Breech Odu, a Process Development Scientist at Merck, discussed adeno-associated virus (AAV) production, with a focus on cell lysis, nuclease treatment, and chromatography. Using detergents for chemical lysis is highly effective in suspending cell culture and is very effective for viral clearance. Odu discussed Deviron C-16 and 13-S9 detergents, which are eco-friendly alternatives to Triton X-100. Benzonase was selected as an endonuclease.

Deviron is new to the market but comparable to or even better than current benchmarks used for lysing HEK293 and Sf-RVN cells. Furthermore, experiments showed that Deviron detergents do not interfere with benzonase activity, maintaining its effectiveness in breaking down DNA. Endonucleases also reduce the virus nucleic acid complex that tends to form in AAV, and they minimise the fouling of downstream equipment. For deviron C-16 and deviron 13-S9…

Regarding clarification, there are two streams of approaches: traditional, which includes centrifugation and microfiltration, and current methods which refer to nominal flow filtration. The traditional methods are difficult to scale up, whereas nominal flow filtrations including Clarisolve and Clarigard produced higher yields and throughput.

Data from Odu’s experiments showed that scalable one-step filtration processes with high yields at small and large scales demonstrated the versatility of Clarisolve and Clarigard.

Moving to the purification stage, it is advised to remove benzonase, DNA fragments, and concentrating material before and after chromatography. The tangential flow filtration (TFF) membranes (100 – 300 kDa) provide better impurity clearance, while smaller membranes improve yield but risk increased impurities. Odu commented: “However, if your product is very sensitive to flux, SPTFF is an option to go for. So with this one, we've carried out some scalability studies between the Pellicon Capsule, which is our single-use device.”

Chromatography was presented as another critical step, with a focus on the Fractogel resin. Unlike traditional resins, Fractogel features tentacle technology, which increases surface area and enhances the binding of AAV particles. Overall, Odu gave a detailed overview of key techniques that balance scalability and safety requirements to optimise AAV production