Hi Evangelia,

What method did you use to purify the vector prep to 10^9 (I'm assuming this is in vg/mL)? What is the total volume you have at this titer? An easy and commercially available resin to purify AAV2 are pre-packed AVB columns (GE Healthcare), provided you have access to an HPLC.

After whatever method you choose for purification, you can further concentrate the prep using an Amicon Ultra centrifugal filter at a 100K mwco (http://www.emdmillipore.com/US/en/product/Amicon-Ultra-15-Centrifugal-Filter-Units,MM_NF-C7715). The limitation is that it can only concentrate your prep about a maximum of 50 fold. To get the 1000-fold increase you are seeking, you will probably have to start with a lot more transfected HEK293 cells (try a Hyperflask), or if your experiment allows, you can switch to a higher producing serotype like AAV8 or AAV9, which produce about 10 times as much virus as AAV2. The transduction and other properties of these vectors differ, so be sure another serotype will transduce your target cells/tissue before switching.

I hope this is somewhat helpful! Good luck,

Anna

Thank you both for the answers. I used the Iodixanol gradient purification kit. I guess I could also try the Ultra centrifugal filter if I am close to the titer I want. Thanks again!!

Hi Evangelia,

Does your idoixanol purification kit including the column? I do not think only iodixanol gradient ultracentrifuge could help to increase the virus titer. If you go through the column after ultracentrifuge, it will help to concentrate the virus.

Interesting discussion, it depends on a number of factors to increase the titer - few of them are having a High producer cell clone, plasmid (quality and quantity) during transfection, good transfection reagent and time period of virus collection and as discussed above by Zheng and Anna, you can always concentrate the virus, but the recovery is limited. So it is better produce high titer of AAV during production for downstream applications.

Thank you all for your valuable help! I actually managed to have a titer of x1013 which is quite satisfying for first time :) I actually doubled the cells I used and did many centrifugations to minimise the volume. Thanks once again for the tips!