Preparing your stem cell cultivation for commercialization

Cultivating quality cells is a key requirement of moving a cell therapy product from the lab bench to the bedside. In this interview, experts from the Cell and Gene Therapy Catapult and Eppendorf discuss, how technology developed in response to research and clinical needs and how the cultivation methods impact scale-up and regulatory compliance.

How is the field of stem cell culture developing in response to changing research and clinical needs?

Sebastian: Stem cell cultivation is coming out of the academic niche and is moving towards clinical research and the manufacturing of therapeutic products. I hope we can apply the learnings made during the commercialization of ‘traditional’ biologics to the field of cell and gene therapy. An important difference is, however, that conventional biologics are produced by cells, whereas in cell therapy the cell itself is the product. This creates new challenges, such as limited possibilities for downstream processing.

Juline: Today most processes in clinical development are done in 2D culture systems, which are labor-intensive and extremely expensive. These challenges need to be addressed in the transition from research to commercialization.

Are there unique challenges associated with products based on different cell types?

Juline: One challenge is the starting material, which needs to be generated in extremely high quality. We need to develop characterization methods to understand the process, such as current work on biomolecules serving as early indicators for iPSCs differentiation. Safety is extremely important because the cell is the product. There is not one process that fits all. For each process we need to figure out what the critical process parameters are and how to measure them, and how to scale-up or scale-out.

Sebastian: The requirements from the FDA for a pharmaceutical product are still the same. Safety, purity, efficacy, identity. The question is how to apply that to a cell product. On the other hand, there are certain process requirements for stem cells that are different to other cells we currently work with. And that’s why we need to develop and establish new processes and maybe need to develop certain supporting products for the bioreactor systems. A lot of work is currently being done on how to expand the cells so that there is not too much left in the process that cannot be removed afterwards.

Therapeutic application needs high cell numbers and therefore large culture volumes. What are the advantages of 3D cultures in stirred-tank bioreactors compared to plates and flasks?

Sebastian: Stirred-tank bioreactors are the current gold standard for biopharmaceutical manufacturing. There are very well established, and researchers can profit from a lot of literature. Bioreactors allow the creation of a controlled environment to provide optimal conditions for cell growth or differentiation. Identification of critical process parameters and definition of critical quality attributes are required for regulatory approval. Furthermore, the scalability of stirred-tank bioreactors simplifies the transition from small to larger scale. One of the biggest advantages I see in the reduction of manual labor. This removes the human error more and more, which is crucial for a safe therapy.

Juline: True, stirred-tank bioreactors are the gold standard. However, especially for stem cell culture, it requires skilled operators. Importantly, with a bioreactor you can increase reproducibility because you are able to monitor many more parameters than in standard 2D culture systems, like temperature, agitation, oxygen, and pH. Furthermore, there are new technologies, like Raman sensors, that can be used to obtain even more information. There is research ongoing on biomolecules which are linked to pluripotency or differentiation. Their analysis can give an early indication, whether the process is developing in the right direction, giving us the opportunity to stop, if it is not, and reduce the costs associated to a batch failure.

Besides their application in the expansion of cells, we can potentially use stirred-tank bioreactors to shorten seed trains. Instead of growing cells in 2D to inoculate the bioreactor, we could try to just use bioreactors from start to end by integrating passages using cell retention systems.

All these innovative technologies can be applied to the bioreactor and can be applied less to 2D systems.

How easy is it to move from a static to a stirred-tank culture and how can this be optimized?

Juline: There are some key parameters to investigate when you move from a 2D static environment to a stirred-tank system. Passaging for example: If you are doing clump passaging in 2D you need an adaptation, which is time-consuming and labor-intensive. If you are doing single-cell passaging, the transition is quite easy. By applying a Design of Experiment approach, you can limit the number of conditions to be tested to find the optimal parameters. And fortunately, we do not need to start from scratch; there is already a lot of data available.

You have to ask yourself questions like: Can we use a suspension culture or are the cells semi-adherent? How does our differentiation process look like? Can we both expand and differentiate the cells in the bioreactor? Or do we need to move back to an adherent system for differentiation? At Catapult, we have been working a lot on hiPSCs and hematopoietic differentiation. We started with a process in 2D and did Design of Experiments and statistics to find the best agitation speed, aeration strategy, and feeding strategy for the 3D culture in stirred-tank bioreactors. Like this we developed a process that was reproducible but also had some drawbacks: When we grew the cells as aggregates in a repeated batch, they grew larger. Therefore, our next step was to look at perfusion technologies. There are many different cell retention devices used in mAB industry. We analyzed, which is suitable not only at small scale but also for a later use at large scale.

Sebastian: You need to understand your process to be able to follow the Quality by Design principle and not just rely on testing the quality of the product at the end. The small-scale model should be your first task. Start simple, for example by testing in a very small volume how your cells behave in 3D. As a first step you can identify suitable process parameters in literature and we advise to follow the recommendations from your vendor and see in iterations, how your cells behave. For example, customers of ours at MH Hannover did exactly that. They started with a 2D static culture, then moved to the bioreactor, and optimized the process using classical process development approaches. They applied technologies and methods like perfusion, media exchange, pH control, and DO control, and optimized one parameter after the other. And in the end, they had a very good process to work with.

Then you have to scale it up and keep the environment constant for the cells. To achieve this, it is important to identify critical process parameters at small scale. We strongly advice taking a systematic process development approach.

How can the choice of cultivation method affect the potential product's progress towards approval?

Sebastian: All process steps need to be compliant to the regulatory requirements. These are currently in development and there is not much experience yet with regulatory approval of cell and gene therapy products. The supply chain needs to be fully compliant, from the raw material to the bioreactor system and any other system in contact with the product. Supplier and pharmaceutical company need to work closely together to understand the requirements and how to meet them.

Juline: In a way we are lucky because stirred-tank bioreactors are widely used in the industry and recognized by the regulatory authorities. Many new technologies will help develop a thorough process understanding and help bringing cell and gene therapy products to the market. However, there are still many challenges, for example if the amount of impurities increases during scale-up. There is a lot to do.

For more information visit: www.eppendorf.group/dasbox-mini-bioreactor-system