Efficient development of nanobody-based affinity chromatography for AAV8 purification.

Area of Science:

• Biotechnology
• Molecular Biology
• Affinity Chromatography

Background:

• Adeno-associated virus serotype 8 (AAV8) is a key vector in gene therapy.
• Current AAV8 purification methods face challenges with low yield and high purity demands.
• Developing efficient purification strategies is crucial for advancing AAV8-based therapies.

Purpose of the Study:

• To create a novel affinity chromatography resin using nanobodies (Nbs) for enhanced AAV8 purification.
• To identify and characterize a high-affinity AAV8-specific nanobody (Nb9).
• To evaluate the performance of the Nb9-conjugated resin (LQ01) for AAV8 capture and purification.

Main Methods:

• Construction and screening of an AAV8-specific nanobody library.
• Expression and purification of the selected nanobody (Nb9) in Pichia pastoris.
• Conjugation of Nb9 to Epoxy activated Bestarose 6B agarose resin.
• Characterization of the LQ01 resin's binding capacity, specificity, and stability under varying conditions (pH, NaCl).
• Assessment of AAV8 purification efficiency using SDS-PAGE analysis.

Main Results:

• Nb9 was identified as a high-affinity, stable nanobody for AAV8.
• The LQ01 resin exhibited optimal binding at pH 7.0 and demonstrated high ligand density.
• Purification trials showed high specificity, purity, and consistent VP protein ratio for AAV8.
• The LQ01 resin maintained significant binding capacity after 20 cycles and demonstrated good stability over time.
• SDS-PAGE confirmed effective AAV8 capture and elution using the LQ01 resin.

Conclusions:

• The developed LQ01 affinity resin provides a scalable and cost-effective method for AAV8 purification.
• Nanobody-based affinity chromatography offers a promising approach for purifying viral vectors.
• This technology has broader implications for biotechnological processes requiring high-purity biomolecule isolation.