What do you want to know about developing a scalable adeno-associated virus (AAV) production process? You might find the answers here, where Cytiva scientists answer some common questions from people who attended webinars on our start-to-finish process.
Transfection, AAV vector production, and harvest
How long does it take to adapt adherent cells to suspension for the HEK 293T cells?
It took about 2 months using direct adaptation. We made 10 cell passages to control cell growth before creating a working cell bank. One tip is to control cell aggregates during adaptation and not exceed 2.5 × 106 cells/mL, because aggregates will increase when viable cell density (VCD) is too high.
Do you expect your PEI-based transient transfection method for AAV will be scalable to 500 L?
Yes, this is what we expect. But so far for AAV2 we have only done the scaling up from 20 mL to 20 L. We obtained similar titers for several serotypes. A customer has a scalable transfection procedure in the Xcellerex™ XDR bioreactor up to 500 L.
How did you select your lysis conditions for AAV2?
We have been doing a study comparing different detergents for lysis of HEK293 cells in an adenovirus process that we published and compared with freeze-thawing. Tween™ 20 showed good performance. It is not on the REACH list (i.e., it is health and environmentally friendly and can be used for large-scale production).
Do you have any recommendations to maximize recovery on Capto™ AVB chromatography resin?
Yes, a few things. Make sure you do not underload the column. Capacity is very high, approx. 1014/mL resin. If the titer is very low in the harvest, try to concentrate it. And try different elution procedures (flows and buffers). Also, the titer in the sample that is loaded can affect recovery (higher titer is an advantage).
How specific is Capto™ AVB resin for different AAV serotypes?
It binds to 1, 2, 3, and 5. But it has also been used successfully for 6, 8, 10, and some synthetic capsids. It does not bind 9 well. We will be looking into more serotype-general ion exchange (IEX) alternatives.
The AAV recovery from the concentration and buffer exchange step was up to 80%. Where was the loss of 20%?
We did analyze the permeate, and no virus was detected there for the 300 kDa MWCO hollow fiber filter. It could be trapped to some degree in the system tubing or filter, or it could also be analysis error. Sometimes we have achieved 90% to 100% recovery. So, we generally get very good recovery over this step.
How does Capto™ Core technology work for AAV purification?
This is a flow-through procedure. The virus will pass through due to the size, and the impurities will go into the pores and bind the ligand (octylamine) inside. We have Capto™ Core 400 and Capto™ Core 700. Capto™ Core 400 is suitable for AAV. The 400 and 700 reflect the MWCO of the outer shell of the bead.
Will Capto™ Core 400 be able to separate full AAV capsids from empty capsids?
No, Capto™ Core 400 is used to scavenge impurities, such as host cell protein and DNA, from the AAV. The inert shell of Capto™ Core does not discriminate between full and empty capsids since they are similarly sized.”
What is the best way to remove empty AAV capsids?
By ion exchange (IEX) chromatography if you want it to be scalable. We are still working on the full/empty separation, but we believe Capto™ Q ImpRes or Capto™ Q is suitable for most serotypes. We also suggest you maximize the % full capsids in your preparation by optimizing upstream production.
What is the binding capacity of the Fibro technology for AAV?
We have only worked with different prototypes, so we don’t have this information yet. In general, the binding capacity is doubled as compared to resin-based AAV capture. The explanation to superior binding capacity is that the accessible surface for virus binding on Fibro is high. Another benefit with Fibro is the opportunity to run the AAV capture chromatography at high flow rates.
What method do you use for measuring AAV titer?
We are using two similar assays: an enzyme-linked immunosorbent assay (ELISA) and a Biacore™ assay for surface plasmon resonance (SPR). We get very similar values that compare to the ATCC reference standard. But we have lower variation (CV%) with the Biacore™ assay.
Did you check residual DNA impurities in your AAV preparation?
So far, we have done PicoGreen™ analysis for total DNA. After the affinity step we could not detect any DNA; it was below the detection limit (1 ng/mL). We plan to perform a qPCR for HEK293 DNA that we have already set up. In the AAV DNA qPCR, we always check background from plasmids and have carefully set up the assay to have full control of any background sígnals from plasmids. We see no or very low background in our qPCR assay.
How do you determine empty versus full AAV capsids?
So far by qPCR:ELISA ratio. However, this is a little uncertain since both assays can vary. We believe a better method is needed, so we are also looking into an HPLC method with fluorescence detection, transmission electron microscope (TEM) analysis, and a few other methods that are easy and quick and that do not consume too much AAV. We do not have access to analytical ultracentrifugation (AUC), which would be good for this but is not quick.
How do people manage viral clearance studies in gene therapy products and processes (e.g., AAV)?
It’s important that the raw materials are not contaminated. But depending on the virus to be purified and what it can withstand, you can also introduce steps with low pH or detergent, so that more sensitive viruses will be cleared.
Looking for more FAQs, insights, and details about the process we developed? Watch our AAV production webinar from ASGCT 2021.