Podcast Transcript

David Brühlmann [00:00:37]:
Every cell therapy begins with a promise: that a single cell survives long enough to become a cure. But between the lab bench and the patient lies a journey most people never think about: freezing, thawing, shipping, and a lot more. Today, we’re joined by Sean Werner, who’s the Chief Technology Officer of Cell Processing at BioLife Solutions, whose career path has centered on protecting that fragile promise.

Welcome, Sean. It’s good to have you on today.

Sean Werner [00:02:27]:
Yeah, thanks for the invitation. I’m looking forward to the conversation.

David Brühlmann [00:02:30]:
Yeah, so do I. To get us started, Sean, share something that you believe about bioprocess development that most people disagree with.

Sean Werner [00:02:39]:
So I think what comes to mind for me off the bat is, within the realm of cell and gene therapy specifically, that distributed or decentralized manufacturing is going to see real growth over the next 10 to 15 years. I think that’s going to be how we find ways to address some of these conditions for which developing a drug therapy isn’t commercially viable. So I think, for me, that’ll really be the opportunity to realize the potential of these novel therapies.

David Brühlmann [00:03:08]:
Before we dive deeper into today’s topic, tell us about yourself, Sean. You’ve built an impressive career. What initially drew you into biotech or cell and gene therapy, and what were some interesting pit stops along the way?

Sean Werner [00:03:22]:
After grad school, my first role was in a group that took me from thinking about cancer biology and that sort of indication pathway to working with a group that was looking into regenerative medicine products. They weren’t necessarily thinking about cell or gene therapies at the time, but were really looking at how hormone cycles versus direct delivery could lead to regenerative processes.

Thinking back, I don’t know how much background you have in developmental biology, but if you look at developmental biology, just the cycles of exposure to different cells can lead to the development of, say, limbs, organ systems, or whatever that might be. And so that was my first exposure to regenerative medicine processes.

From there, as you noted, I moved from that kind of biotechnology-based research into a medical device company, where I was working for Cook Medical. They had an autologous muscle-derived cell therapy program, which they still have today. I worked very closely with that team as well.

That allowed me to continue working in regenerative medicine while also gaining much broader exposure to medical products, both on the regulatory and approval side and on the development side. I was able to put those two things together: the cell therapy program and the question of how you bring products to market. I think that wasn’t exactly a straight path to working in cell and gene therapy, but that’s how I got there.

Over the years, while working with that company, we spun out a new division focused on evaluating new ideas, handling initial commercialization, and then potentially finding homes for those products. All of those products were specifically focused on concepts related to regenerative medicine.

We were working on medical devices intended to deliver cells without damaging them, or devices specifically designed for direct delivery to targeted locations. We were also developing supplements for cell culture processes and packaging solutions specifically for cryopreservation. By building that company, I transitioned from the therapy side of things more into the tools and processes used in regenerative medicine and cell therapy development.

In 2019, we spun that division out of Cook as an independent company, Sexton Biotechnologies. Then, in 2021, BioLife acquired us. I’ve been there ever since, now heading up product development at BioLife Solutions. It’s been a really great transition over the last five years.

David Brühlmann [00:05:51]:
Your company believes the promise of cell and gene therapy starts with the integrity of a single cell. Tell us more about that.

Sean Werner [00:05:59]:
That’s right. So if you think about potency in biotech or traditional pharma, it’s really easy to characterize at this point. I’m not saying it was always easy, but at this point, we have a pretty good understanding of how to characterize or identify the critical quality attributes of those products.

But for cell therapies, even though, depending on where you start, we’re 10 years into a full drug development and commercialization cycle, we still don’t really have a good definition of how we know that a cell is going to perform the way it’s supposed to. You really don’t know if it’s going to do all those same things based on what you’ve measured.

So for us, we really think that one of the keys is that you can do all the development work and all the in vitro analysis, but the best way to try to make sure that you haven’t done anything to change the course of that cell’s function is to really lock down and minimize any damage that happens during the cryopreservation step.

And so, if you’re going to do that, you might as well take the time to understand how to optimize that process. Because there are good ways to do it, there are bad ways to do it, and there’s a tone of variability along the way.

David Brühlmann [00:07:15]:
Let’s unpack that, because cryopreservation is key, and it’s absolutely essential. What is the exact challenge if we look further into what happens inside a cell when you freeze and thaw it? What are these challenges?

Sean Werner [00:07:32]:
So there are a couple of ways that cells can be damaged, and everybody generally has an idea that ice formation is a critical part of what damages cells. And that’s absolutely true. There are internal and external pressures, physical pressures, and biophysical pressures caused by ice formation. That’s critical.

But there are also other things that people don’t realize. For example, as the cell freezes, think about what happens to the water. All of a sudden, you’ve got very high osmolality right there at the surface of the cell because all that water is disappearing from the outside. The water content and size of the cells change because of water transfer, movement, and changes in osmolarity. That’s something people aren’t necessarily as familiar with.

That’s where the original technology we developed was focused on making sure that the cells were ready to handle that osmotic damage, as well as controlling ice crystal formation.

There are a couple of other aspects that are less well-known or understood. One of them is the ice nucleation process itself. Everybody kind of has in their mind that ice forms at 0 degrees, but in reality, that’s not exactly true. In fact, you can get supercooled water. People are familiar with that and have seen all the videos of people throwing a pot of water up in the air in super-cold temperatures.

I think that’s one of those things people recognize, but they don’t necessarily understand that there’s a really critical element to it when you’re cryopreserving a cell. As the temperature drops, you get that supercooled state. Once the ice starts nucleating, that’s an exothermic process as well. And if you go too far into a supercooled state, that massive spike in temperature at the point when the ice finally nucleates is really damaging to the cells.

People have developed processes to induce ice nucleation at a specific point during their cycle when using a controlled-rate freezer. But there’s a pretty common misconception that the protocol results in the nucleation you expect.

Sometimes, people will, for example, insert a probe, a thermocouple, to test a sentinel vial or a sentinel bag. But depending on how you place that thermocouple, it can itself initiate ice nucleation. The rest of your cell products may not undergo nucleation until a much lower temperature, which can be extremely damaging to the cells.

So you’ve carried out your process and think it’s working correctly. In reality, you’re damaging your cells much more than you expect because you didn’t perform the measurements correctly.

I think the last aspect, which is relatively new in terms of our industry’s efforts to address this, is a phenomenon known as ice recrystallization. That’s a thermodynamic process in which, as the frozen product increases in temperature, whether it warms up or undergoes temperature cycling, the ice crystals can change. Thermal cycling can occur during storage when you open the freezer or the cryogenic storage unit or liquid nitrogen tank.

As the temperature rises, the ice changes from small crystals, which are less damaging, to larger crystals. It’s a natural process, but when it happens, it can really damage cells as well. For example, if you have slow thawing or a non-optimized thawing process, significant recrystallization can occur and cause a great deal of unexpected damage to your cells.

That’s something to consider when you’re working on your product and carrying out development. You may not encounter these problems because you may be very good at controlling the temperature. But I think a lot of people recognize that once a product reaches the clinical setting, people are busy. They’re trained to handle all these different types of products, and making sure they handle a product exactly as required is a real challenge.

To address that, we and others are looking at technologies to inhibit the ice recrystallization process. So it’s basically an additional step to protect against specific damaging processes.

David Brühlmann [00:11:17]:
To manage this whole process in a lab is one thing, but as you said, once you’re in the clinic, it’s a totally different ballgame. The slightest temperature variation, mechanical stress, or anything else could influence that process.

So my question, Sean, is: can you really control this process by measuring what I’d call, quote-unquote, external parameters, such as temperature and so on? Do you really know what’s going on inside the cell every single time?

Sean Werner [00:11:49]:
Well, I think “no” is the easy answer to that. But that’s why we really believe you need to take the time to figure out how much protection against those variables you can actually build in.

If you have the right cryopreservation formulation, the right cryoprocess on the front end, and then figure out how to protect against damage during thawing, you can do a better job of defining what matters, rather than just telling a clinical user, “Yeah, just leave this out on a bench or thaw it in a water bath.”

If that works, you’ve already defined the process. If it doesn’t, then you need to figure out your protection methods and provide solutions so that users aren’t unintentionally damaging the product. It’s a challenge, but I think you have to build those guardrails in during the development cycle.

David Brühlmann [00:12:34]:
In bioprocessing, we talk a lot about manufacturability, or, in other words, starting with the end in mind. Make sure that you can actually manufacture what you want to manufacture. So what early cryopreservation choices should you make to succeed?

Sean Werner [00:12:53]:
To me, one of the most obvious ways to introduce control and reduce variability is to go through an optimization process.

A lot of people take what may have been used in an early research lab. Maybe it’s still coming directly from an academic lab, or even within the industry itself, people have a default idea of what an optimal cycle looks like. There’s a pretty common belief that freezing at -1 degree per minute is the goal, and that you can apply a single temperature drop at a specific point to nucleate the ice.

In reality, that’s not correct, and it’s not optimal for every cell type. That protocol was developed for a very specific cell type. So I think the first thing I’d recommend is taking some time to think about this early on, because the optimal process isn’t necessarily going to match what’s already been done.

David Brühlmann [00:13:46]:
And how often does a problem that appears later in the clinic actually trace back to these early assumptions or incorrect choices?

Sean Werner [00:13:54]:
I think that’s a hard thing to identify, to be honest. A lot of the time, when you’re looking at clinical programs and outcomes, it’s really hard to determine whether a specific part of the processing went wrong. I don’t know that it’s easy to identify cases where cryopreservation errors directly led to a failure. I think it’s more likely to become apparent during in vitro work as you’re developing your process.

But the challenge with cell therapies is that many companies have really aggressive timelines and milestones to reach the clinic and generate clinical data so they can secure their next round of funding.

At that point, they may not have developed good potency assays or really good methods for analyzing the cells. So it’s challenging to know whether your cryopreservation process is significantly damaging your cells when you don’t really know how to distinguish a damaged cell from a well-performing one. I think identifying that as the key issue is a real challenge right now. Better analytical methods will be needed to help us sort this out as we move forward.

David Brühlmann [00:15:01]:
What are some common cryopreservation assumptions or pitfalls you see people falling into?

Sean Werner [00:15:07]:
As we discussed a little earlier, one assumption is that a -1 degree per minute freezing cycle, using that default cycle, is going to be good enough. I think that’s one of the key issues. I really advise people to put more effort into making sure that’s actually what works best for their cells.

The second issue, in my mind, is how people evaluate their cells after thawing. People will typically perform cryopreservation, thaw the cells, and assess viability. They might also look at apoptosis or other measures. But many people only do this immediately after thawing, just to see how the cells are doing at that point.

However, if you take the cells, grow them out, and then perform additional measurements—not just viability, but also expansion rates and phenotypes—you may discover further effects. We’ve seen cases where protein production or cellular function can be affected a week or even two weeks later, depending on how the cells were damaged during the process.

So I think one of the big assumptions people make is that they can determine whether a process is working well by looking at simple measurements immediately after thawing. We definitely recommend looking further ahead and assessing how the cells perform over time.

David Brühlmann [00:16:20]:
The technical or scientific challenge is one thing. But we work in a regulated industry. What do regulators say about these protocols or about how to best manage freeze-thaw cycles?

Sean Werner [00:16:33]:
Not a lot, to be honest. Now, I will say that my direct understanding of the conversations between sponsors and regulators is limited to what we can find by looking at, for example, complete response letters or review memoranda for approved products. When you go through all of that, I’d say that, in general, regulators want to see that your process is qualified, validated, and controlled.

I don’t know that they weigh in specifically on the processes themselves. As with other parts of the manufacturing process, they really focus on whether you’ve done a good job of bridging if you’ve changed the process at some point.

That’s where I think most of the challenges arise. If you’ve made a change later in development, it can lead to a lot of back-and-forth and delays in getting approvals if you don’t have sufficient data comparing the process before and after the change. So I don’t think this is terribly different from most other unit operations in manufacturing. It’s just another challenge to manage.

David Brühlmann [00:17:29]:
Now we know the problem. We see that there’s a challenge, and it’s also difficult to control everything fully. My question now, Sean, is: what can we actually do to optimize the process? More specifically, what should we focus on? Should we focus on potency after thawing? Should we focus on viability? Should we focus on both? What’s the best strategy?

Sean Werner [00:17:52]:
I think it’s important to be able to measure some level of performance that you believe is critical to the effect you’re hoping the cells will have in vivo.

If you look at viability, and you have lower viability after the thawing process, there are some benchmarks out there that are, quite honestly, a bit arbitrary. For example, the idea that you should have 70% viability. That doesn’t necessarily relate to anything specific.

Apoptosis is similar. If you measure it early, it’s obviously going to account for some of the cells that you initially counted as viable when you come out of thawing. And that’s really obvious when you put them into culture.

But I don’t know that the cell number specifically post-thaw is a measure of whether or not that cell product is going to be functional because you’re going to have some cells and unless you really know that to have an effect, you need some specific concentration of cells that are going to perform their duty, I don’t know that that tells you much other than was there some unintentional damage.

I really do think that you need to get cells in culture, look at how they come back, do they thaw out and do they grow at the similar kind of rate that you expected? Do they perform the way you expected? Are they producing the impact that you want them to? And so from that perspective, I would say getting to a measure of what you believe is a critical potency-related outcome is the thing that you need to do, and not immediately. Look at it after they grow out.

David Brühlmann [00:19:15]:
And something I’d like to add, because perhaps some of our listeners work in the biologics space, where they focus on viability and that’s often good enough, is that the cell itself isn’t the product. Here in cell therapy, we’re playing a completely different ballgame, correct?

Sean Werner [00:19:33]:
That’s right. And I think what I was mentioning before is that we’ve only recently started to see that the cryopreservation process may actually affect how quickly those cells recover and start performing.

So, in the biologics space, what we think that may mean is that if you have an optimized process, you may actually get better producer cells after thawing, and they may start producing the product better and faster. So you may end up with higher titers during production.

It’s still early days, so we’re not 100% sure what the impact will be. But we do think there’s a role for this, even when you’re looking at what the cells produce rather than the cells themselves.

David Brühlmann [00:20:13]:
There’s a lot more ground to cover with Sean Werner, from DMSO-free cryopreservation to the regulatory scrutiny that can catch teams off guard downstream. We’ll pick that up shortly in Part 2.

If this conversation is giving you a new appreciation for everything that happens between the bench and the patient, consider leaving a review on Apple Podcasts or your favorite platform. Thank you for tuning in today, and we’ll see you in Part 2.