AI-Supported Quality Assurance in Stem Cell Bioprocessing: An Interview with Dr. Klaus Graumann

Our authors:

Benjamin Obexer

Lead content writer, life science professional, and simply a passionate person about technology in healthcare

Elisa Opriessnig

Content writer focused on the technological advancements in healthcare, such as digital health literacy and telemedicine.

Klaus Graumann holds a PhD in Biotechnology and worked for approximately 20 years in the biopharmaceutical industry (Boehringer Ingelheim, Novartis/Sandoz). In 2018, Klaus left Big Pharma and started his entrepreneurial career as a Biotech Consultant and CEO of Phoenestra GmbH. Phoenestra is developing platform technologies for stem cell-derived products.

Getting to know Klaus Graumann

Benjamin: Hello Klaus, I hope you are doing well. It is a pleasure for us to have you here. Thank you for taking the time. I would like to ask if you could introduce yourself.

Klaus: Hello everyone, thank you for having me. It is a great pleasure to be here. My background is in biotechnology, and my specialty – a long time ago – was the downstream processing of proteins. Then I spent 20 years in the bio-pharmaceutical industry at Boehringer Ingelheim and Novartis. My first job at Novartis was in a lab head position, and I ended up being responsible for the drug substance development of Novartis Biologics. 

After about 17 years, I left Novartis and decided to start Phoenestra together with a bunch of people. I’m now the CEO of Phoenestra and, as we are a startup I am doing everything basically that is needed. So, I’m also looking into project management and business development, and sometimes I am even in the lab, horrifying my team (laughing).

Benjamin: That means you have quite an in-depth background. What motivated you to launch your own business or lead your own company?

Klaus: As you said, what motivated me to leave my quite lucrative job at Novartis in Big Pharma was the desire – that was already starting a long time ago – to run my own business. I wanted to start something as an entrepreneur, to build a successful business. Specifically, I think stem cell biology got me more than 15 years ago when Professor Yamanaka invented iPSC technology. So, there was a defining point in time when I got interested in this area, and I was happy that the opportunity found me in the end.

Introduction to Phoenestra and Processes

Elisa: Since it is a startup, can you provide an overview of Phoenestra’s mission and elaborate more on its operative field?

Klaus: Phoenestra was designed with the idea to address topics that are related to manufacturability, scalability, and product definition of stem cell-derived products, which are very complex biologics, and not only the cells but also products derived from stem cells are in our scope.

Elisa: So, is this the gap you want to fill with your technology and products?

Klaus: Access is also one of our targets in the gap that we see. We know that some of these therapies that are out there, and gene therapy especially, are hugely expensive, and this is not sustainable in our view. 

Elisa: Which platform technologies is Phoenestra currently utilizing for stem cell bioprocessing?

Klaus: It is essentially three pillars we are working on. First is the induced pluripotent stem cell (iPSC) technology. The second pillar, I would say, is mesenchymal stromal cells (MSCs), which have also gotten a lot of interest over the last few years as potential therapeutics, and we have, in collaboration with the company Evercyte, access to stable MSCs. They are telomerized, so they are overexpressing a fragment of the human telomerase enzyme. And the third pillar is cell-derived vesicles, which also have gotten a lot of interest recently.

Elisa: I see. I think some of our listeners might not be aware of the main difference between iPSCs and MSCs. Could you briefly summarize that, please?

Klaus: So iPSCs are induced pluripotent stem cells, which are generated from adult cells. They could be from any body cell. In our case, we are harvesting cells from urine. Usually, people also take skin cells, fibroblasts from the skin, or cells from the blood. With some tools, we can put these cells in a state that resembles the embryonic state of stem cells.

The MSCs, mesenchymal stromal cells, on the other hand, are specialized cells and can be found in many tissues or even in all tissues of the human body. They have a particular function there. Adipose tissue, chondrogenic tissue, or bone are the cell types into which they can be differentiated.

Benjamin: That means that iPSCs do not have any limitations in terms of differentiation. You can differentiate them into every cell type or tissue, right?

Klaus: That is correct. They are replacing, from our perspective, embryonic stem cells, which have ethical issues and are hindering the field. They have some genetic question marks. Ensuring the preservation of genetic integrity is essential for these cells because it is crucial to prevent any genetic aberrations during cultivation that could have negative effects on the process or the final product.

Benjamin: You also mentioned something very interesting. You receive the samples, somatic cells from urine, or human urine samples.

Klaus: That is correct.

Benjamin: And what are the next steps? As far as I know, or as far as I can remember, somehow, you need to introduce stem cell-associated genes into those cells, right, so that you can transform them into iPSCs. What technologies do you use there?

Klaus: So, we expand only a subset of the cells that you can find in urine, for example. And then, we introduce certain genes into these cells. We do this in a virus-free manner.

There are different ways of doing it. We use episomal vectors, which are transfected. This works very well in our hands and has the advantage that we do not have to handle viruses in our laboratories. These episomal vectors are also – after a few generations – removed from the iPSCs. So, at the time point, when we bank the iPSCs, usually, these vectors are not there anymore.

Benjamin: And the transformation process we covered already for the iPSCs. Is there the same approach for the MSCs or a comparable one? Do you introduce genes or transcription factors into the cells then?

Klaus: That is a fragment of the human telomerase gene. How is this exactly done? I do not know all the details. It is the know-how of Evercyte to generate stable cell lines from tissues in a virus-free manner. This is certainly something that is not available from a lot of companies worldwide. So Evercyte is one of the very few companies in the world, which can do that successfully. We have a small library of cell lines from them, derived from different tissues, tested for biological activity, and so on.

These cell lines have undergone thorough testing and comprehensive documentation. They meet the requirements for GMP manufacturing, which is one of our primary objectives. Our plan involves conducting laboratory studies and process development, as well as successfully transitioning these processes into GMP to produce clinical study materials utilizing these cell lines.

Elisa: Right. MSCs, as you mentioned, are much more limited in their use. So, if iPSCs could be used for any purpose, why do you still have MSCs?

Klaus: That is also a good question. Certainly, MSCs have typically been tested in numerous conditions due to their inherent function of maintaining tissues and organs. They also secrete noteworthy vesicles that possess regenerative capabilities. It is this regenerative potential that makes these cell types highly sought-after in research and also investigated for therapeutic applications.

Elisa: You already touched upon it briefly, but specifically regarding these two types, how does Phoenestra tackle stem cell expansion? And what major challenges do you currently encounter in this process?

Klaus: First, it is critical to know that we work strictly under serum-free conditions. So, we don’t use any serum or animal components in our media, for example, or in our cultivations overall. And in the end, animal-free for regulatory and safety reasons makes a lot of sense. 

Our experience has shown that induced pluripotent stem cells (iPSC) are easily expanded due to their high activity and rapid growth. They greatly benefit from our pH control system, particularly in our bioreactors, as they tend to produce abundant lactic acid or lactate. By using our bioreactors, we can regulate the pH, provide additional media, and efficiently nurture the iPSCs for a specific period, enabling them to multiply and reach exceptionally high cell densities.

The MSCs need a surface to which they can adhere. This means we have to use so-called microcarriers. These microcarriers are floating in bioreactors. And there might be some mechanical stresses, which the cells do not like. So, we had to find a way to have enough surface in the bioreactor still to have them grow, but on the other hand, have limited or low mechanical stresses.

Elisa: That is interesting. When you arrive at the end product, what methods do you usually employ to isolate and purify the cells?

Klaus: This depends very much on the product itself. What is our product? Is it the cells? The iPSCs, for example, grow in spheroids. So, it is effortless to harvest them in this state. Of course, one obvious thing is that we do cell differentiation. We perform multiple steps with the cells including washing and concentrating them, for instance, using filtration methods such as tangential flow filtration.

As previously mentioned, the production of extracellular vesicles involves the separation of cellular components and cell debris. Filtration serves as an initial method for this purpose. We utilize tangential flow filtration to segregate various sizes of the secretome of cells, allowing us to fractionate it into different particle dimensions and beyond.

Benjamin: Yeah, that is a great achievement. I also want to shed a bit more light on the culturing process. You mentioned a lot of buzzwords like serum-free, xeno-free, and purification. Why is it so important to have a highly purified end product?

Klaus: There are different aspects to it. I believe that the level of purity of the product is not a major concern for me. The utmost priority should be to ensure its safety. We must remain vigilant in identifying and mitigating any potential hazards that could pose a risk to patients. These hazards may arise from the cell lines or substances utilized in the production, and even from the by-products generated during the manufacturing process.

Second is that we have to define our product. The authorities want us to understand what is active and why it is active. This is the primary reason why we currently do a lot of purification or separation of, for example, extracellular vesicles, to understand what components are active in biological assays and meaningful functional biological models.

Quality Control and Monitoring

Benjamin: Right. So, we learned a lot about the processes and platforms to use. I want to go more in the direction of quality control and monitoring. In the context of your iPSC bioprocessing, how do you currently address quality control and monitoring?

Klaus: We monitor pH, temperature, and oxygen concentrations, and supply gasses as needed to the bioreactors. Additionally, we regularly check glucose levels and measure key metabolites usually at line.

Microscopy is a very important piece of the analytical puzzle. We regularly evaluate cell morphology, purity, homogeneity, viability, and overall cell count. These factors impact productivity in cell and vesicle production.

We also need to look at this to re-confirm the identity of the cells and to measure the right marker proteins, for example, stem-cell markers for the iPSCs and MSC typical markers. For the iPSCs, we’ll especially look into genetic integrity, and also, perform HLA typing.

Benjamin: I am very interested in cell counting and characterization of cells. You want to bring AI into your processes, right? How would you envision implementing an image analysis platform like IKOSA to enhance your quality control?

Klaus: That is something we have been already exploring together with KML Vision for quite a while. We need to monitor the state of our cells. For example, what are the viabilities and what is the cell growth? So, I think there are ample opportunities for AI lead image analysis.

In my opinion, the implementation of the tools will be driven by specific factors. Currently, there is a growing trend towards differentiating iPSCs into various cell types, which takes place in a bioreactor process. We must closely monitor the cell state, differentiation process, and the transition from iPS cells to progenitor cells, and eventually to the desired cell type and its quantities. For instance, we need to determine the amount of non-transformed cells remaining and address other related specifications. Also, the reproducibility of these processes raises important questions.

In my opinion, image analysis is undoubtedly one of the crucial supplementary tools required to achieve this. It plays a pivotal role in showcasing our capabilities and assuring regulatory bodies that we possess the necessary control over the process. It enables us to replicate our work consistently and meet specific requirements.

Elisa: I am curious about the use of a platform like IKOSA. Would you see it as a tool that comes in handy during production quality control, or when you start using it later for product efficiency, to see if it is what you were aiming to have in the end?

Klaus: I believe it would be appropriate to start developing such a tool even earlier than previously stated. It is logical to initiate the development of this tool simultaneously with the creation of a product or the establishment of a process to produce the product. Investigating further into manufacturing, I would argue that these tools hold significant importance. In the biopharmaceutical industry, we have been engaged in process modeling and modeling practices for over 15 years, if not longer. We have been using the ‘golden batch’ principle to understand and control our processes better.

I believe there will be a similar development in the field of cell and gene research. Process modeling has already found its way into the realm of Cells and Gene. I think image analysis holds great significance in this domain, particularly for distinguishing between various cell types with distinct shapes. Microscopic examination can also track different features. These tools can play a crucial role.

Elisa: I think there is always this balance, you try to keep because you mentioned costs and risks, or safety measures. But on the other hand, you need productivity, like a good workflow. Do you see a tool like IKOSA aiding with that to keep that balance or even shifting it to more productivity, fewer costs, and fewer risks?

Klaus: Yeah, I think the expectation for IKOSA tools is also to be able to detect early when processes, for example, deviate. And when an intervention might rescue a batch, right? This is particularly important in manufacturing as batch failures can be expensive. The same applies to the products we strive to produce. Therefore, it is clear that if we can rescue batches by detecting deviations early and fixing them, this tool will be invaluable in controlling manufacturing costs.

On the other hand, as I said before, fast analysis and a better understanding of what is going on and, ultimately, faster development can be arguments. By employing supplementary tools, one can enhance the results obtained from an experiment. It is widely acknowledged that the human eye has limited perception capabilities. Additionally, analytical methods solely assess specific aspects for which they are designed. Hence, incorporating orthogonal methods in the evaluation of our intricate products holds immense significance. In this regard, image analysis serves as a vital component of these orthogonal techniques.

Benjamin: Yes, whatever you’re interested in, you’re going to annotate it in the images, train your application with it, and then analyze it. So, there is a great chance to also gain insights or data, which you don’t know yet, right?

Klaus: Yes, exactly. I believe the expectation is that one plus one equals three, not two. However, it is essential to involve the expertise of the KML Vision team. I am confident that by working together and combining our problem-solving skills we can generate thrilling opportunities.

Benjamin: Let’s maybe dive a bit into the process of quality control. If you introduce the cells into the bioreactor, what would the workflow look like? When do you take the first sample?

Klaus: Typically, the initial sample, known as the seeding or inoculum, undergoes a thorough evaluation, including viability and specific cell markers. Subsequently, daily samples are taken to assess the metabolites and nutrients daily. The bioreactor-controlled parameters are of course continuously monitored and regulated. Should there be any deviation in pH levels, it is immediately managed online.

Other things are done offline. We look at different aspects of how we produce extracellular vesicles, the count of particles in the supernatant, and so on. And this could be on a daily or bi-daily manner. It is not necessary to do this every day but regularly. You will check your culture and calculate your productivity from that.

Benjamin: You talked about assessing the viability of cells. If you are taking a sample are you taking the phase contrast images? How do you assess the viability then or how do you count cells?

Klaus: Usually, we do staining and fluorescence microscopy. We use Calcein for live cells and Dapi for dead cells. There is a life-death ratio and I am happy to say that usually, we see very, very few dead cells, meaning very little blue, and a lot of green in our cultivations.

Benjamin: That sounds good. Additionally, I can envision the possibility of using IKOSA to evaluate the yield or final product, presenting another potential use case.

Klaus: Indeed, I agree. I believe that understanding our cultural progress, achievements, and sustainability is a crucial area that piques our curiosity. To accomplish this, we acknowledge the necessity for supplementary engineering tools or avenues. However, it’s worth noting that we are receptive to exploring this area and we are aware of potential collaborative partners who may also be keen on developing novel ways of analyzing online cultivations.

Benjamin: What we didn’t clarify yet, is which cells, if we compare iPSCs and MSCs, are easier to process, easier to expand?

Klaus: Certainly the iPSCs, as they grow very well in our hands. As previously mentioned, they can grow in spheroids, negating the need for a surface. They exhibit self-organization by forming small aggregates that gradually enlarge. These aggregates possess empty interiors, which means they have no restrictions on nutrient availability within the diameters we are working with. Additionally, they can be expanded by approximately 15 times within a short period, resulting in a high concentration of millions per milliliter. This remarkable capability serves as a solid foundation for conducting various processing procedures, such as differentiation.

On the other hand, the MSCs, as I said, are very delicate. Our team calls them ‘Divas’ sometimes because they do not like mechanical stresses, and they like surfaces. This is a little more of a challenge to develop a platform here. But we are thrilled to say that we now have a proprietary setup that is very productive for cell expansion, and also for extracellular vesicle production, which we are also filing for a patent currently.

Benjamin: How long does it take, let’s say, from the start of inoculation to the time when you want to yield it or until you reach the end volume of cells?

Klaus: For the iPSCs, it is just four to five days, then we have expanded the cell mass and they slow down their growth. We could probably expand this a little bit with different feeding strategies, but so far, there was no need for that. 

For MSCs, we’re seeing a cell expansion phase that goes over seven to ten days. And then usually we have reached a maximum on our support and harvest cells or go into perfusion mode for prolonged EV production.

Benjamin: It sounds quite quick to me.

Elisa: Indeed, although it may be a rapid process, the ultimate objective is to achieve a clinical translation. Given your characterization of MSCs as the “Divas”, the more fragile entities, would you still regard them as potentially falling behind in attaining future clinical translation or usage, for evident reasons?

Klaus: Not necessarily. For me, the crucial question lies in determining the location of the activity. How can we precisely define and create a therapeutic approach? To be frank, when it comes to MSCs, I have a strong belief in the significance of vesicles rather than the cells themselves as a viable product. Of course, the cells still hold potential, but based on numerous clinical studies, their success seems quite limited.

I think what now comes more and more is that the extracellular vesicles or products you derive from EV preparations can have very similar effects as the cells themselves. EVs are maybe easier to manufacture and also safer to apply. And all this still needs to be proven in different indications.

There was a recent press release I think from a US company that has tested extracellular vesicle fractions out of their proprietary process, which was tested in a phase two study for acute respiratory distress syndrome (ARDS) (Direct Biologics Announces Publication of Significant Survival Benefit with ExoFlo™ in its Phase 2 Randomized Controlled Clinical Trial in the Journal CHEST | Direct Biologics). And they could show a dramatic decrease in mortality in the treatment arm of the study. This shows the potential of these vesicles.

I think a big argument for these vesicles is that there are no cells involved. So, teratogenicity is not a topic anymore. However, I have to say that MSCs and the telomerized MSCs we use are regarded as safe and non-cancerogenic or have any other safety issues.

Elisa: Interesting! Are any efforts being made for this? Are there plans to collaborate with other companies sharing the same goal, to gather more data and create a comprehensive registry?

Klaus: Of course, it is very important. We have a network of partners that help us master this complexity that we’re working on. Similar to how KML Vision and Phoenestra work together.

I think a small company like Phoenestra couldn’t do this alone. And so we work with several other partners who have excellent opportunities or capabilities available to make steps forward to collect data on these very complex products. And stepping more into the biology or the medical questions, it is even more important to collaborate with partners, who have special know-how and those tools available.

Elisa: You mentioned regenerative medicine, are there any other therapeutic areas that you consider equally important or that you’re targeting?

Klaus: Regenerative medicine is a broad term. We aim to explore two conditions where our vesicles and cells, with their anti-inflammatory and anti-fibrotic properties, can make a positive impact. Our objective is to demonstrate this concept in a preclinical setting. To advance this research, we need to prioritize and find partners. Given my experience in the biopharmaceutical industry, I understand the necessary competencies and resources for therapeutic development.

Benjamin: Certainly! I completely agree with you. The aspect you highlighted holds immense significance, particularly for startups and smaller companies like ours. Collaborating and pooling our resources is essential to thrive in this competitive landscape. One major advantage we possess as a small company is our unparalleled flexibility, don’t you think? I believe that this could prove pivotal in achieving significant success.

Klaus: Absolutely. And, you know, what I always feel is that some people think, Oh, why should you be successful? Right? Why? Why aren’t others doing this already if it is promising? But as I said, we are the innovators in the industry, Big Pharma is not the innovator, they buy innovations. We are flexible. We are agile. We can create synergies by joining forces. So I fully agree with your statement. And I think this is why I also believe we are fully on the right track.

Benjamin: Exactly, yes. What would you say are the most exciting developments and breakthroughs on the horizon in stem cell and extracellular vesicle research space?

Klaus: I am of course biased here. I think looking at the iPSCs, they show great potential which is still not harvested, right? Why is it not harvested? I believe we need iPSCs that are engineered so that they can be applied directly or in the form of differentiated cells to patients without immunosuppression. 

So, you asked about the next exciting thing, I think it’ll be a universal cell line or a universal iPSC library. It will not be one cell line but a certain number of cell lines, which can make cell therapies successful without immunosuppression. This is number one. 

The second option is heavily focused on the vesicle field. Extracellular vesicles are naturally secreted from cells or mechanically generated nanovesicles. I strongly endorse nature and prefer avoiding complete engineering from scratch or overloading these vesicles. However, I believe a more effective approach lies in leveraging the vesicles’ natural capabilities, enhancing them, and utilizing them for diverse therapeutic applications. This approach could potentially replace viral gene therapies or non-viral gene therapy. Only time will reveal the outcome, but these are the two avenues I place my confidence in. It’s important to note that my perspective may not be entirely objective.

Elisa: Yeah, certainly an interesting field. So, as we wrap up, is there any additional information you would like to share with us about Phoenestra’s work, goals, or broader landscape of stem cell processing?

Klaus: No, I think I have already said a lot. I feel that we have started to address certain topics systematically. I think systematic work pays off, but it may not be funded well. It is not always easy to sell these types of projects to investors. But I am happy to say that it bears fruit and that we found support from investors. Also, I think we have to be thankful to, especially Austrian funding bodies who believe in us and who support us as much as they can.

I believe that the most crucial aspect is having an exceptional team. I am pleased to announce that we possess a skilled, driven, dedicated, and enthusiastic team. They are responsible for advancing our work both within our organization and alongside our partners. Consequently, there is plenty of interface management involved. However, it is truly the team’s qualities that bring me immense happiness and confidence in our prospects for success. Additionally, we enjoy fantastic collaborations with an exceptional network of partners, which ensures that our journey is never dull and always enjoyable.

Elisa: I appreciate your optimistic perspective as I believe it is crucial to possess a visionary mindset. As you mentioned, having a strong team and reliable partners makes achieving goals much more feasible compared to the absence of these key elements. Therefore, I share your excitement about the future and extend my gratitude for your time.

Klaus: Thank you very much for making all these efforts. It was fun talking to you! I wish you all the best and we will keep in touch.