Application Note

Full Adeno Associated Virus (AAV) Capsid Enrichment Using Mustang® Q Membrane

Source: Cytiva

AAV is well suited as a gene therapy vector as it is non-pathogenic and has an acceptable safety profile. Production of AAV particles is dependent upon enzymatic insertion of the ssDNA encoding the gene of interest (GOI) into a stochastically assembled capsid shell. However, the efficiency for producing functional AAV is highly variable resulting in low quantities of full AAV (capsid containing gene of interest) relative to empty AAV (capsid containing no DNA). Given only the full AAV particles can deliver the therapeutic agent, the empty capsids can be viewed as a process impurity and removing them presents a challenge to the manufacturing process.

This document describes a method for the enrichment of full AAV capsids that contain a DNA payload. Removal of empty capsids that do not contain DNA is achieved through elution from Mustang Q membrane. Instead of traditional linear gradients, we show a novel method employing small (~1 mS/cm1) conductivity steps. The small steps result in a series of discrete elution peaks, simplifying assessment of the purification and thereby accelerating process development. Employing Mustang Q membrane along with this step elution strategy is shown to provide ~5-fold enrichment in full capsids for AAV serotypes 5, 8 and 9 which makes it suitable as a platform process step.

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