Usually if you have good enough transfection of 293 (https://altogen.com/product/293-transfection-reagent-emb-kidney-crl-1573/) you can get the virus to be filtered with simpler methods. I would look at incubation protocols and Darrick's answer for more information.

We routinely use TFF (it really isn't that expensive to get a system setup), but you can and precipitate the AAV using PEG. Let me know if you need details (the PEG precipitation is the same process routinely done when making AAV from cells).

J

If you want to concentrate your virus from culture media you can use mangetic nanoparticles such as Mag4C Kits. This is a very easy and rapid magnetic concentration method. You can get information in the link or here directly http://www.ozbiosciences.com/magnetic-virus-concentration/47-mag4c-ad-magnetic-adenovirus-capture.html

 Best Wishes

http://www.ozbiosciences.com/magnetic-virus-concentration/47-mag4c-ad-magnetic-adenovirus-capture.html

Thanks  Jonathan. Yes, Currently we don't have TFF system now. and I do need the details about how to use PEG to precipitate AAV from media. I read Guo et al J. Virol Methods 2012 183(2) 139-146. They use 40% PEG 8000 and end up with 8% PEG 0.5N Nacl to precipitate virus. My question is what kind of buffer I use to make 40% PEG? and after precipitation, what buffer should I use to resuspend virus?

Hi Tong,

We make up a 40% PEG 8000, 2.5 M NaCl solution (400g PEG8000, 146.1g NaCl, make up to 1L with water) and use this for the precipitation.

We resuspend our virus in PBS + 0.001% poloxamer (F-68).

We also perform the 2 hr pre-incubation of the cells with 0.5M NaCl prior to harvesting of the media to increase the amount of vector in the supernatant.

http://www.ncbi.nlm.nih.gov/pubmed/20497038

I hope this helps.  Good luck!

J

Thanks, Jonathan, that's really help.

Hi,  Jonathan, I have one more question, In the whole purification step, how do you keep your product sterile? Do you filter your final virus product? or, by ultracentrifuge, we get rid of all the bacteria contamination?

Hi Tong,

in principle, if you work carefully there shouldn't be any bacteria in your product. Nevertheless, the easiest way is actually to sterile-filter your AAVs at the very end of your purification protocol. We prefer 0.22 µm filters for this purpose.

Thanks, Benjamin. Also, I want to know when using PEG8000 to precipitate AAV, I found not all of the pellet can get into solution. I resuspended virus in Tris-NaCl-MgCl2 buffer which I used in gradient buffer for ultracentrifugation. Did I lost some virus in insoluble pellet?

Hi Tong, we resuspend the pellet using a pipette (but not everything will go in solution straight away). We then rotate this mix overnight at 4°C in a tube rotator. After that everything should be dissolved. In the following centrifugation step, it is normal to get a pellet, but most of the AAVs should be in the supernatant, which you then use for your gradient.

Hi Tong,

If you want to avoid ultracentrifugation, precipitation, re-suspension  and chemicals troubles, my suggestion will be to use a magnetic-nanoparticles based concentration system as mentioned before. Let me know if you need further information. Good luck

Olivier, do you really offer a magnetic nanoparticle-based kit for AAVs? I can just find Adenovirus kits on your company website...

Hi Benjamin, yes we have this kit see page below:

http://www.ozbiosciences.com/content/18-Magnetic-virus-concentration

we receive positive feedback from customers that are now using the kit for adenovirus also for AAV http://www.ozbiosciences.com/magnetic-virus-concentration/47-mag4c-ad-magnetic-adenovirus-capture.html

I hope this can help you if you need information you can contact me

All the best

Olivier

Thanks, Benjamin. I tried yesterday. Next week I'll have result. and Olivier, your magnetic kits is for AD or AAVs?

Hi Tong,

Originally, the Mag4C-AD Kit was developed with Adenovirus but scientists asked if they could try for AAVs and based on their feedback it look like it is also working for AAVs. If you have a magnetic separation rack, I can send you a free sample of the magnetic nanoparticles. Just let me know at [email protected]

Good luck Olivier

Hi Tong did you get good results with either PEG precipitation or the magnetic kit?. 

I am getting consistently high rAAV titers when I purify cell pellet extract via Iodixanol. I am just truly disappointed that the purity is not good at all as observed by SDS gel with silver staining. I have tried several protocols without much improvement of purity. I don't want to switch to CsCl or add extra purification steps involving chromatography. Any advice would be much appreciated. Regarding the paper that Jonathan and Benjamin mention where only the sup of transfected cells is used to load the iodixanol gradient: The paper mentions to transfect cells for 5 days in serum-free media. Are they using special or adapted 293 cells? I worry that my 293 cells will crap out for so long without FBS. Also the "secretion" of AAV to the sup after 5 days and addition of NaCl seems to change quite a bit between serotypes. Is this an issue?

I would be happy to try this protocol as the sup is for sure a much cleaner starting input to load in iodixanol gradient as opposed to working with whole cell extracts....

thanks all in advance for the valuable input!

We have developed some AAV purification/concentration kits that might be useful to you. Currently, we have 2 different scales for AAV purification: mini and midi. Our mini kit can purify from between 0.5 mL to 10 mL of cell culture media, cells, or the two mixed together. The midi kit can purify from 10 mL to 50 mL of similar input. I have found that as little as 0.5 mL of mixed cells/cell culture media input can lead to visible expression of an alkaline phosphatase transgene in vitro.  Only standard laboratory equipment ie, a swinging bucket centrifuge, microfuge is required, no ultracentrifugation necessary. The process is rapid (1 to 1.5 hours) and multiple purifications can be done in parallel to accommodate large screening experiments. We are also working on larger scale purification (50 mL to 1 liter or more).

Please send a message or visit our website for more information:

https://norgenbiotek.com/category/virus-purification

Good luck!

Dear Darrick

The norgenbiotek kits look great. The incubation of post-transfected cells out to 7 days seems too long, isn't 2-3 days more typical? (Concerned that you'll lose virion activity at 7 days). Are you able to confirm that the lysis buffer is triton-based?

Have you ever run typical eluate on a gel to assess purity? If you are able to share an image, please email one to d.farleyAToxfordbiomedica.co.uk assuming you can't load one here.

Thanks

Dan

Usually if you have good enough transfection of 293 (https://altogen.com/product/293-transfection-reagent-emb-kidney-crl-1573/) you can get the virus to be filtered with simpler methods. I would look at incubation protocols and Darrick's answer for more information.