Multimeric fusion single-chain variable fragments as potential novel high-capacity ligands

Area of Science:

• Biotechnology
• Biochemistry
• Protein Engineering

Background:

• Affinity ligand design is crucial for high-capacity applications in biotechnology, particularly for therapeutic molecule purification.
• Existing methods face limitations in capacity and efficiency for downstream processing.

Purpose of the Study:

• To establish a proof-of-concept for using multimeric fusion single-chain variable fragments (scFvs) as high-capacity ligands in affinity adsorbents.
• To evaluate the impact of ligand format and density on binding capacity and utilization.

Main Methods:

• Design and production of mono- and di/tri-scFvs with Pro-rich linkers.
• Immobilization of scFvs onto 6% cross-linked agarose beads.
• Frontal binding experiments using a 50 kDa target protein.

Main Results:

• Achieved up to 20 mg·mL⁻¹ dynamic binding capacity and 82% utilization yield at 100% breakthrough.
• Demonstrated that ligand format and density significantly impact binding site utilization.
• Showed that pore size limitations of the adsorbent were not the primary factor affecting utilization.

Conclusions:

• Multimeric fusion scFvs are effective high-capacity ligands for affinity adsorbents.
• This technology enables lean process design and improved alignment with process specifications for biopharmaceutical manufacturing.
• The findings offer a new strategy for developing advanced affinity purification systems.