An industrial purification platform for AAV
This poster presents fully scalable non-affinity purification strategy that has been proven to be effective for all AAV serotype tested to date. Cell lysate is directly subjected to column purification after removal of cell debris without requiring a concentration step using tangential flow filtration. The process consists of three chromatographic steps. Hydrophobic interaction chromatography on a CIMmultus OH monolith is used for initial virus capture and purification. Precipitating salts are used at 1.0–2.0 M to achieve virus binding. Most of the small molecule contaminants and proteins are eliminated in the flow-through. AAV co-elutes with a highly reduced population of contaminating proteins. DNA-protein complexes are very strongly retained and require NaOH for removal. Intermediate polishing is performed with a CIMmultus SO3 cation exchange monolith. The AAV fraction from the capture step is titrated to a pH value of 3.5—5.0 and diluted to binding conditions. Sugars and surfactants are added to suppress non-specific interactions with tubing and containers, and the product is eluted in a salt gradient. Final polishing is conducted on a CIMmultus QA anion exchange monolith which separates empty capsids from full capsids. This is achieved in a salt gradient at alkaline pH. For more information please refer to BIA Application note A048 (www.biaseparations.com/applications).