When using AAV vectors have you used ultrafiltration for virus stocks and if so what do you recommend and how does it compare to traditional methods?


Yes AAV vectors stocks are often concentrated and polished using ultrafiltration and this generally works well. Commonly we seen ultrafiltration as being best used after a microfiltration step (clarification of larger cell debris) and prior to another purification step such as column or membrane chromatography (to remove similar sized HCP molecules, etc). In this workflow the ultrafiltration step aids in a further clarification by removing particles that are 10x larger or 10x smaller than the chosen MWCO*, whilst also concentrating the virus stock to a more workable concentration. However, ultrafiltration is also used in vector workflows for buffer exchange steps prior to chromatography and final polishing steps after chromatography.

In terms of recommendations it depends upon what is best for your specific process and end goals, happily we have a regenerative medicine team that can provide proposals specific to you should you be interested in more info. However in my opinion ultrafiltration with a 50K MWCO used in conjunction with a membrane chromatography step tends to be the most efficient for producing stock quickly whilst maintaining titres. Whereas methods such as microfluidization can have higher shear stresses and may need more optimisation, although I’m not an expert in these areas.

*Ultrafiltration can be used for separation of molecules based upon size, but due to the biochemical variables involved in ultrafiltration the rule of thumb is that true fractionation can only occur between molecules that have a 10 10x difference in molecular weight or diameter.


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