Im trying to optimize my downstream process for AAV processing and I've been having quite a bit of trouble for some reason, I described my process below:
I lysed cells in production media with 0.5% Triton X-100 for 45 min, checked titer by qPCR, added 0.5x cOmplete protease inhibitor, spun the debris down, left supernatant at 4C overnight. The next day I added 1mL of AVB resin to the supernatant, shook for 1.5hrs at 37C, processed resulting slurry over drip column. I collected flow through, wash, and elutions and checked all for my AAV. Somehow it completely disappeared... its not seen in any of the above fractions. I started with 6e10 vg, I know its not a lot, but I was hoping I could use ~1e11 vg/run to optimize the purification. Also, I didnt check the strip to see if it ended up there since my second elution is, I assumed, pretty harsh (0.1M H3PO4 pH1.7 + 500mM NaCl).
Ive previously tried using the resin by flowing my sample through the column/pre-packed resin but everything ended up in the flow through.
I have three questions for anyone thats used the GE AVB resin before:
1) Have you seen that purification requires excessively long residence times?
2) Have you found a more optimal set of elution buffers than the recommended buffers?
3) Are there any negative effects of Triton X-100 on AAV binding to column?
Thank you in advance!
I know this isn't really answering your question, but have you tried using the AAV-X column? We've used it for AAV2/2, 2/8 and 2/9 and it's really super
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Column