July 08, 2021

Which method for cell culture: adherent or suspension?

By Byron Rees, Senior Manager, Research and Development Cytiva

Each production method can offer specific advantages to gene therapy manufacturing. However, as there are so many different considerations for each gene therapy product, we recommend performing a risk analysis. Learn which factors to consider in this blog.

New gene therapies frequently start their development journey being produced in adherent cell lines, usually for good reasons. All you really need at this stage is an incubator, and as the amounts required for early-stage development are very small, cell maintenance and production is relatively straightforward. It’s no wonder that many academic and research facilities prefer adherent cells. However, once feasibility is established and you decide to move into the next phase of the development process to produce larger quantities for analysis or preclinical material, it’s worth considering whether to keep your process in adherent cells or look at moving to a suspension platform.

Speed to market is key when developing a new gene therapy and it is often seen that the fastest route is adherent. Provided the lab-scale process is delivering in terms of desired yields and quality, why introduce a change that could delay getting your treatment to the patients who need it? The case for this approach is strengthened when you consider that there is proven technology that will get you there. Licensed gene therapies are already manufactured in adherent and suspension bioreactors, so from a regulatory perspective, either can be acceptable. There are also aspects of process development that can take advantage of having your cells fixed to a surface. For example, it’s easier to fully change the medium in the bioreactor, providing the opportunity to optimize growth and production phases. Also, perfusion is a more accessible option, and can be employed for processes producing less stable products such as lentivirus.

Yet while there may be benefits in speed to market, adherent manufacture may not be the best option for all processes. While the current adherent bioreactors on the market can provide up to 500 m2 of surface area for cell growth, this still may not be sufficient to produce the titers needed for gene therapies requiring a high dose per patient, or for indications where the target population is high. The obvious solution is to scale out the process and absorb the cost of additional equipment and space in clinical manufacturing. Alternatively, you could consider moving your process to suspension production.

A suspension process has the advantage of being easier to operate than an adherent one. Adherent processes require cells to be disassociated from their growth surface for routine maintenance, scale-up, and counting. Dispensing of this step greatly simplifies the process. Once your cells are no longer dependent on a surface for growth, scale-up becomes much easier and the range of technology to support manufacture increases. If your therapeutic can be produced in a stirred-tank bioreactor, then your options for manufacturing capacity open up, as this technology is widespread at many contract development manufacturing organizations (CDMOs). Processes of up to 2000 liters in single-use systems are common, though this does introduce other potential issues such as performing transfection at large-scale. As your culture volume increases, so does your transfection mix volume. Plasmid DNA and transfection reagents must be mixed and added to the bioreactor in a consistent way in each production run. Variation here could lead to changes in complex formation, and this in turn could affect transfection efficiency and yields.

The move to suspension could have an impact on yields and this should be evaluated during development. The majority of adeno-associated virus (AAV) processes use HEK293 cells, which can be obtained already adapted to suspension. These can be tested quickly with your plasmids to assess yield at the laboratory scale. If you do notice a decrease in yields, then you may need to consider adapting the adherent cell line where better productivity was observed. There is no guarantee however, that this will remedy the issue and adaptation can take several months, adding further time and cost to the development. A move to a different cell line or adapting an existing one is also a good opportunity to change the production medium, whether it’s to go fully chemically defined or boost cell growth.

It is important to consider the wider process implications of your chosen production method. The composition of feed material going into downstream processing can vary from adherent to suspension production. For example, if cell lysis is a requirement, the lysis strategy can vary between the different production methods. Also, the loading onto the clarification step can differ. The feed from a secreted therapeutic produced in a fixed-bed bioreactor is relatively clean, and most of the cells will be retained in the bioreactor. This could allow for a reduction in depth filtration area compared to a suspension process, which will need to be sized to remove more of the cell debris too.

These points lead to the cost per dose of each production method. While there are clear differences in the cost of consumables between the production processes such as fixed-bed single-use bioreactors being generally more expensive than single-use stirred tank biocontainer bags, there may be areas where cost reduction is possible at other operations in the process. The important factors are yield and quality for each production run, and how many productions runs are being targeted to meet the patient population demand.

To summarize, each production method can offer specific advantages to gene therapy manufacturing. However as there are so many different considerations for each gene therapy product, we recommend performing a risk analysis considering some of the factors discussed in this blog. Going through this process will help define your development plan and contingencies if you don’t see the results you desire.

Learn about AAV solutions from Cytiva.