Hello,
I'm looking into buying a new HPLC machine for purification of AAV preps. Has anyone had a good (or bad) experience with a particular model?
Thank you.
Sure, this is commonly done with adeno-associated viral vectors (AAV) and if you do a keyword search on the web (i.e. Google, Bing...), then you will find many papers, articles, reports and information. Invest in a research grade, (not academic grade) HPLC system with DAD (e.g. Agilent 1100, 1200 or 1260-series system with Quaternary pump with AIV, column compartment, Low volume A/S and std DAD plus 3D software).
BTW: I provide consulting services in this and many other fields and one the preferred methods for most AAV's is to use ultracentrifugation (though it can be far more expensive). This can be done on both an analytical scale and production scale using a continuous flow system.
Good Luck with your project.
Hi William.
I am quite familiar with various AAV production methods. I just wanted to gather personal experiences from people using a specific machine/brand.
I'll have a look at the Agilent products, thanks.
As long as you purchase a high quality Research Grade HPLC system, you will be fine. Once that is done. it is all about the training more than anything else. A basic level of proficiency with HPLC using takes several years, with mentoring, to achieve. It is not something learned in a day, week, month or year. At least AAV HPLC methods are some of the easiest to run which means fewer problems, but more emphasis on hands-on skills and troubleshooting. Besides learning the technique, you will also have to learn the hardware and software, as each brand and model is different.
Hi Giulia,
I just saw your question, and I hope my answer is not too late (and is also helpful)! I have used an AKTA pure (https://www.gelifesciences.com/en/us/shop/chromatography/chromatography-systems/akta-pure-p-05844) in my PhD and now Postdoc labs to purify AAV using AVB HiTrap columns (GE healthcare) with resounding success. The AKTA is very sophisticated and may be overkill for your needs if you are only doing AAV purification and not other proteins, but it is fully automated to the point that you can run it overnight (and monitor it remotely if you like, but I trust it). It gives easily interpretable readouts in real time, so you can determine when your washes are clean, and exactly which fractions contain eluted vector (and roughly how much vector you’ve yielded). The AVB columns don't work for every serotype, but POROS resin may be a good substitute and I think you can get pre-packed columns of it. Of course, the disadvantage of affinity purification is that empty capsids are not removed as in centrifugation based methods, but I have used AKTA/AVB preps in animal studies successfully, and their purity/quality is high enough for cryo-EM studies on the particles themselves.
I also have experience doing batch-loading based purifications with these resins without the use of any machines, and have optimized this as a cheaper (albeit more labor intensive) option than buying an HPLC/FPLC for when I start my own lab and have no money to buy equipment like an AKTA yet, haha. Happy to share tips on this or using the AKTA if this would be helpful to you.
Cheers,
Anna
Hi Anna,
thank you for your message.
I ended up getting the ATKA go, the compact version because of limited bench space, and using the AAVX columns as we produce different capsids. We also have an ultracentrifuge (which I still personally prefer).
Thank you again and good luck with your work!
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