Despite the fact that the number of therapies utilizing rAAV has increased, there are still significant barriers to the use of AAV, such as low viral amounts and impractical purification processes.Here, we report the optimization of rAAV production in suspension HEK293 cells in SFM media via triple transfection approch. Titers >2e11 VG/ml were observed at harvest. Furthermore, optimization enabled several AAV serotypes & transgenes to exhibit a 3-4 fold titer increase. A scalable enrichment process was established to achieve >50% full capsids at DS. Development (10L), pilot (200L), and production (1000L) were used to determine the process's scalability and robustness.

A scalable and cost-effective production process is crucial to making recombinant adeno associated viral vector (rAAV) gene therapy more widely accessible. In this study, multiple factors were optimized through a design of experiments (DOE) approach using flatware to improve the yield of early clinical stage programs using HEK293 adherent cell system. A model was established to systematically analyze the impact of each factor on titer response and the interaction of multiple factors using multivariate regression analysis. Conditions optimized in flatware were scaled into the iCellis Nano fixed bed bioreactor to test optimized conditions against a validated scale down model.

There is an industry-wide need for  improvement in upstream yield across adeno-associated virus production to meet eventual clinical and commercial need. Despite this, there is still a lack of information around the cellular processes and pathways involved in the manufacture of AAV. This presentation will describe our efforts to improve the yield of our upstream processes while simultaneously determining what cellular processes contribute to this increase in yield.

In this presentation, we will describe strategies applied in upstream and downstream process development to increase the vector genome (vg) titer and achieve high full particle enrichment of AAV6 particles using a two-chromatography step approach. With these improvements, the final AAV6 products showed higher concentration (>1E13 vg/ml) and better quality (70% full particle content) with reduced DNA and host cell protein (HCP) impurities.

Accessibility and affordability remains one of the biggest challenges in AAV manufacturing, calling for a high-yielding, robust, scalable, and cost-efficient process. At Ultragenyx, we employ a HeLa producer cell/helper-virus based infection process for AAV production, which costs less and scales more readily than a transfection-based process. Furthermore, we developed a modified batch process with perfusion to boost productivity to > 5E11 GC/mL in up to 250L SUB.

The demand for lentiviral vectors (LVs) for R&D and engineering cell therapies stems from their efficacy to deliver genes into targeted cells. Current LVs' production bioprocesses vary widely, significantly impacting quantities, quality, and costs. We have used a holistic approach to address challenges of upstream and downstream bioprocesses to increase titers and recovery.

Lentiviral vectors (LVV) have demonstrated noteworthy clinical success in patients during ex vivo CAR T as well as stem cell therapy gene therapy (GT) applications. The third-gen SIN LVVs have shown improved safety profiles for a conceivable durable treatment of rare disease and cancer indications. However, it’s application as in vivo therapy option is severely limited due to manufacturing, safety, and quality challenges. In this presentation, we will highlight several development and manufacturing considerations for generation of LV vector suitable for in vivo GT use.

Implementation of a wide range of AAV capsid variants has provided a unique challenge to process development groups, as manufacturing attributes of the AAV differ drastically between serotypes. The Alexion team has developed a manufacturing process with the goal of improving the consistency of the productivity and the quality of AAV produced in the bioreactor, as well as providing flexibility in the purification process to handle performance differences between serotypes.

This presentation will discuss scale-up of a suspension cell-based process for manufacture of AAV from 5 to 200 liters. Results, including cell growth, titers, and impurities, were consistent across 5 L and 200 L scales and will be presented along with several challenges related to scale-up and manufacture.

In this investigation, we employed AEX chromatography to simultaneously enrich full AAV capsid and eliminate endotoxin from the final drug product. By optimizing the column conditions, we achieved a high level of AAV capsid purity, as well as a significant reduction in endotoxin levels. This strategy offers a promising solution for the production of safe and effective AAV-based therapeutics.

Manufacturing strategies and progress towards platform processes for adeno-associated virus (AAV) production have seen substantial advancement in support of the impressive clinical success for this modality. This presentation will provide a case study for the development of a robust, high yield downstream process for the production of AAV serotype 5. Key findings will be discussed in addition to pitfalls and challenges in this development work.

High throughput purification development has become an integral part of purification development by providing an order of magnitude reduction in material and time requirement. It can be especially valuable since material limitations are often a challenge for AAVs. An initial screening of several process operating parameters using small amounts of material can be performed to narrow down the development space to focus on and further optimize the process. Here we discuss the various high throughput approaches utilized in Sanofi for developing several downstream unit operations including clarification, affinity chromatography, polishing AEX chromatography, etc.

Recovery out of harvest is the least understood step in downstream purification of AAV vectors. Complexity of lysed cell culture coupled to relatively low protein concentration of AAV product makes it very difficult to analytically investigate this process space. Here we evaluate the ability of dynamic light scattering (DLS) to serve as analytical characterization tool that would allow to investigate AAV capsid aggregation in a complex matrix of clarified harvest.

This talk may cover topics including selection of appropriate purification methods, process optimization, and quality control measures to ensure high-quality plasmid DNA for gene therapy applications.