Cell therapies have the potential to treat and even cure disease for which therapeutic options are lacking. This potential has led to increased investment and an increasing number of clinical trials. To date, much of the focus has been on autologous cell therapies that are created using a patient’s own cells. To advance the industry to be able to treat a broader range of indications and a larger patient population, that focus has recently shifted toward allogeneic therapies that could be produced at larger scale.

Allogeneic therapies provide solutions that autologous therapies can’t because they are produced from donor cells, can be manufactured in advance and at larger quantity, and can be stored and ready for use as patients need them. This opens the door for broader use, but also presents unique challenges. In this Ask the Expert session, we spoke with Whitney Wilson and Antonio Fernandez-Perez, both Field Application Scientists with Corning Life Sciences, about the incredible potential of allogeneic therapeutics, ways to solve the current challenges they face, and what the future may hold.

Our questions and answers are below.

High Quality seed culture

How do you define high quality source material?

First, it is important for companies to define high-quality source material prior to starting their process development because this is the foundation for several decisions that will need to be made. Companies will want to consider where the source material is coming from and whether they are using the correct tissue that they are going to reprogram or define their iPSC culture. Depending on the application, they may or may not want to use biopsy material. Another important consideration is the age of the material. When selecting source tissue material, they will want to think about the donor in terms of age, background exposure to toxins, etc. in addition to physiologically where the starting material to generate the iPSCs is coming from.

How do you select the best donor population for your application?

Thinking about the end goal is very important when selecting the donor population. For example, if you start with a more plastic population, you will have more potentiality to create multiple cell types. However, if you want to make an immune cell, it may be better to start with and reprogram immune cells. Understanding the biology and implications/limitations of different cell types will have a big impact on what companies select as their starting material. There are pros and cons for different donor sources as well.

Another consideration would be the convenience and ease of acquiring the source material. For example, it can be very difficult to acquire neural stem cell tissue. There are considerable medical and ethical considerations around sampling brain tissue vs. taking a skin punch biopsy. A skin punch biopsy is a relatively routine procedure from which fibroblasts can be isolated and used to make iPSCs. It’s also a procedure that is very easy to get approved through regulatory bodies within the hospital.

Robust Cell expansion and ECMs

Can you talk a little bit about ECMs and iPSC expansion?

This is another area where the end goal is very important. Some extracellular matrices (ECMs) have a better propensity to differentiate iPSCs into different cell types, so that’s something to consider. The same is true for media. Corning offers three different matrices to ensure there is an option that fits individual customer needs: Matrigel®, rLaminin-521, and a vitronectin derivative called Synthemax®. These are the three most commonly used substrates in the field. It is project specific as to whether an animal-free solution is required, and which matrix will provide the most efficient differentiation substrate for the end product.

For example, with iPSC, you want to be able to maintain pluripotency and genomic stability. Your matrix and media formulation will have a big impact on this. If you are expanding your iPSC population, you will need to keep the integrity of the cells in mind and consider whether the media formulation or the ECM is robust enough to maintain chromosomal integrity as the culture is expanding and the surface area is increasing.

Matrigel is a gold standard in the industry when it comes to growing iPSCs because it provides so many of the components that the cells need to grow and maintain the pluripotency and potentiality. So, as companies are expanding the process and defining source material, they need to determine the sensitivity of the culture to those components. They may want to start with an ECM that they know will work well and will be robust. Later, they may want to move to more defined components and can start testing rLaminin-521 or Synthemax to replace the Matrigel.

Cost of expansion and cell banking

What kinds of considerations are important when it comes to cost during the expansion campaign?

This is an important question because as clinical treatments, these therapies need to be reimbursable by insurance companies. This won’t happen if the product is exorbitantly expensive. Therefore, there is substantial pressure to reduce the cost of production for cell and gene therapies. As companies start to expand production, cost is a critical consideration, in addition to making sure the product is safe and efficacious.

Cost of production considerations include the production platform and the culture components. What degree of quality and product definition is going to be required, is a good question to start with. If it is an R&D project, then companies can be more flexible about products that are being used, but when a company moves toward therapeutic applications, they will need to have well-defined conditions.

If you are talking about ECMs, then you may need more robust ECMs in R&D, depending on the cell type, or you may want to experiment with defined ECMs during R&D, if the goal is to create a clinical or commercial production process.

In addition, you need to consider the source of the ECMs, as that will have an impact on cost. As the industry progresses, we will have more understanding about the process, and that will allow for some reduction in R&D costs. It also opens the opportunity to produce these more specialized ECMs in bulk at a lower cost, which could also help to reduce production costs overall.

How do you go about building a homogenous cell bank?

Very carefully. As you expand you need to continually test to make sure that the cells are expressing the profile that you have identified and are expecting. You also need to check for genetic stability. As the cells expand, you can have some problematic cells that appear in your population, so you need to be constantly checking to ensure that it doesn’t negatively impact the efficacy or safety of your product.

Another thing that companies need are SOPs (standard operating procedures) consisting of the proper protocols. Sometimes when working with stem cells we call it an art, because cells can be finicky and may not respond the way you expected. The operator is going to be the driver of the quality of the cell bank. One way to make the process more consistent is to have robust SOPs in place with defined metrics that operators can implement and collect data on. Information on population doubling time, the phenotypes, etc., should be tracked over time as cells grow. This will ensure that the process can become better defined over time. It also helps to answer questions about scale. For instance, will I need 100 vessels to make my bank, or can I do it in 3-4.

What platform choices are there for balancing needed quantity of cells, scalability, and cost-efficiency?

Right now, the industry has the most data on growing iPSCs and human embryonic stem cells in adherent 2D culture platforms. This works well at smaller scale, and we can culture these cells quickly.

However, if the goal is production of a cell therapy, we need to be able to increase the scale from a well plate to a flask to even larger platform solutions to accommodate larger demand.

Corning has developed a diverse set of platforms designed for this purpose. The Corning® CellSTACK® and HYPERStack® vessels increase scale by increasing the surface area while maintaining similar culture conditions. So, it is an easy transition to scale up to larger surface area platforms.

As we scale up it is important to understand how much volume is needed and what vessels will be required to meet those demands, all while still maintaining the quality required. Over the past 20-30 years, much work has been done in the field to understand the phenotypic and biologic profile of these cells and so far, it has been shown to be safe to grow iPSC in a 2D environment. In a 2D environment, we can be sure that the iPSC phenotype isn’t changing and that we can identify any areas of differentiation. When you move into a 3D platform, you no longer have the visual cues. Instead, you need to rely on assays, like flow cytometry, to identify if your cells are maintaining pluripotency or exhibiting spontaneous differentiation. .

It is also easier to eliminate parts of your population that aren’t behaving as expected in a modular 2D environment. For example, you may see that one stack has differentiation, but the other stacks are fine. In this case, you can dispose of the one stack that is problematic and continue to grow the stacks that are fine. In a 3D environment it is more difficult to eliminate any cells that you don’t want to expand.

Time and cost are also big considerations. Do you have the time and resources to conduct the required process development to translate your 2D culture to 3D and achieve the high cell number that you are looking for. Also, can you achieve the same level of cell quality in a 3D environment?

How impactful can coatings/surface treatments be?

The matrix is really one of the main drivers for keeping your iPSCs potent and primed for differentiation. You may have to optimize coatings or use a mix of ECMs. These are important questions that need to be answered during R&D and the impact of this is huge. There are a lot of resources being spent to understand this process, and groups are achieving scalability.

Safety and Efficacy

How do you ensure continued safety, efficacy, and high quality as you scale up?

It is key that during your process you are continually testing to make sure that there are no abnormalities within the DNA of your cell population, and that the cells are maintaining their stemness without becoming cancerous. It is a key consideration when working with iPSCs because cancer develops when an adult cell has become stem cell like, basically unregulated and acting out of turn. So, with iPSCs and any stem cell product it is important that companies make sure they understand these cells and that they don’t become oncogenic. Due diligence is key here and SOPs and documentation are an important part of that. Tracking how the testing is being done and what types of tests are being conducted is important for understanding quality. Looking at phenotype and ensuring that it is congruent with current scientific understanding and is properly documented throughout the process goes a long way to achieving consistent quality.

Safety and efficacy are also product specific, and bioassays need to be developed to be sure these specifications are being met. Before that happens, companies need to determine what constitutes safety and efficacy of a product so that the bioassays can be used on a regular basis both in process and for release testing.

How to manage and monitor potency and differentiation?

It is important to have the right potency assays and bioassays. For example, let’s say you have an immune cell that has been derived from iPSC, and you have a target tumor cell population. When you combine the two in vitro in a dish, the immunotherapy kills the cancer, but is that the context that is going to exist in the patient? Is it a solid tumor where there is a physical barrier that the therapy must penetrate, for example? You must develop an assay that mimics or tries to recreate the conditions in the patient. So, understanding your target, your biological product, and coming up with the right set of assays is the goal.

Rejection of the of the cellular therapy.

Another key consideration when we think about allogeneic therapies is rejection. This is because with allogeneic therapies you are getting a product from a donor, whereas in autologous therapies the product is produced using the patient’s own cells. Because of this, allogeneic therapies are susceptible to rejection similar to what you would see with an organ transplant.

The great thing about mesenchymal stem cells is that they have an immune evasion aspect. So, you can create a mesenchymal based product from a donor, and it will be able to be given to any patient without rejection. However, when the product is developed using iPSCs that is not the case. When companies are developing their cell bank and choosing starting material, they must consider how they will deliver a product that is going to be accepted by the patient without having to use immunosuppressive drugs, which can create a cascade of other issues. This means possibly generating starting cell banks that are haplo-identical, so that they have a lower risk of rejection. There is an effort right now to have an iPSC cell bank of the different haploid combinations globally that would be able to treat most of the population. This would help to address the problem of rejection and make allogeneic therapies more viable.

Fill/Finish and Drug Product (DP) delivery

What decisions about drug delivery need to be made early in your process and why?

This is a very important consideration to address early in the development of a therapy. Is it going to be a fresh product, meaning that the cellular product is created and goes directly into the patient, or will it be cryopreserved and available off the shelf? This decision will have implications for how the product is generated.

If it is a fresh product, then the product will need to be continuously produced, and the scope of the product will be limited in the number of patients that can be treated. There are also regulatory considerations. For example, how long is it considered fresh, and what storage conditions are required?

If the product is be cryopreserved, there are multiple options for packaging the therapy, typically in bags or in vials. Other important considerations include stability, preservation, transportation, and sensitivity to fracture temperature, as well as how it will be stored, delivered to the clinic, and what equipment will be required to take the stock product and translate it to patient delivery. In terms of storage and transportation, for example, do you need a minus 180-degree Celsius container and if so, how do you ensure that you can maintain the temperature through the entire process shipment and delivery process? Lastly, it is important to test whether the product retains efficacy after cryopreservation as not all cells do. These factors need to be well understood prior to process development because they will have a big impact on product manufacturing, shipping, and patient delivery plans.

Future

Future developments to watch?

Right now, mesenchymal stem cells are the most popular and closest to clinical stem cell products that we have, excluding bone marrow transplants. There are two other main types of adult stem cells that are used therapeutically, one of them is hematopoietic stem cells, which generate all the different immune cells in your body. Neural stem cells are the third type and the most difficult to obtain. You don’t have many of them and they all exist in the brain, so they are very difficult to source. Thus, generating neural stem cells from iPSCs is a popular way to get that cell type to use therapeutically. The reason we are passionate about neural stem cells is that they may solve a problem that otherwise has not been effectively addressed.

Mesenchymal stem cells are often used for wound healing applications, but we have other therapeutic options for wound healing They may not be as good, but they exist. When it comes to neurodegenerative diseases, we have fewer treatment options. For example, we don’t have any options when it comes to spinal cord injury. Because of this, neural stem cells could be the ultimate solution in terms of their ability to provide treatments where there are no other options. This could be a big turning point in the cell therapy field.

Another area to watch is improved scalability for cell therapies to reduce cost and make therapies more widely available on a large scale.

For more information, please see the Corning whitepaper, “Guide to Seeding, Expanding, and Harvesting Stem Cells.”

About the Experts

Antonio Fernandez-Perez, Ph.D., Field Application Scientist, Corning

Antonio Fernandez-Perez is a Field Application Scientist at Corning Life Sciences. He received his Ph.D. in Genetics, Development, and Disease from the University of Texas Southwestern Medical Center. In previous roles he functioned as a Senior Scientist of Process development of iPSC-derived cellular immunotherapies for patients with cancer and autoimmune disorders. Antonio now works extensively with academic researchers, biopharma companies, and advanced therapy manufacturers to optimize cell culture assays and cellular scale-up conditions using Corning technologies. These efforts help to advance the fields of cancer and disease research, as well as drive commercialization of life saving therapies.

Whitney Wilson, Field Application Scientist, Corning

Whitney Wilson is a Field Application Scientist at Corning Life Sciences. She received her B.A. in Molecular and Cellular Biology with an Emphasis in Neurobiology from the University of California, Berkeley. Prior to joining Corning, Whitney spent 12 years at the U.C. Davis Institute for Regenerative Cures and was the Director of the U.C. Davis Stem Cell Core. Whitney works with process development groups, optimizing production capabilities and cellular scale-up conditions for processes ranging from viral production to cellular therapeutics, as well as 3D cell culture environments.