Preferential Crystallization of fullrAAVs in a Hanging Drop Crystallizer as an Alternative Method for Downstream Purification of rAAV-based Gene Therapies

Technology 

This technology leverages preferential crystallization to purify the full rAAV capsids from the empty capsids. The capsid mixture is mixed into a crystallization solution with pre-determined set of conditions including precipitant concentration, salt concentration, and pH. to induce the crystallization of full capsids at concentrations of >90% while leaving the empty capsids in solution. Then, the mixture is incubated for a sufficient period to allow the full capsids to form crystals, while the empty capsids remain uncrystallized. After crystallization, the full capsid crystals are separated from the solution, effectively purifying them from the empty capsids. These purified crystals can be stored at controlled temperatures, with or without cryoprotectants, before being later reconstituted with a pharmaceutically acceptable excipient for use in gene therapy treatments. 

Problem Addressed  

This technology addresses the problem of purifying rAAVs, which are often contaminated with empty capsids that lack therapeutic genes and increas the immunogenic load on the patient. Due to the similar physical properties of empty and full capsids, separating them is difficult with the primary options being the more costly and less scalable methods of chromatography and ultracentrifugation. This crystallization method offers a simple method of separating the full capsids from the empty using crystallization and phase separation. In addition, the new method cuts costs, processing time, complexity, and product losses, alleviating the purification bottleneck for clinical grade vector production. 

Advantages 

• Simple crystallization method for >90% concentration of full capsids  
• Lower cost and scalable approach than current purification options 
• Enables concentrated, stable intermediates for easier storage and transport 
• Reduces patient immune responses by minimizing empty capsid load 
• Can be adapted to purify multiple rAAV serotypes 

Publications 

Bal, Vivekananda, et al. “Selective enrichment of full AAV capsids”. arXiv (2024): https://arxiv.org/abs/2412.06093  

Bal, Vivekananda, et al. “An integrated experimental and modeling approach for crystallization of complex biotherapeutics.” arXiv (2024): https://arxiv.org/abs/2412.09821 

Bal, Vivekananda, et al. “A phase diagram for crystallization of a complex macromolecular assembly.” arXiv (2025): https://arxiv.org/abs/2501.18104