I am working on AAV purification, and we are facing the problem that the host cell DNA of the final product is relately high, more than 100ng/dose.we guess that the host cell DNAs is enveloped inner of the AAV particles according to our experment . The problem is that how to lower the host cell DNA concetration. I think this is a problem that many companies may encounter, however few references are available . Can anybody give me some suggestion.
Dear Li Lejun
we perform iodixanol gradient purification. Are you using the same method or some other strategy? You can consider including a Benzonase treatment step of the cell lysate to help with removing excess DNA.
best wishes
Shambhabi Chatterjee , hi，Thank you for your suggestion. we do Benzonase (Final concentration，0-100U/mL,37 ℃ ，1h)treatments before every process of purification，however, It has little use for the final product. we also perform iodixanol /CsCl gradient， however it seems not work.
Dear Li Lejun ,
we use a different benzonase concentration (250U/ml) and it requires the presence of magnesium as well for optimal results. Trying a higher benzonase concentraion might improve your results (i am not sure if you meant 100 U/ml or 1000 U/ml).
you can read some more details about bensonaze and magnesium along with other buffers in this publication- https://www.cell.com/molecular-therapy-family/methods/fulltext/S2329-0501(16)30101-2
Also I am not sure why you think that the host DNA is being packaged into the AAV? I would rather guess that you have empty AAV capsids and/or host DNA contaminating the final viral suspension. This can be improved by specifically collecting the 40% iodixanol phase and avoiding collection of other phases (which contain empty capsids).
Hi,
We also use 250U/ml, and I don't have free DNA.
I would like to take this opportunity to ask what is the yield in your productions, have you used desalting devices (removing the Iodixanol) or concentration devices?
Best wishes,
Dear Luisa Fernanda Bustamante Jaramillo
Yes, indeed we use PBS washes and Amicon filters to remove excess iodixanol.
In my experience, the yield depends on the size of the construct being packaged (when comparing within the same serotype) but we regularly produce AAVs with titres in the range of 10^12- 10^14 U/ml for our constructs.
best wishes
Shambhabi
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