New purification method produces higher-quality gene therapy vectors

A research group led by Specially Appointed Associate Professor Yasuo Tsunaka, Project Assistant Professor Risa Shibuya, Assistant Professor Yuki Yamaguchi and Professor Susumu Uchiyama at the Graduate School of Engineering of the University of Osaka, was the first in the world to clarify that the unidentified fraction, separated during the purification of adeno-associated virus (AAV) vectors for gene therapy, contained structurally altered particles with lower functions. The findings have been published in Molecular Therapy Advances.

While AAV vectors for gene therapy are expected to serve as next-generation pharmaceuticals, the lack of purification techniques for realizing mass-production of high-quality formulations has remained a challenge. Although the density gradient ultracentrifugation method can yield AAV vectors with high purity, it is difficult to scale-up, and it cannot separate particles that are structurally different but have similar densities. Meanwhile, although an unidentified particle fraction has been observed following anion-exchange column chromatography, the nature of these particles and the mechanism underlying their formation remain unclear.

The research group combined anion exchange column chromatography with particle surface charge analysis, mass spectrometry, biosensor analysis and gene transduction experiments. They revealed that the unidentified fraction contained structurally altered particles in which a protein composing the vector shell is partially externalized, resulting in reduced transduction efficiency. Furthermore, the group has established a purification method in which the unidentified fraction is selectively removed by anion exchange chromatography to obtain AAV vectors with higher quality and purity.

This achievement raises hopes for the development of new purification and quality assessment techniques that lead to the advancement of manufacturing processes for producing AAV vector formulations with improved quality, safety and efficacy.

Uchiyama said, "This achievement is the culmination of our laboratory's multilayered efforts in development of quality analysis techniques and manufacturing processes of AAV vectors. We have demonstrated that low-quality vectors, which were difficult to distinguish by conventional methods, can be separated and removed. This achievement will greatly advance the development of high-quality, high-performance gene therapies using domestically produced AAV vectors. We believe that the technology originated in Japan is about to enter the phase of becoming a leading technology in the area of vector manufacturing."

Journal Information
Publication: Molecular Therapy Advances
Title: Anion-exchange chromatography separates structurally heterogeneous and low-potency particles in adeno-associated virus manufacture
DOI: 10.1016/j.omta.2026.201812