therapeutic applications.

Main Methods:

• Development of peptide-functionalized affinity resins and membranes with varied material and structural properties.
• Evaluation of binding capacity, productivity, and contaminant removal efficiency.
• Implementation and assessment of a four-step purification process including clarification, affinity capture, polishing, and filtration.

Main Results:

• Peptide-functionalized resins and membranes demonstrated high capacity (up to 5 × 10⁹ TU/mL) and productivity (up to 2.9 × 10⁹ TU/mL/min).
• Achieved substantial enrichment of transducing LVVs and significant reduction of host cell contaminants (110-170-fold).
• The four-step process yielded 33%-46% LVV recovery with residual host cell protein levels below 5 ng/mL.

Conclusions:

• Peptide-based affinity chromatography offers a highly effective and scalable solution for LVV purification.
• The developed resins and membranes significantly improve LVV isolation efficiency and purity.
• This purification strategy supports the advancement of LVV-based gene and cell therapies.