A challenge in the production of recombinant AAV is the presence of capsids that lack the required gene of interest: and therefore cannot carry out their role in gene therapies.
A novel anion exchange chromatography elution method is set out by researchers: who say the method could also provide insight into development of elution methods for other AAV serotypes.
Search for scalable methodologies
AAV holds potential as a delivery vector of therapeutic genes for multiple diseases. FDA gene therapy treatment approvals for Luxturna (for retinal dystrophy) and Zolgensma (for spinal muscular atrophy) have accelerated AAV based research and this is highlighted by numerous clinical trials involving AAV.
However, with the challenge of AAV supply, there has been a push to increase capacity and process efficiencies. This includes the development of more scalable methodologies for AAV purification.
In manufacturing gene therapies, the encapsidation process results in virus particles with the desired genome (full) and virus particles without any genome (empty) or fragmented DNA (partial). Empty AAV particles often outnumber full or semi-filled particles by up to 30-fold. Without the gene of interest, the particles are not inherently functional for gene therapy and may be considered a process related impurity.
In order to produce a consistent product, the ability to control the ratio of empty to full particles is necessary in the purification of viruses.
Problems with current methods
A range of methods for separating empty and full AAV particles currently exist: including analytical ultracentrifugation which relies upon density gradients, typically cesium chloride or iodixanol.
"This process often yields highly purified AAV particles making it a reliable approach in terms of product quality. The drawback to ultracentrifugation, however, is application to a manufacturing environment where operational costs and limited instrument size do not scale with material load volumes," note researchers.
Chromatography methods including affinity, ion exchange, and size exclusion have been adapted for multiple AAV serotypes in downstream purification. However, the order and conditions of the methods used vary greatly among different groups.
A novel anion exchange chromatography elution method for enrichment of full AAV particles is set out in the study. A step gradient with small conductivity increases of around 1 mS cm–1 provides more efficient separation of empty and full AAV serotype 5 across membrane media as compared to conventional linear gradient method.
This 1 mS cm–1 step method has been invented by Adam Hejmowski; and outlined the potential of the process in the study with fellow Pall employees. A patent for the process has been filed.
The use of this approach in creating a simpler method for manufacturing processes and scalability to a larger chromatographic volume is also explored in the study.
"With this approach, the authors achieved greater than 4-fold enrichment of full capsids, to give a total of ≈50%–60% full capsids, using a 25 mM Bis-Tris Propane pH 9.0 buffer system with NaCl as the eluting salt. Results suggest that this elution method can be implemented into a scalable process and can provide insight into development of elution methods for other AAV serotypes,” notes the study.
Source: Novel anion exchange membrane chromatography method for the separation of empty and full adeno-associated virus: Biotechnology Journal, December 18, 2021.
Hejmowski, A.L.; Boenning, K.; Huato, J.; Kavara, A.; Schofield, M.
