Better Media, Better Outcomes

David Sheehan: Well, thank you, Rachel, and super excited to have everyone here. I think these poll results… Can everybody see these poll results?

Rachel Gauvin: Yeah.

David Sheehan: Okay, great. So I think these poll results align with what we believe also that media can have a significant impact on your therapy. And 92% of respondents felt that that was the case, and that’s really what we’re going to talk about today is better media and better outcomes and what is the relationship. And I think in a lot of ways, we’ve evolved in cell and gene therapy, and now we realize the importance of media and the importance of controlling the media to drive your therapeutic efficacy.

This is our panelists today. My name is David Sheehan. I’m the President and CEO of Nucleus Biologics, and we’re a leader in custom media and reagents. And we believe that media is a critical part of your cell therapy ecosystem. And I’ll let the other panelists introduce themselves. Roddy?

Roddy O’Connor Ph.D: Hi, I’m Roddy O’Connor, a Research assistant professor at the Center for Cellular Immunotherapies at UPenn, and very interested in this webinar today because my focus is on CAR T cell metabolism.

Alex Klarer: Hi, my name is Alex Klarer. I’m the Head of Cell Therapy Development at BioCentriq, an emerging cell and gene therapy CDMO out of Newark, and we’re excited to be working with Nucleus and the other partners here to talk about the advantages of custom media.

David Smith Ph.D: And, finally, hi, everyone. David Smith, Director of Research and Development at Hitachi Chemical, so a CDMO devoted to cell therapy and really challenged, I guess, in that manufacturing space to say, how do we manufacture these future therapies? We know how we do it now isn’t really working particularly well, and, so, I was intrigued to dive into some of the challenges we’re facing and some of the solutions that you like to bring.
Overview and Start of Presentation

David Sheehan: Yeah, great. Thank you. And maybe we’ll get started and talk about what we’re going to cover today. So, clearly, our industry is in a rapid growth phase, and we’re not only going through this rapid growth, but we’re also in a period where there’s a race to get these therapies into the market as quickly as possible. And, so, time to market and the ability to, to cut time out of your development cycle is absolutely critical.

Media and reagents are a hidden source of variability. We know that. Whether you’re talking about classical media, and you’re going to learn about it today, or proprietary media, and propri-, — by proprietary, I mean media that is sold kind of as a black box. You buy it from a company, and it’s designed for a specific cell type, but you don’t necessarily know what’s the constituent components. And, so, both of those are a source of variability, and individual components in your, in your media can have a profound effect on your therapeutic efficacy. We’re going to demonstrate today just some very simple examples that both prove the ingredients in your media can have an effect in terms of tumor killing capability and in terms of efficacy.

And, up until now, there really haven’t been any great choices. Custom media is extremely difficult to cut, to create, and it’s a long lead time and, in many cases, cost prohibitive. And really what we believe is that, as a scientist, you need to control your media. When you control your media, you have knowledge of the ingredients, you have the ability to troubleshoot results, you get your — you can choose second source options. The media can become part of your company intellectual property, and that can not only impact your therapy but improve your ROI as a, as a corporate entity.

And, finally, it’s maybe a question that we’ll loop back to at the end of this webinar is, if media can impact your therapy, can it be a competitive advantage? Can it be more than just what you’re growing your cells in when they’re outside the body? And our, our view is the answer is yes.

So, if we look at this industry, we’re at this cusp of the very rapid market growth, and the panel on the upper left is basically the growth in terms of billions of dollars in cell and gene therapy market, and we’re at this point right now where the market is growing at an 80 to 90% clip. And, so, you know, that creates an underlying tension and, and pressure in the industry.

But the system’s imperfect because if you look at the lower right panel, $28 billion a year is lost on irreproducible research. And this is from PLOS Biology in, in June of 2015. But what’s most relevant about that is 36% of the time, it was reagents and reference materials that were the source of the lack of reproducibility. Again, I’ll say it again: 36% of the time, reagents and reference materials. So we’ve got this rapidly growing market, and then we’ve got this imperfect research model, and, now, we’re taking that into manufacturing.

And, you know, another data point is that an estimated 10% of cell therapy manufacturing fails. And, you know, those failures, many times, are not just that you’re under the therapeutic dose, but it’s problems with reagents or the manufacturing process. And the economics of failure are really pretty profound for a therapy company. And, so, there’s many sources of this variability. And what I love to do is have David step you through some of those sources of variability that lead to these problems.

David Smith Ph.D: Oh, thanks, Dave. And, yes, they’ve pretty much set up perfectly there –reproducibility, control, variability, all things that we really tackle with and talk a lot about actually. I think anyone that’s been to any in-person conference last year, any virtual conference this year would have heard a lot of the sort of, we need a better workforce; we need to look at automating to increase our reproducibility; technology’s a great solution, how do we work on that; logistics of getting products to each other; a focus on quality for all of this; and, now, is products designed to go sort of more towards the commercial avenue, where the scalability come in?

But I think there’s still a number of features that have such a profound effect on manufacturing that still haven’t really come to the surface. We still don’t talk about it. And, for me, you know, raw materials is one that really sticks out there. David mentioned that the failure rates, the number of sort of deviations we have every single time because of the variability into our process… And yet we go down sort of root cause analysis, and we can really point to a failure of technology, a failure of the workforce but don’t necessarily understand why that failure exists.

And, and I think if we’d sort of dive into raw materials and look at how much do we really understand about our raw materials, there’s a number of buckets we put into. We’re all aware, we know, especially in this industry, the variability that we get from our cells, that initial input. You’ll hear, obviously, that the groups like Heed My Care, Be The Match are trying to understand how they can provide a more consistent starting product, and that, really, variability starts there. It has a profound effect thereafter.

The consumables that we use, there’s variability within those, whether they’re sort of other reagents, beads. Single source in the supply chain is still so fragile in our industry and such a stressful area in order to try and operate and produce a therapy. Cost is something that maybe isn’t too much of a factor early on but really starts hitting the point. And then bioburden and particulates, what’s that even mean? Particulates we could spend an entire webinar on. They’re all things that add variability that we really don’t have a good handle on.

Media is another one that, for me, is maybe more profound than these. We’re looking at single source. We’ve seen issues in the supply chain in the last 12 months even prior to COVID, where we can’t get our hands on a single source supplier suddenly, especially — this is a market looking to go commercial, looking to scale up to really get bulk volumes, and our supply chain isn’t set up for that. Our manufacturers aren’t necessarily set up for that.

What does stability look like for these things? Another big issue, especially as you — the logistics, are they maintaining temperature? Are we recording temperature that our media is being held at? Lot-to-lot variability, and we’ll come onto this one. Do we understand the variability? Is there something we should be measuring in there as we go commercial? Do we really understand that? And then some of these other things that really just fall off the radar. How do you troubleshoot media, if the media has gone wrong? Can you go to some of these providers and change your media? You know, add a bit more glucose if you need it, to provide you formula that you don’t even know what’s in it, sort of thing?

Where does the traceability come from? If you’re buying from some of these large distributors that maybe have warehouses elsewhere that are stuck on a shelf at some room temperature maybe, maybe cold. There’s a lot of factors in here that we truly need to understand. So I think one of the prominent question that we asked at the start, do you believe that media can impact your therapy outcomes? It was great to see that of sort of the nearly 100 participants we’ve gotten here, that the majority are saying, “Yes, I think I do.” But then I think the follow up question, you know, there’s, there’s sort of cell culture ecosystem you control in that. You think media can have a, a big outcome on your therapy, but how much do you understand? How much do you control in your media therefore? Are you buying off-the-shelf media and throwing it over to the large companies out there and saying, you know, “We’re just going to use it.”

We know it’s important, but we’d have no way of controlling it. And then should we control it? Do we understand that variability? Is there variability in your media? Is there lot-to-lot variability, as we start to look to go commercial and then keep on coming back to it? We need to understand that. Believing what’s on the C of A isn’t necessarily good enough.

And, so, this is a list of just, you know, understanding a few of the factors that prominent features, that are in your media, that make it up. You know, water is a large part of it. There so many different ways of water that could have a profound effect, not even mentioning the amount of glucose or something, all the different proteins, cytokines, growth factors. There’s a lot that goes into the media. There’s a lot of variability between each of these components and even where the components come from themselves.

So, now, you’re looking at — you may understand that your media manufacturer has a great quality system, but where were they buying the components from? What’s the quality system that goes back? Go all the way through your supply chain to understand that. That’s a huge undertaking. So right now, we go, “Oh, we’re going to trust what comes on the C of A.” And I think we see as we go into the next slide if that’s something we should be doing.

So, you know, using actually the Rebel device from 908 Devices, an off-the-shelf, easy to use device, it took five different vendors of DMEM, the easiest basal medias that you can get, 14 out of the 24 components were different than the specification. Let me repeat that because everyone believes the C of A. 14 out of the 24 components were different to the C of A, and no two of these vendors are the same.

So does it matter where you get your DMEM from? I think it does, doesn’t it? Does it matter which lot you’re using? It probably does. You can pick out any of those individual media components and see the variation that some of them have between vendors, you know, going from zero up to sort of a normalized value of 2.5 on some of those. Does that have an effect? Maybe we’re barking up the wrong tree, and it doesn’t have effect. But I think there’s something in there that says, you know, the variation exists. Maybe we should understand if that is having an effect on our product. I think, with that, I’m going to hand over to Roddy actually to go a bit more into the science and understanding of how important these internal factors are.

Roddy O’Connor Ph.D: Thank you very much, David. Thanks. Yeah, so for us, you know, very interested in — I suppose we feel it’s very underappreciated how the properties of the medium can impact the therapeutic potential for, you know, cell-based therapies. And, of course, we’re very interested in T-cell-based amino therapies. And things like, you know, our collaboration with Nucleus and this webinar, it really highlights a renewed interest in how, you know, the, the media formulations can impact therapeutic effectiveness and efficacy.

So we’re going to discuss some specific examples here. And the first is from — it’s really seminal work from Roger Geiger’s lab, and this was a Cell paper showing how really subtle variations in one amino acid, concentration of a single amino acid, L-arginine, that was adjusted in the media formulation… And they looked at how this influenced outcome or the impact in a preclinical model of melanoma T cells that — they did as we do in the clinic — that were expanded ex vivo over a number of days.

So, you know, for this experiment, they established these melanoma xenografts in their mice models, and at the same time, they’re culturing tumor reactive T cells in a conventional media or in media, which, like I say, it’s a subtle adjustment; they’ve just increased the availability of L-arginine. And then once the tumors are established, they’re going to infuse the, you know, different groups of T cells grown in the very medium conditions.

So we can follow tumor growth on this graph here on the x-axis over the days, so days elapsed in the experiment, and on the y-axis, we’re really looking at an index of, you know, tumor burden. So we can see in the gray line, literally no T cell treatment at all, there’s an exponential growth of tumors, which would be expected. And, then, in the group that were infused with T cells that were expanded with conventional media, the T cells, you know, they have a remarkable anti tumor function in the melanoma preclinical model. You can see that there is partial control of tumor burden where the tumors come down in size, but by day 25, there’s kind of a relapse. So, once again, the tumors escape, and they emerge once again and grow exponentially.

What’s really interesting is that the group that received the higher arginine during the ex vivo expansion phase, that the T cells really outperformed any other groups. So they were superior anti tumor function. We can see that the, you know, the tumors are, you know — reach miniscule levels by day 20, and this persists over a long time. So the performance of the cells is a lot better than any of the other groups. So a subtle variation in the media component had a dramatic impact on outcome in this model.

So we’re going to discuss another, little closer to home, so from a recent paper we published in collaboration with Nucleus. And here we’re interested in, once again, it’s just a subtle change in the media formulation. We replaced the protein source, so human serum, which may become declining in years to come with Physiologix, this alternative from Nucleus Biologics, and, in it, similar kind of preclinical model. We’re looking at a model of neuroblastoma, where there’s established — we established xenografts in these immunocompromised mice and the tumors grow over a number of days and we prepared CAR T cells.

So it’s a CAR T cell this si-, — this time, redirected against a specific antigen found in neuroblastoma, the GD2 antigen, and the CAR T are expanded ex vivo either in media conditioned with human serum or Physiologix. And, following infusion into the xenografts, we can see that the, the mice that were or the T cells parts that were cultured with the Physiologix at varying doses, even, you know, when the doses were lowered to really challenging levels, to 0.75, that the tumor size was significantly reduced even at low doses, hinting at an increase in potency — and not just a general outcome but actually potency effect of changing one variable in the media formulation, so, so, you know, tremendous and immediate translational kind of relevance from just a simple adjustment.

And we are going to talk as well about — we can’t kind of talk about the metabolism issues without mentioning Erika Pearce’s work. She led the charge in a lot of how metabolic reprogramming influences T cell fate and anti tumor function, so, once again, returning to xenograft model of melanoma. And she introduced the concept of glucose restriction, so with her ex vivo phase of expanding, once again, tumor reactive T cells ex vivo, that with transient glucose restriction. So here it is. It’s intermittent fasting for T cells. It’s finally made it to the cell culture model.

But it really had a remarkable effect whereby if we look at the, the preclinical model of melanoma and the tumor size, that all the controls are there, again, where we see exponential growth if there’s, you know, nonreactive T cells present or infused, and the T cells where there’s, you know, transient glucose restriction — and, of course, it’s more than just a general caloric restriction because glucose has a very kind of indirect and direct roles in T cell effector function — that there’s a dramatic outcome, once again, from changing a simple parameter or variable in the media, the glucose levels that the T cells are exposed to during this kind of conditioning phase, which is highly relevant to clinical applications and all cell-based therapies.

So I’m going to kind of hand the baton to Alex now, and he’s going to continue our webinar here.

Alex Klarer: Thank you, Roddy. So, as he was able to lay out, your media is going to have a substantial effect not only on your manufas-, fan-, manufacturing success rate but on your therapeutic success rate as well. And, when you’re making a decision on such a critical part of your therapeutic ecosystem, you’re really stuck between these two choices. You can go with proprietary off-the-shelf media choice, or you can go down the path of custom media development. And I think, largely, the choice right now is people go toward off-the-shelf media, where you’re engaging with typically a sole source supplier who owns the formulation and who you need to trust is going to consistently and reliably produce and have available that media for you. But, on the flip side of that, you’re getting a readily available form of media formulation that you can purchase off-the-shelf and have available for you early on in your clinical development.

Then, on the other side of that, you have your custom media, where you can really define exactly what’s in that formulation, know every ingredient, and try and maximize some of those benefits that Roddy was pointing to with transient glucose restriction or the potential for certain amino acids to increase therapeutic potency. However, there — our current — the current avenues for generating that custom media aren’t conducive to the regenerative medicine industry, where a lot of the therapy developers are fairly small, are really pushing a lead candidate through clinical trials in an accelerated timeline, where you can’t work with a large media manufacturer and their six-month lead time in order to — and still meet your clinical, clinical timelines. And, then, if you start to see some efficacy issues during your development, it’s very difficult to iterate with that supplier and say I want to make small point changes on this formulation and still keep that sus-, supply available and ready for your clinical trials.

And, so, we see this happening in your clinical environment right? You’ll make these choices in a vacuum. As much as we all like to feel the focus purely on the science of it, you have to decide when you’re betting on a certain media formulation and what the environment is — that’s — that you’re going to make it within.

And we’ve kind of broken it down to these four major buckets that you might be looking at deciding on, on certain process changes. So you have early Process Development, where you’re defining the therapy and you’re really focused on generating the therapeutic product and having, having good understanding of your method of action, or then you move on to Pre-IND, where you’re preparing for GMP manufacturing and human trials, right? And then you’re moving on to Early Phase, where you’re focused on characterizing the drug product, the dose and potency of that therapy, and finally you have your Late Phase Product, where you’re optimizing the manufacturing process and getting ready for commercial manufacturing.

And, when you look at the, when you look at the comparability concerns with each of those pha-, each of those phases, people focus in on the Pre-IND phase to make these major media considerations. So you have a 10 to 13 month period where you’re developing your GMP-ready manufacturing process and the regulatory bodies are expecting some changes to the cell journey or some higher level comparability shifts between what you were doing in the Discovery Process Development phase and moving into your Early Phase clinical trials, where now you’ve filed your IND and your manufacturing process is a little bit more constricted. But, as we talked about before, you’re trying to hit these clinical timelines and you’re really time-constricted to make that choice and you might be focused more on the speed of getting these process development changes made, which is really going to drive toward the fastest option as long as it provides a reasonable level of efficacy. And, so, if we, if we expect people to be able to move toward custom media or have a better control and ownership over their culture media formulation, there needs to be a solution to that six-plus month lead time on culture media formulation.

And, so, to summarize what all of our participants have been talking about today, we are in a rapidly growing industry and the time to market is key for all these different therapeutic modalities because we’re all racing to develop hugely s-, — hugely efficacious therapies and modalities that haven’t been available previously. And we’re seeing a lot of variability, you know, and we’re keying in on a little bit more as we start to understand the method of action a little bit better. But we’re still facing a lot of variability, and media and reagents are an unexpected source of that variability when we’re using this kind of standard proprietary media that we’re seeing coming off-the-shelf in the industry right now.

And there’s such a high impact from each individual component within that media. You know, we need to be able to exert a higher level of control with — on each of those different components and until now, there hasn’t been a great option that would allow you to both keep with that short time to market that a lot of new therapy developers are dealing with and also address some of these smaller individual component variations that you want to control within your media.

And, as — I’m sure a lot of you scientists want to do is create your — is — own the knowledge of the ingredients, own the IP, be able to troubleshoot, and have more sustainable reagents and manufacturing process in the long term, so you can control the impact to your therapy over the lifetime of this product. So, if you are not customizing your media, you may start to lose a competitive advantage in your therapeutic area as some competitors start to take advantage of these new, new developments in this space.

David Sheehan: Thanks, Alex, and thanks to David and Roddy too. Really wanted to, you know, make sure we understand this is a, a broad group here covering everything from the development through — and the research all the way through to the manufacturing side, so you’re getting insight from people that are close to both ends of the spectrum, from the pure process development all the way through the manufacturing. And what I would love to do now is open it up to Q&A, and I’ll let Rachel take over at this point.

Q&A Session

Rachel Gauvin: Thank you, everyone. Please do type any questions you may have in the Q&A box here. We’re happy to answer them. Anything you want our fantastic panelists to comment on, please feel free to type that in as well. Don’t have any at the moment. We do have a couple of notes to us panelists saying — just validating how important custom media is and, and thanks for kind of driving this home. But one question we’ve just got: What kind of added cost is involved with custom versus off-the-shelf media?

David Sheehan: Yeah, maybe I’ll take that. I think, actually, custom media is probably, if you get into larger lot sizes — is going to be less expensive, and the reason why is because you’re only paying for what you’re putting in there that is unique to your ecosystem. And, so, a lot of custom media manufacturing is based on lot sizes, and if you get into larger lot sizes, the cost per litter is less for custom media than it is for a lot of the proprietary off-the-shelf medias.

Rachel Gauvin: Great. Next question is How long is the wait time to get a tumor specific media to use in cell culture?

David Sheehan: Um…

Rachel Gauvin: Take that one, again, Dave?

David Sheehan: Yeah, I guess I’ll take that one again. I think there’s — if it’s pure formulation development, which we do a lot of formulation services work, it’s typically around three months to develop a formula for a specific cell type. In terms of the manufacturing of the actual media, you know, our lead time is 12 weeks. And we’ve done it as — in as short as six weeks. And, so, it really depends on what your requirements are, but that gives you an idea of the time frame that we’re talking about. So, if you’re starting with a media right now, there’s — it’s a lot easier to, to create an improved formulation or a xeno-free formulation, if you need it.

Rachel Gauvin: Great. There’s one for —

Roddy O’Connor Ph.D: — I can add in. I can add something there. You know, we used to have reps that stopped by the lab and in passing, they’d always mention, “Oh, yeah, we can do a custom media.” And, then, you know, when they go back and you do a few back and forth emails, it’d be, you know — it would be like buying a house kind of thing, the cost, and they want, you know — there’s litters and litters you have to buy, and the time frame is just — it — it’s untenable. So there’s definitely, you know — highlighting the work done, you know, by you guys, Dave, it’s just fantastic for, for the opportunity to make customized media in a short time.

David Sheehan: Yeah, I think that was the idea behind NB-Lux. It was really to create a tool that put the control of the media back in the hands of the scientists and be totally transparent about cost and lead time. So you can go onto that portal, you can configure exactly what you need, whether it’s the testing, the packaging, or the individual component concentrations, and you’ll get a quote right then that you can place an order against.

And, so, the idea is, we don’t — we, we know media is important. We can take the mystery out of the media. And it has to stop being secretive because if we want to advance the science, we all need to work together to put these tools into the hands of the people that are developing these therapies.

Rachel Gauvin: Great. There’s some great ques-, questions coming in. I’ll continue to read through them. Do you think custom media formulations are more realistic for allo therapies that are HD derived rather than auto patient derived due to heterogen-, genei, heterogeneity? Sorry, guys.

Roddy O’Connor Ph.D: Heterogeneity. That’s, that’s an excellent question, Cody, and I think from the preclinical work, there should be a broad impact to all. So we can’t overlook it for anything. So it should have an impact, and it should be broadly applied to, you know, autologous and allogeneic.

David Smith Ph.D: I think — at the — we haven’t done enough work yet to even sort of really even touch the surface on that, I think. You know, we know that there’s heterogeneity within sort of, I guess — more within the auto patient derived, but, you know, should we be doing custom media based on your product? So is the CAR T media very different from an NK media? Very different from… Really get into the depths and understanding what each of these cells need, what amino acids are the ones that’s really driving it, that’s going to give you the quality, the efficacy, the potency, the cell number growth, and things. And I think, for me, there isn’t enough research yet out there to say, I think, you know… Custom media, I think, jumps out that it makes sense to go down a load because you suddenly need a lot more media, potentially. But I think, given that, I would start arguing a lot of the allo therapies that are in clinical trials right now aren’t that different in size to autologous. You’re still looking at single digit liters, maybe just get into double digits, right? You’re not looking at hundreds of liters that the biologics world is in, where it really starts playing out. And I think that, for me, has always been one of the sort of hurdles to overcome, is that, generally, it’s a low number. And maybe without taking over Rachel’s job here, I see one for David to answer. There’s a question there. It says, “What would be the minimum order for the customized media?” I think it’s an important one to dive into.

David Sheehan: Yeah, actually, on NB-Lux, you can order two liters, so two liters of custom liquid media. And you can also order powder of any size or any amount. So there is — the idea is to make this really easy. You don’t need a lot of media early, early on. Roddy talked about being told that you had to have these massive minimums. And I think it’s about what’s the mindset. The mindset is, industry is here to support the science. And, if we really look at it through that lens and everybody embodies that in their product offering and their pricing strategy, then we’re going to move this, this science faster. We’re going to get these therapies out there faster. And that’s really the philosy — philosophy that Nucleus has. It’s how do we make it easier for the scientists to iterate quickly to get to an optimized outcome?

And I think one thing that kind of ties a lot of this together is every single therapy provider has a slightly different ecosystem. They might be using a different bioreactor. They might be using a different cell. They might be trying different — to get different critical quality attributes. All of those have an impact around your media.

Rachel Gauvin: Yeah, that’s great. And Diana is asking if we have any case studies we can discuss. And I know here at Nucleus, we’re working on a few, but I don’t know if any of the other panelists know of any that they could share.

All right. Then, I’ll say hang tight, and we’ll get you some case studies shortly.

David Sheehan: Case studies in terms of —

Rachel Gauvin: — Um…

David Sheehan: Maybe — I’m sorry. I’m not seeing that because my screen is the one that’s sharing. What are the case studies?

Rachel Gauvin: Just asking if there are any case studies that we can discuss?

David Sheehan: Well, maybe, Roddy, you can talk. You’ve placed an order on NB-Lux. Maybe talk a little bit about your experience. What was it that you were interested in studying, and why did you use that tool?

Roddy O’Connor Ph.D: Yeah, so for us, we do a lot of — for the metabolic reprogramming studies, we do a lot of, you know, carbon tracing and isotopically labeled amino acids. So I gave the example of the arginine. And, you know, for those kind of studies, you have to kind of supplement an arginine-free media with, you know, an isotopically labeled or a carbon 13 labeled arginine as a tracer, so you can, you know, trace the contribution into central metabolic pathways, and, you, you know, try to get things like an arginine-free media or for other amino acids, if you’re interested in… So we presented recently at ASTCT on branched-chain amino acids. So it’s very challenging to, to, to, you know, obtain these medias. They’re expensive. So that was one thing that really, you know, was of interest to me, to have a really efficient platform to, you know, look at kind of novel amino acids that we could adjust very simply and would be, you know, made very efficiently and reliably for us.

The other was, you know, we work on the Seahorse platform a lot, and that’s kind of unique media in itself. So — and it can be quite expensive sometimes and maybe not readily available, so it’d be nice to, you know, once we, you know, understand the components of it, to be able to, you know, make it ourselves even and adjust it ourselves and tweak it to our own, you know, unique formulations or specifications. So that was why I think the platform is ideal for us for our purposes.

Rachel Gauvin: Thank you, Roddy. The next question is, Is it important to patent a novel cell culture media?

Roddy O’Connor Ph.D: Yeah, only if you include us.

All: [laughter]

Roddy O’Connor Ph.D: No, I think — I — I think you’ll find these challenges, right, Dave? Because you can’t control what other people put in the media, right? So, if you disclose what’s in it, and you found something great, it’s hard to — isn’t that, right? That you can’t control it?

David Sheehan: Yeah, I think —

Roddy O’Connor Ph.D: — Challenging.

David Sheehan: I think what the industry has done in general is avoid patents and kept them as trade secrets, and by trade secrets, it means that it’s not publicly disclosed. The challenge with medias that you patent is you will be forced to list not only the components but the concentrations. And, so, maybe as a therapy provider and as part of your system, it might make sense to go and file a patent. As a media provider? Unless you have found some novel compound. And I think it’s one of the things that I want everyone to realize is, you know, you, you buy these off-the-shelf proprietary medias from these companies, thinking there’s something novel in it. It’s really not that novel. It’s — a lot of times, it’s standard, classical, basal media that’s been supplemented with something so the — there’s not mystery. We didn’t create new amino acids that don’t exist. We’re using the same base building blocks. And, so, that’s why it’s important to have this transparency, to have this disclosure, and get out of this paradigm of secrecy that’s holding back our ability to get therapies into the market quickly.

Rachel Gauvin: Absolutely. The next question is How is this custom media different from Plasmax or the Tardito lab or HPLM from Sabatini lab?

Roddy O’Connor Ph.D: Yeah, so it’s kind of, you know, similar theme, right? David Sabatini’s work at MIT, you know, it’s, it’s — really highlights that renewed interest in an optimal media formulation. So he uses his kind of, you know, physiologic levels of all amino acids or metabolites like glucose in his mediums. That’s his media formulation. And, you know, very relevant to, you know, work in that area would be if you’re doing, let’s say, studies on intracellular signaling pathways. It’d be a lot more relevant to what’s happening in the body if you have a media formulation that recapitulates, you know, the concentrations found in tissues or found in plasma.

So his, you know, simulates or, you know, mimics what’s found in physiologic environments, and that’s very helpful for signaling studies, instead of having overly saturating doses, which is what’s present in media today. And, you know, that was designed just to support kind of proliferation or cell propagation, but it’s very limited in terms of looking at, you know, outcome in, in cells. So it’s that renewed interest. It highlights a similar theme.

Alex Klarer: Yeah, and I think we’re interested in opening up that media discussion from — to kind of beyond what it has been typically, where you might choose a couple basal medias and supplement different concentrations in order to test what you might want your media formulation to be carried forward. And, you know, maybe you’re going to base it on one of these predefined media. And, now, with some additional tools, you can actually tailor it a little bit more precisely and create a design space for your media, as opposed to relying on this design space that have been preordained for you.

Rachel Gauvin: Thank you. Next question is Have you seen any quality or functional differences between human source growth factors like the recombinant substitutions — and their recombinant substitutions, like transparent, etc?

David Sheehan: Anybody want to take a stab at that?

David Smith Ph.D: I’ll nominate Roddy.

Roddy O’Connor Ph.D: [laughs] Oh, yeah. We haven’t kind of actively studied that. The, the, you know, recombinant growth factors are kind of readily available. Yeah, we’re not — it’s not, per se, our active interest, but it isn’t, you know — it is — without saying that isn’t an interesting question because so many people just buy, you know, recombinant growth factors… So they should consider that.

David Sheehan: Yeah, and I think —

David Smith Ph.D: — I think it comes [crosstalk] Go ahead, David.

David Sheehan: Go ahead, David.

David Smith Ph.D: Sorry, I was going to say I think it comes a lot down to, you know, you’re relying on what the media company is telling you. And, so, a lot of what we see from the manufacturing is all what we’ve swapped out, we’re now using. Instead of sort of a, a human source growth factor, we’re going to use the recombinant substitute. Oh, and it’s better. And it’s going to be better in your product. But they don’t have your product. They don’t know what your product is. They don’t know exactly what you’re doing, and, so, without doing that test, you’re not going to know.

And I think it comes back to a lot of believing what the media companies are telling you versus really evaluating it. I think, you know, everyone on this call is probably taught to question absolutely everything, and yet, it comes to media, we believe what’s in front of us, and I think — and it took, to Roddy’s point — I don’t know that enough work’s been done to actually say if there’s a difference. I’m not aware of people necessarily doing that work. And there could be other drivers, I think. There’s other drivers to recombinant. Maybe it is less variability. Maybe it is lower cost. Things like that that are drivers. It isn’t always about necessarily getting the, the best product that’s possibly out there because there are other drivers in our industry: cost, time, speed, those sorts of things. But I think it’s an important question and a good one to ask.

Rachel Gauvin: Yeah. Another quick question here is Before the decision is made to the right formula, kind of like beer testing and tasting in the pub, could you make some different — provide some different formulas in small quantities to make the decision for the right one? And I think —

Roddy O’Connor Ph.D: — What a great question.

Rachel Gauvin: Yeah.

Roddy O’Connor Ph.D: The pub.

David Sheehan: [laughs]

Rachel Gauvin: I always love a good analogy.

David Sheehan: Yeah. I mean, I think the answer is yes to that, for sure. And, you know, again, speaking for Nucleus, we do a lot of formulation work in the cell and gene therapy space, so we have a library of, of compounds that we’ve developed or that we’ve tested on our own, independent of the contract work we do. And what’s novel about the way we do our formulation services work is you own all the IP. We don’t own it. So I think that’s — gives you the flexibility to know that one, your formula is secure, and it’s something that becomes part of your therapy IP.

Rachel Gauvin: Absolutely. And, like I said about NB-Lux, you know, it’s lots as well. It’s two liters, so you can order a variety and, and test yourself. Lots of great options.

Next question is, In a slide shown today, the actual concentrations of amino acids were different than the specifications. How can customers control the media if manufacturers do not meet the specifications?

David Smith Ph.D: That’s a great question. Again, I think there’s a part of it of holding your manufacturers of your media to the highest standards. And I think that’s throughout the industry we’re talking media, today. But it’s across absolutely everything, all of your consumables, the particulates in them, the supply chain of them, where the industry need to hold companies like Nucleus Biologics to a much higher standard than we have today. We’ve always got away, we’ve always trusted, we’ve always believed them, and we don’t push back. We accept what we’re given, I think.

There’s a certain amount that the regulation hasn’t pushed on this either, right? I think the regulators right now have given this industry a huge amount of flexibility in which to operate, given the results that we’re seeing from some of the products. And, so, I know, from us as a manufacturer, where we buy media in, we trust in the C of A. The reason we trust the C of A is that we’ve gone and we’ve done a quality audit on the manufacturer and said, “What they see is on the C of A and we believe that and we trust that.” Now, this data and as we become more powerful with data, we can really start to question that and as you get further towards commercialization, the regulators are expecting you to do that, right? You can’t trust the C of A as you get down commercialization. You’ll pull in from each lot. You take, you know, hundred bottles that you’ve ordered. You pull a bottle. You’re going to measure what’s in it. If it’s not to specification and a specification that you’ve agreed upon with the manufacturer that’s documented, you don’t accept it. You send it back. You don’t pay for it, right? And you, you hold them to that standard. And I think that, for me, we’re starting to see that now. We’re starting to see some of the more crucial elements, some of the raw materials coming in that people are setting up supply agreement with specifications in. And you know companies we mentioned, 908 Devices, right? They’re very easy to use instruments, where you can do a quick verification — pass or fail, accept, decline — and go down that route.

And I think, you know, we don’t need to do it for clinical trials. But the need is definitely growing, and the regulators are starting to push down on this a bit more. And it’s all part of — I think Alex mentioned it. They’re your design space. Learn everything you can. We can now measure all of the scientific information, so let’s utilize it. We actually have the power. We don’t have to buy anything off the media companies, right? They need us to buy their media. So we need to start sort of holding them to a much higher standard than we have today.

David Sheehan: And maybe just to piggyback on that, one of the services we offer to customers is if they want, we will do a chemical fingerprint of the media once we make it. We do have a 908 Devices. It’s a great product. It gives you a pretty accurate reading of all the amino acids and so-, soluble vitamins that are in there and the concentrations. And, so, you will know how it stacks up against the spec. And, so, that’s one thing we’re trying to do, to, to David’s point. We need to be driving the standard. We need to be proving to the consumer that this product is what we say it is.

Rachel Gauvin: Absolutely. The next question is How does Nucleus Biologics avoid media manufacturing delays as you mentioned frequently happens with larger media processors?

David Sheehan: Well, I think one thing that we try and do is control the entire supply chain. So we do our own milling and blending in-house, and we’re buying our raw materials directly from the manufacturers. That’s probably different from a lot of other — a lot of the big companies do that, but not many of the small companies do that. So, by controlling the supply chain, knowing the provenance of the raw materials, it’s really important. It gives us also the ability when we want to do quick iterations or uncommon combinations. We have that flexibility because we have all the raw materials in-house.

And, so, they’re still — I want to make sure everybody understands. Our industry’s going through this incredible growth phase right now. Global supply chains will tighten, and if you’re on the procurement side, you’re seeing it right now. Whether it’s bags, tubes, bioreactor, whichever it is, global supply chains will tighten. And, you know, picking who you want to partner with will become very important to your — ultimately, to your success. And, so, what we try and do is form collaborative relationships with our customers and make sure that we have everything in place to support their needs.

Rachel Gauvin: Great. Roddy, this is a question for you. Going back to your recently published paper comparing HS and Physiologix media, was the consumption of other standard media components, for example, amino acids, different between the cultures? Or were the changes solely due to the composition of the supplements?

Roddy O’Connor Ph.D: Yeah, that’s a good question. Actually, didn’t have the Rebel 908 device on hand at that time, so we didn’t actively look at that. We are formulating a new, even better media, and we’re actively exploring it. So it’s, it’s one of those kind of stay tuned questions, but we’re certainly taking a more comprehensive look at that question with the new technologies we have on hand now, so…

Rachel Gauvin: Great. Next question is, Since all the CAR T, NKs may be — have different metabolic needs, some CARTs may not need specific nutrients than the other. How do you troubleshoot this issue, or thoughts on this? Any timeline for this?

Alex Klarer: I think this goes back to — you know, what we’re talking about is figuring out what the design space for your cell — your particular therapy in your particular cell type is going to be. I think what we want to move — potentially move away from as an industry is, you know, relying on media manufacturers to provide all encompassing solutions for what are, like, pretty variant cell population, and especially with what we’re seeing with the definition within T cell and NK cell populations, where, depending on the type of lymphocyte that you’re trying to culture, you’re going to have slight or major differences in that metabolic need.

So I think it’s helping your, your industry partners or your development partners understand what your needs are and use the solutions like, you know, rapidly customizable, small batch media formulations to augment the already in place media analysis you might be doing and moving past the media analysis as just assessment of certain basal medium or media with protein supplements or some additional supplements.

Rachel Gauvin: Thank you. The next question is Can you give some thoughts on using animal derived components such as FBS or BSA and CGT media versus pure chemically defined alternatives?

David Smith Ph.D: That might be a “How long have we got?” [laughs] And I can go extremely high level and, you know, not saying anything that we don’t know around where are we going to source enough animal derived components as our entire industry goes commercial. Or are we going to get stuck? But, on the backhand, what we see, they work better. We, we use chemically defined media from large suppliers, and we add serum to it, even though they say you don’t need to add serum to it. Even they will add serum to it when they start putting out papers and posters and things like that, right? Is that to date so far? That’s where we stand.
Now, there’s always new things coming on the market, and I’m sure Nucleus can talk more to sort of — where they stand on this issue. But, to what we’ve seen is that, you know, we’ve moved away from FBS, given all the tight regulation around the supply chain of FBS. Is that going to be the same for human serum in a, in a few months, years time sort of thing? Are we going to go more chemically defined?

I think, if we really want to understand variability, well, we know there’s huge variability in serum, right? We know it’s a big issue. We know it’s a, a can of worms if we start going down there, so people don’t want to do it, I think, you know. I think it’s a, it’s a really tricky one that cells seem to have a better quality with it [sounds like, 00:54:08], to date, and that’s changing and evolving before David jumps in. But I think, you know, we need to see a way that we can remove it. We need to find a way to remove it if we really want to have a better control over our manufacturing.

David Sheehan: Yeah, agreed, and I think we’re in a unique position because we started as a fetal bovine serum company. And, you know, we prided ourselves on great lot-to-lot consistency, but there was still variability. And I think, you know, most of the formulation work that we’ve had over the last year has been moved. We’re moving animal origin products from medias and finding viable substitutes. And, somebody had asked earlier. For sure, recombinant growth factors are less potent than, you know, the human derived versions or the composition proteins, and I think part of that may be the recombinant manufacturing process. It also may be that there’s more signaling molecules in those composition proteins. And, so, I think where the industry is going is towards recombinant proteins. And I don’t see that — I see that trend accelerating, not slowing. And, so, you know, to David’s point, FBS has been around for 40 or 50 years, so it will always have a place in — on the research side. It just won’t have a large niche or a large position in cell and gene therapy because the regulators don’t want to see it.

Rachel Gauvin: Okay. And one last quick question. I know we’re running up to the top of the hour. Can NB disclose XS — XF Physiologix components?

David Sheehan: Yeah, absolutely. And, in fact, I’ll say it across the board: you know, we’re developing a T cell media with Roddy right now. We will disclose the components, so if you want to reach out to us at any of the information on here, I’m more than willing to get on a phone call and tell you what, what is in Physiologix and, and also kind of some of the other work that we’re doing.

Rachel Gauvin: Great. And I know there are a couple of questions we didn’t have time to get to. If you still have those, please contact us here at any of the emails you see here. Or you’ve got some emails from us about this webinar, so you feel free to respond to those emails. We’ll definitely address each and every question personally there. But I want to thank you all for your time in joining us today. I hope you got something out of it. And thank you to our panelists.

David Sheehan: Thank you, Rachel.

David Smith Ph.D: Thanks, Rachel. Thanks, everyone.

Alex Klarer: Thank you.

David Sheehan: Thanks, everybody.

Rachel Gauvin: Have a great day, everyone.

David Sheehan: Okay, bye now.

Rachel Gauvin: Take care.