As gene therapies advance in both clinical successes and regulatory approval, there is a need to focus on increasing manufacturing efficiency.  A key area to focus on is increasing viral vector titers, which ultimately will enable the industry to meet increasing demand.  One area that manufacturers are moving toward is the increased use of adeno-associated virus (AAV) in suspension HEK293 cell lines.

Of course, a key aspect of improving the productivity of any cell culture-based process is to optimize the cell culture medium. Media optimization is a very time consuming and resource intensive process and with speed to market considerations for these therapies, there is often little time for these activities. Thus, utilizing media panels to select and optimize HEK293 suspension media for AAV production can accelerate the process and drive better gene therapy processes through clinical evaluation to commercialization. Media panels comprise a range of nutritionally diverse media formulations for manufacturers to test in their process with their cell line, expediting the selection process and allowing them to progress through media optimization much quicker. Since media panels offer so many benefits to production, we asked Céline Martin, PhD, Senior Product Manager for Thermo Fisher Scientific to serve as our expert for this session. She answered frequently asked questions about AAV media panels.

What aspects of AAV manufacturing can be improved with optimal media?

Finding the optimal medium for an AAV manufacturing process is usually solely focused on physical titer increase by screening various formulations at small scale, and indeed, medium can drive some titer improvement. However, there is a lot more media can improve: cell line development, viral particle quality, or scale-up. On cell line development, depending on how you generated your cell line—adapted it from adherence, engineered it or subcloned it—the media used at those stages indirectly creates a selection pressure over the cell population and plays on the cell’s diversity & metabolism you’ll ultimately have in your Master Cell Bank. Regarding particle quality, similarly to what we’ve seen with mAb development where the field focused first on titer the later on glycosylation patterns, nowadays very little is known about which factors may improve empty:full ratios, post-translational modifications, driving more particle secretions. Similarly to CHO, medium and sometimes its trace level contaminants may have an impact on quality. Regarding scaling up your process to a bioreactor, an optimal medium should be able to support an efficient seed train and larger scale bioreactor culture with limited foaming and enough surfactant to limit bubble burst stresses to the cells.

What should AAV manufacturers be considering when evaluating and selecting media for their process?

Media for AAV processes should be consistent as these processes are already seeing a far greater variability to what is usually considered acceptable in the biopharmaceutical industry, medium doesn’t need to be an added source of variability. Thus, it is always good practice to validate a medium two-ways: with enough replicates within one medium lot to truly assess its performance, but also amongst several lots. Pro-actively screening media that are or will be available in multiple formats and packaging is also a good practice as few developers already know what their production facility will look like during the early stages of development.

What are common pain points when optimizing media formulations?

Media formulations are especially complex entities, they are composed of multiple components that play a role not only by their presence but also by the range of concentrations they are present at and their actual bioavailability to the cells. Fully optimizing a formulation for a cell line and process can be particularly lengthy and that doesn’t account for qualifying the manufacturability of the medium itself. With the speed of the industry, very few developers have the luxury to dive deep into the key drivers behind the performance they are seeing, and this isn’t facilitated by the relative lack of characterization of the HEK cell lineages, the cellular mechanism behind transfection, assembly of recombinant viral particles and how we can indirectly influence them.

How does the transfection parameters impact media selection and optimization?

The most widespread method to produce AAV relies on triple-transfection, so it seems quite evident a medium should be selected for its compatibility with the transfection process to avoid cumbersome and expensive medium exchange steps. At the stage of transfection, testing whether diluting the culture with fresh media is beneficial can be media-dependent. This operation is usually implemented for practicality at small scale but can be difficult to implement in production, so it’s definitely a factor to look at. Finally, depending on the make-up of your transfection complex, stability and efficiency can be impacted by the buffer or medium used to make-up the final volume of your DNA/transfection reagents mix, especially when larger volumes of transfection complex are used compared to the initial culture.

Why do AAV manufacturers frequently opt for a catalog media rather than developing a custom media formulation with a media supplier or in-house?

Catalog media are a great option to start from for small-scale developments, they are readily available off-the-shelf and some may have already been referenced in the literature or even been used for clinical or commercial manufacturing. They are the ideal solution for a speedy approach to process development.

What is the downside to selecting a catalog media over something more customized?

The downside of selecting a catalog media is that they’ve not been designed for a specific cell or process unless they are part of a complete system. After developers have invested time screening from several suppliers, and selected one for their early proof of concept productions, a time usually comes when they consider a second version that may be more tailored to increase performance, which can involve simple modification to a catalog medium or more complex mixture and supplements. While moving from version 1 to version 2 of a process, especially when it comes to media, more factors may have to be considered, like the status of your cell bank, the efficiency of the seed train or the scalability to the production bioreactor.

What are media panels and how can they be used to speed the media selection process?

Media panels are small library of diverse media formulations purposely designed to provide a large range of nutritional composition to the cells while having previously demonstrated performance in multiple cell lineages or clones. The Viral Vector HEK Media Panel contains 5 formulations developed to be compatible with transfection and able to support the growth of HEK cells while being diverse enough to adapt to multiple processes. Having access to a media panel, as opposed to screening multiple catalog media, gives developers more assurance they are screening diverse formulations using a manageable experimental design, but it also gives them the opportunity to identify drivers behind performance if they have the desire to expand their development beyond a simple screening.

How can the use of media panels improve productivity?

Some HEK cell lines have very specific nutritional requirements depending on their history, how the cells have been adapted to suspension and banked in serum-free medium, also the processes run for AAV production are themselves quite unique with a long seed train and a very short production time. What we’ve identified is that a panel can improve productivity directly by just getting a formulation more finely tuned to your cells, but it can be particularly useful to tune media not just to the cells but also to the process, giving more flexibility to developers to customize their media formulation.

What should companies consider when selecting a media supplier?

Even though the field is moving fast, I believe it’s important to consider the long-term supply of media. A lot of investment is made to develop processes and working with a reliable supplier operating under a robust quality system can de-risk implementation down the line by making sure the supply chain will be there when they are ready to start manufacturing at larger scale.

How can media panels improve gene therapy development overall?

There are so many process development opportunities in gene therapy as we’re creating the building blocks and biological understanding to create robust manufacturing workflows. With the panel being easily customizable, I’m looking forward to them  supporting the next generation of AAV processes, testing how they can be modified to support N-1 perfusion, high-performing engineered HEK clones, producer cell lines, and in general, supporting streamlined AAV platforms.