Path to Media Mastery, Part 1

Rachel Gauvin: Welcome, everyone, to the Path To Media Mastery webinar. I’m Rachel Gauvin, Head of Human Potential and Program Management here at Nucleus Biologics, joined here with some wonderful women and colleagues, and I’ll let them introduce themselves momentarily.

I just want to summarize and remind you all what we’re doing here today. We have three leading experts here to demonstrate their path to media mastery using our proprietary tool NB-AIR™ to help create custom media formulations. So they’re going to walk you through their protocols for experimentation and then use NB-AIR™ to create some custom formulations live in real-time. So you’ll get to see that.   

Throughout the webinar, I want to encourage you to send your questions and comments through the Chat and the Q&A box. We’ll have some time at the end to address those, so anything that comes up during the webinar, you can send as it arises. I’ll let everyone say a little something about themselves. We’ll start with Asma. 

Asma Ayari, PhD: Hi, everyone. I’m Asma Ayari. I’m a scientist at Nucleus Biologics. I work in the R&D team, and one of my main tasks is formulation service to develop custom media for cell and gene therapy. 

Rachel Gauvin: Great. Thanks, Asma. And, Lori? 

Lori Noffsinger: Hi, my name is Lori E.B. Noffsinger. I work with Cognate BioServices in the Baltimore team. Cognate is a Contract Development and Manufacturing Organization, a CDMO, and Baltimore represents the D in the CDMO. So majority of the work that we do in Baltimore is going to be the assay and process development. I thank Rachel and Tiana and their team for inviting me today because I think this — I’m really excited about learning more and figuring out what this is all about. 

Rachel Gauvin: Great. Thanks, Lori. And, Saba.

Saba Ghassemi, PhD: Hi. First, thanks for inviting me for the webinar. I’m a Research assistant professor in Center for Cellular Immunotherapies at Penn, and my lab works on optimization of generation of CAR T cells for adoptive immunotherapy. We would like to have the most efficacious CAR T cells, and that’s why I think the media is very important because that would be a fuel for CAR T cells and their function. 

Rachel Gauvin: Great, thank you. We do have a series of webinars that we recorded prior to this that are on our website that alludes to what Saba is talking about, about how important your media is and owning your formula and all of that. So, if you’re curious to learn more about custom media, I definitely encourage you to check out our website and our Education page, in particular. So thank you. 

We’ll dive right in. Asma, let’s share your protocol with us.

Start of Presentation 

Asma Ayari, PhD: Okay, so, for this, I am going to test this protocol, which is for proliferation of  T cells. So the idea for me and following my protocol is to activate T cells using Dynabeads™ in a small scale in a 96-well plate and then to assess viability and cell counts across the whole experiment from day zero to day nine. Then, to end up the experiment, that day nine, assessing the phenotype, mostly with CCR7. 

For the media that I’m planning to work on, it’s going to be — I will have my positive control, which is going to be OpTmizer™ supplemented with human serum, and then I’m going to use the NB-AIR™ to generate two to three formula to test and compare it to the positive control. 

So now I’m on NB-AIR™, and as I mentioned before, I’m going to use OpTmizer™ as a positive control. For the supplement, I’m going to supplement with human serum — use human serum as supplements. For my target cell type, it will be T cells. Once I select T cells, you can see that there’s a lot of information that is available on NB-AIR™ regarding T cells. 

If I go Next here, I’m able here to select among all these different CQAs, and one of the CQAs that’s most important for me is viability. If I select here Viability, you can see that there are three levels of impact on this CQA. If I select Decrease, I will have all the information and the papers that will show what decreases viability. Most of the time, these are the components that we want to avoid and not have in our media. If I select Maintain, I will find all the components that are known to maintain viability. If I select Increase, I will have all components that are supposed to increase my viability. 

For my second CQA, I’m going to select the Proliferation, and then, again, we have here these three levels. You can see here we have the ranking. Proliferation is going to be the second on my ranking. And, again, you can see here. We can have data pulled on based on Decrease, Maintain, or Increase Proliferation. 

The third CQA for me will be cytotoxicity, which is the CQA that I want to maintain. So I’m going to select Maintain here. As you can see, here it pulled automatically all the data that are related to maintaining cytotoxicity. So the good thing here is that what you can see, for example, for proliferation, for arginine, we have a score attributed to this component. This is built based on all the publication that is published out there supporting the idea that arginine is going to support proliferation. This means that there’s a lot of data supporting this information.

The nice thing here is that I have a link that will take me directly to the paper mentioning how arginine is going to improve proliferation, in case I want more information about that. If I go Next, after selecting these three CQAs with their different level and the ranking, here, this page, will help me select my basal media as a first step, and then, as a second step, I will supplement the basal media with the different components that were suggested by NB-AIR™ based on the CQA that I selected. 

So, for the basal media, you can see here that we have NB Recommends, which is the media that based on our experience in the R&D department at Nucleus Biologics, we saw that IMDM works better for this type of cells and for these CQAs. And then there’s also this information about what type of media is mostly used in papers that we used to generate this information. You have also the second media mentioned. So, as you can see, we have here RPMI, we have here DMEM, and we have here IMDM. I’m going to go ahead and select IMDM.

To select the different components, as you can see here, we have them in different category. We have cytokines, growth factors, inorganic salts, amino acid, dipeptides, and proteins. And there are other components: nucleotide, nucleoside, antibiotics, vitamins, and carbohydrates. We’ll also have this possibility to add all the components in case you’re interested in adding any other component that is not listed by NB-AIR™ and if you want to test it.

If I go back here, under cytokines and growth factors, for me in my experiment, I’m not going to supplement with these cytokines. So I have to deselect IL-7. And then what I want to test is sodium selenite as inorganic salt. So, when I select sodium selenite, we have all the data related to sodium selenite under the CQAs that I selected at the very first step that it’s showing here. Then, what I would have is the L-Alanyl-L-Glutamine, which is GlutaMAX™. As a protein source, what I would add is human serum albumin. I would also use Physiologix™. It’s going to be all for me, so I’m not going to add any other components more than that.

If I go Next, after making all this selection, the nice thing here is that NB-AIR™ is going to — I think I’m missing one component. Yes, I’m sorry for that. I just want to add human serum albumin. If I go here to NB-AIR™, you can see that it has the capability of suggesting different combinations of all these components that I’ve selected.

So, for me in this context, I would like to have the media that has all the components. As you can see, for each component, NB-AIR™ is suggesting a certain level. If you click here, on Information, you can have actually a range. You can actually change the level under this wrench. All this is to guide the user to make a media that works the best for him.

If I go here and then I select this one, I can save this formula. So I have all the components that I selected. If I hit confirm, it will automatically take me to NB-Lux™. From there on NB-Lux™, I can find my formulas saved in the cart. If I check here on View Formula and I want to check more on my formula and then to give more details on what I really want in this formula, if I want more details on the pH, on the osmolality, sterility, we have a lot of tests that are available here.

It also has the possibility to choose from different packages. As I’m going to do — on a small scale, I don’t need more than two liters. So I have to hit Confirm here and then Confirm here. Then, here, I also have the possibility to check again if all the components that I have selected, which are the IMDM, and then with the supplementation of all the components that are added. Then I hit Confirm here. From there, I can place my order, add my address, and then Confirm. This way, I will have the formula that has all the components that I’m interested in.

If I go back here to NB-AIR™, I can still select another formula from here. So the second one for me will be the same one as the previous one, except that it’s not going to have any sodium selenite. As you can see, it’s the same thing. So the idea for me is to compare the previous media that I have selected to this one. If I do the same thing, it’s also going to take me to NB-Lux™, and then from there, I can place my order. 

Rachel Gauvin: Thank you so much, Asma. 

Asma Ayari, PhD: Your welcome. 

Rachel Gauvin: We will move on and hear now about Lori’s protocol. Lori, take it away. 

Lori Noffsinger: This opportunity lined up with a request from our analytical development team, and they wanted some Tregs, enough Tregs, to do a flow assay on. When I get a request like that, that’s what defines really my CQAs. So I need enough, and I need to increase proliferation so that I can get enough, and I need to maintain my morphology or my characterization — and you’ll see as I walk through that, show that they’re still Tregs at the end of the day — and maintain my viability. 

And I’m keeping this simple. I’m not looking at this point, this assay, for any suppressive function or anti-inflammatory requirements. If I were, this is a perfect place to use this NB-AIR™ because — say I wanted to add rapamycin, or if I was looking at the cytokines, I would change different cytokines. Since I am keeping this simple and because it’s just a flow assay, I’m really interested in proliferation of my Tregs, as well as maintaining the viability and characterization. 

The method is just your standard method out there. I’m going to do my isolation, and I’m going to activate similar to Asma’s protocol and then expand split. And mine’s going to be a 12-day comparison. I’ll just have the single activation step. 

So, when I look at my medias, I actually — the standard protocol is with the TexMACS™, which is the Miltenyi. Since that’s what I’m used to using, I’m going to put that in there as a control, but I want to get away from it because I’d like to be able to manipulate these if I’m going onto different assays where I’ve got to do a little bit more manipulation. So my standard will be the IL-2 at about 500 units and the human serum albumin I will put across my formulas. 

Then, we can go into the software. I’m going to have Rachel help show you guys how I would do this. So it’s T cells, and I’m mainly interested in proliferation and maintaining the morphology as well as the viability. When I go down and I look at the list, a lot of different things come up. Certainly, the glutamine or the L-Glut I will use. I often notice when I went in there the TGF. The glutamine I’m used to seeing in a lot of different protocols. I’m going to put the glutamine and my human serum across the formulations, but what I like to do is — and here’s where I get the TGF Beta. I looked at the paper, and I got a few different ideas. I think that this is going to work for my Tregs. So, taking into consideration that I’m activating Tregs as opposed to a normal T cell activation means that these papers really are insightful and, again, with a good focus towards What do I want to get out of these cells at the end of the day? 

So I’m going to choose TGF Beta as one of my variables. I’m also going to tweak the IL-2. So I know that I want IL-2. But I’m not positive how much I really need based on the protocol that I’m using or on the paper. So I’m going to look at those two different variables. 

When I went all the way over to the end and I wanted to add a component, one thing I saw when I was looking at the papers was the BME. So I’m going to add in here the 2-Mercaptoethanol as well. And so one of my variables will be the BME and the TGF Beta. 

Rachel Gauvin: Remind me, Lori. You also wanted L-Alanyl-L-Glutamine and human serum? 

Lori Noffsinger: Correct. Human serum, L-Alanyl, IL-2, TGF Beta, and BME.  

Rachel Gauvin: Right, and we skipped most importantly: Which base media would you like to use? NB Recommends or the Most Cited?

Lori Noffsinger: I’m going to go with the Most Cited and go with the RPMI. 

Rachel Gauvin: All right. We’ll select that. We have our components. Let’s move on. 

Lori Noffsinger: Okay, again, just the same as with Asma, there’s nothing all the way over in the Formula One, and then Formula Four, I will go to, and that will have all the tweaks that I want to make. On my TGF Beta, I’m going to go with I think what the website recommends, which I think is 0.005. For the IL-2, the increased dose is going to be the 0.002. For the human serum across the line, I’m going to go with the 5%, which is 50. With the L-Glut, I will go with the recommended amount, which is going to be when it normally will pop up, at 434.44. Yeah. So, going back, that’s all my tweaks. That is everything I want to throw at it. 

When I go backwards, my other one, I just want to look and see what the IL-2 increase does by itself. So, Formula Three is just the 0.02 IL-2. I’m also keeping the heat — the serum and the L-Glut in both of them. That’s correct. My Formula Two, I’m going to look at the normal 0.01 IL-2, and I want to have my variables be the TGF and the BME. Oops, I forgot to put BME in my everything. I was throwing out that too. That’s 0.78. 

Rachel Gauvin: ll right, how does this look for you? You have your negative control here, this experimental setting. That look right? 

Lori Noffsinger: I wanted the L-Glut in there or the GlutaMAX™ in there as well. I was going to say. 

Rachel Gauvin: That’s right. And then your second experimental setting, you’re testing the increase in IL-2.

Lori Noffsinger: Right.

Rachel Gauvin: And then we have —

Lori Noffsinger: — but not the…

Rachel Gauvin: But not the TGF?

Lori Noffsinger: Or the BME and then everything thrown at it in the last one.

Rachel Gauvin: Yeah, great. Asma did hers one by one, but Lori, since you want all four, we can actually just save all to NB-Lux™. You can rename them. If I rename this as Lori’s control, we can rename all of them across the board so that when it populates in NB-Lux™, we can show that, but simply, since you want all four, we’ll just load all to NB-Lux™. Needs a moment. A lot of information processing.  All right. Just like with Asma, it’s going to take us right over to our account, and you’ll see that there’s formulas, Lori’s control, and then 2,3,4, like we had. And, just like Asma was able to do, Lori, tell us what you’re looking for.

Lori Noffsinger: I’m just going to go with the minimum amount. I’m fine with them in a one-liter bottle. So I would go with two one-liter bottles of all of them.

Rachel Gauvin: Just a reminder that you can see the base media and all the components. If you wanted to edit anything from the base media, this is where you would do that. And then we can confirm that and get you ordered. So you can review everything here as well. One more check there, and we’ll get your media moving. 

Lori Noffsinger: All right. I don’t want my team to sound like a bunch of nerds, but this is so fun. 

Rachel Gauvin: [laughs] Love it. It is fun. All right. You have a nice little depiction of your protocol here. 

Lori Noffsinger: Yep, and that is the end of it. It’s basically just a picture of what we kind of already went over and — 

Rachel Gauvin: — with all your fun formulas. 

Lori Noffsinger: Yeah, my fun formulas, and then I’ll test at the end for my CQAs and see what works. 

Rachel Gauvin: Well, we’re excited to see the results. All right. Saba, let’s hear from you. 

Saba Ghassemi, PhD: Hey. If you go on the next slide, I’m just going to give a little bit of a background because I’m just going to pick a complex media. Because I work on the CAR T cells and adoptive immunotherapy, want to explain that there are several factors that come together to contribute to the overall efficacy of CAR T cells. It’s quite complex. But, here, we’re only focusing on the one central question: How does media formulation impact CAR T cell fitness and CAR T cell function? Because it’s like a fuel optimization for CAR T cells.

The other factor that I’m interested in is the differentiation of CAR T cells. So, because of that, I developed a process that we eliminate the activation steps, which is common for many CAR T cell, basically, product when we generate CAR T cells, and that expedite the process, and it actually preserve the maximal potency of CAR T cells.  

If you go to next step… One of the problems that we have with the media currently is that it is designed for proliferation, and it’s not really designed for preserving the stemness and quality of CAR T cells. So what I’m looking for is a media that vary in component, that it can support the gene delivery for my non-activated T cells, and I would like to limit the differentiation of T cells and retain the full potency.  

So, basically, here is my design. I would use T cells, and I just add Lentivirus vector to these T cells, and then I would wait a few days. At the end of, let’s say, day four or day five, I will check the viability of T cells, and then I check how much CAR T Transduction I have, and then I will check their effector function, but I want to use some conditioning factors to boost basically my process.

If you go to next slide, Lori… I actually watched your last webinars, that one of the components that you guys discussed was that excessive glucose could impair the mitochondrial function because glucose — as you know, this is a snapshot from your website. The paper that you put in the NB-AIR™, it shows that glucose produced a lot of interferon gamma that induces differentiation. So, usually in the media that — right now, we use 25 millimolar, but here I would like to reduce the glucose level to 10 millimolar.

And I would like to add IL-7 and IL-15 cytokine to my media. These are homeostatic cytokines, and it keeps cells alive for a long period in the lymph node. And also for the non-activated T cells, it increased the transduction, makes T cells permissive for gene delivery basically, and then it’s very important for cell survival and for the quiescent T cells.  

Next slide. Also, instead of usual FBS or human serum, I would like to use Physiologix™. And this is basically with the collaboration that we had with Nucleus Biologics. We showed that Physiologix™ enhanced lentivirus gene delivery, and it also enhanced anti tumor function of CAR T cells. This is a paper that is in the website, in the NB-AIR™. And, yeah, we can take a look at it, but based on the paper, I’m going to use 2% final concentration of Physiologix™ instead of the usual serums. 

Next slide. One of the parameters that I would like to add is arginine. Again, based on a paper that I found in the NB-AIR™ website, it shows that T cells that are grown in four millimolar arginine, they demonstrate superior anti tumor function in a mouse model of melanoma. So I’m just going to add arginine into my media and to see if it boosts my T cells’, CAR T cells’, function.

Next one. The last parameter that I would like to add is carnosine. Again, in a study that we’ve done, we profiled factors in the media, and we identified carnosine as a relevant candidate that it — in a higher concentration of carnosine, it has — there’s higher concentration of carnosine in the optimal media. Carnosine is important because it controls the pH of the media, and that’s important for lentiviral gene delivery. So, based on the recommendation of the paper, I’m just going to use eight millimolar final concentration of carnosine.  

So, now, we can go to the website, and then we can implement all the component here. If you go to the beginning… I will use T cells, yes, as my main cells that I would like to work with, and then for my CQAs, one of the important parameter I would like to have, Viability, and I would like to maintain Viability because when I get the cells, they’re viable anyways. I would like to maintain it. And then I would like to choose Activity. Also, I would like to maintain that. Another CQA I would like to pick is Cytotoxicity, and I would like to increase the Cytotoxicity. 

Rachel Gauvin: This is where you got those screenshots from your slide, right? 

Saba Ghassemi, PhD: Exactly, yeah. That’s how I actually picked because I did this before, so I picked the papers and then took a look at the suggested concentration. As Asma explained it meticulously, it gives you a lot of different components, and then they’re all linked to published data. NB recommended IMDM; just would like to kind of compare it with the usual media that I use. If I want to choose my cytokine, as I explained in the slides before, I will use IL-7 and IL-15. So that’s IL-7. IL-15 didn’t show up, so we can actually manually add IL-15. Because, usually, some of these components are good for generally proliferation or other CQAs that I didn’t pick, but we can actually pick whatever that we’re interested in.  

If you go back, we have glucose, we have the cytokine. Nucleotides? There’s no nucleotides. I’ll pick Physiologix™. And then here, if you go down, no, I won’t use anything. And, no. Nothing? Okay. Here, let’s add arginine. Okay, the first one? Yeah. And then also carnosine. Yes, and then the GlutaMAX™ basically.  

Rachel Gauvin: L-AnalyL-Glutamine or GlutaMAX™?  

Saba Ghassemi, PhD: Either way, right? Okay. Sounds good. 

Next. Here, basically, I think I’m going to use maybe Formula One, just like I will manipulate all the components, right? For the glucose, 4500 is the usual concentration, but I would like to decrease the glucose to 1800. Then, the IL-7, I usually use 10 nanograms per ml, so it would be — two milligram per liter — 0.01. Same as IL-15. For Physiologix™, you could put 20 ml per liter. For arginine, which is four millimolar, it would be 690, the conversion. Yes. For the carnosine, it would be 1970, and GlutaMAX™ would be 470.  

So this is the media, and it’s very specific media. I would like to just use this media and then compare it to the media that I often use here and see, if I use this media, can I improve the cytotoxicity of my T cells and then also their viability and the gene transduction to my non-activated T cells?  

Rachel Gauvin: So, for you, we’ll just save this one. I renamed it for you, and you can take a peek. It has all your additions as well as the base IMDM. Again, we’ll pop over to NB-Lux™, review that, load it into the Configurator to finalize it. How many liters? Two?  

Saba Ghassemi, PhD: Two liters, yeah, minimum.  

Rachel Gauvin: Quantity testing?  

Saba Ghassemi, PhD: I actually would like to test the pH, yeah.  

Rachel Gauvin: All right.

Saba Ghassemi, PhD Yes, that’s very important for gene delivery. So I would like to see how much pH we’re working on and test it in different batch when we get a different media.  

Rachel Gauvin: Great.Great, and one-liter bottle is great for you?  

Saba Ghassemi, PhD: Yes.

Rachel Gauvin: Fantastic. And then, again, you can go and see all of your additions and your base formula here. But I think we know what —  

Saba Ghassemi, PhD: — This is great because you can actually kind of double-check if you have — because many of my concentration, I have it in millimolar, so there’s option that on top, you can switch between millimolar or… so this is a great way to just double-check yourself. Then, we can just confirm that and place the order.  

Rachel Gauvin: Place your order. Simple as that.  

Saba Ghassemi, PhD: It’s actually very exciting to be able to pick and make your media of interest with — because, generally, we have to deplete medias to just get rid of the stuff that we don’t want and add manually. So this is — from get go, we can pick the components that we’re interested in, and we don’t have to go do extra work to deplete stuff and add different components.  

Rachel Gauvin: Yeah, both these tools are really built for iteration and for you guys to test and kind of keep tweaking, so we love that.  

Well, thank you, ladies. That was wonderful. I want to open it up for Q&A, but I do want to just remind everyone in case it wasn’t clear that there is a second, follow-up to this webinar that I will put in the Chat box the link to register to that, where we’re actually going to get the results of these protocols. So we would love to have you there and get the insight, and these women will get their media, they’ll run their experiments, and we’ll have all that by April 29.  

Q&A Session  

We have a few questions — never apologize for basic questions. We’re happy to answer them — What do we mean by Decrease, Maintain, and Increase with the Critical Quality Attributes?  

That simply is if you’re trying to increase your proliferation, and you want them to grow more. I don’t know if any of the panelists want to take it on any further and maybe elaborate on one of your examples.  

Asma Ayari, PhD: I can take this. For the Maintain, Decrease, or Increase, the idea is that you will select a CQA, for example, which is, for my case, proliferation, and if I want to increase proliferation, in my case, I will select Increase. This way, I will have all of the components that are known from the papers to play a role in increasing proliferation. If I select Decrease, it’s to have all the components that I don’t want to have in my system, and I want to avoid having them in my system because I want to improve proliferation. Especially to have this information — if you click on Increase, you will kind of select only the components that are known to increase proliferation and same for Maintain.  

Rachel Gauvin: Great. Thank you, Asma.  

Asma Ayari, PhD: Your welcome.  

Lori Noffsinger: I want just to add that there is a time when you would want to decrease. Say I want less IL-17 because I’m going for an anti-inflammatory or something like that, so I want to decrease a — there is a situation where you would want to have less of a cytokine during your culture period because an increase in that cytokine might decrease one of your attributes that you’re looking for. I never thought of it that the way Asma said, which is what’s the opposite of what you want if you’re looking to increase it, and that’s one way to look at it. But another way to look at it is there are cytokines that you would want to decrease. There are times where you would want to decrease a certain cytokine or anti-inflammatory response or something like that. That’s kind of the gist of it.  

Rachel Gauvin: Thank you. All right. Another question is Are the lots of cytokines and hormones kept consistent between the formulations within an order? Yes, within an order, the lots would be the same. So there’s a quick answer to that one.  

Another question is When should the scientists expect to receive their media? What’s the turnaround time?  For these small research lots, it can be as short as one to two weeks, which is pretty exciting, to be able to get your media and then be able to run the experimentation. And, like I said, these women will get their media and be able to run their experiments prior to the April 29th webinar. Any other questions or comments anyone wants to share?  

Well, I’ll leave this open for a minute or so. But, again, we have the follow up webinar. I put the link in the chat and you’ll find it on our — if you’re   registered, you’ll get our emails and our social media posts about it as well. But we’d love to have you there and have you in on the fun and hear what their experiments resulted in.  

Again, I’ll stick around. In case anyone has any final questions, we’re happy to answer them. But I do appreciate everyone’s time today, panelists and attendees included, and I hope everyone has a wonderful day.  

Asma Ayari, PhD: Thank you.  

Rachel Gauvin: Yeah. Thanks, panelists.