Hi, I am Anjli Venkateswaran and it is my pleasure to introduce Amanda Clark, who will be joining me today on this podcast. Mandy is the senior program director of laboratory sciences at Biomere in Worcester, Massachusetts. Mandy received her doctoral and bachelor’s degrees from Boston University prior to joining Biomere. Biomere is a preclinical CRO with locations in Worcester, Richmond, California, and also has multiple sites in China. Their portfolio includes comprehensive discovery, AME, and non-GLP and GLP toxicology services. If you’re interested in learning more about Biomere’s service offering, check out their website at www.biomere.com. Thanks for joining me today, Mandy, and let’s get into today’s topic on multiplex analysis methods to analyze disease and PK biomarkers. Thank you so much for joining me today. Thanks for having me. Yeah, so let’s get right into this. So, you know, plate-based assays like ELISA and MSD have diverse applications, right? And they’ve been, I think many companies offer them and they’ve been used for years and years. So, let’s get a little specific right off the bat, how is Biomere using these assays in preclinical client programs? 

At the lab at Biomere, basically our job is to support the in vivo work that we do, so the animal work that we do here. We run a lot of plate-based assays after animals have been dosed just to see what’s going on after dosing. We’re looking for a lot of inflammation markers and cytokines, any kind of drug interactions, things like that. We’re using all these plate-based assays to quickly get an answer of what’s happening. Right, right. And so, you guys, do you guys have a preference, or do you primarily use ELISA or MSD or both depending on what the study is? Yeah, I think it depends. The trend has been doing more MSD just because it’s faster, we get more information out of it, but we can go into that in more detail as well. Yeah. Cool. Well, you mentioned different inflammation and cytokine panels. So, can you tell us a little bit more on the kinds of the most popular panels that you guys are seeing and our clients requesting? Yeah, so it’s a lot of inflammation markers because again, we want to see if there’s some kind of response. So when you get dosed with anything, you want to see something a little bit, of something, but not a lot. So you know, “stranger danger” is a good thing, but having inflammation continue can cause bad things to happen. So we do a lot of things that look for any kind of an immune response or an allergic reaction or some inflammatory response. So a lot of like interleukins, interferons, TNF alpha is a big one. Just a lot of the cytokines. Right, right, right, right. And are there any favorites or any top contenders? You mentioned TNF Alpha, what about IL one beta? Is that a popular choice? Yeah, IL one beta, IL 2, 4, 10 are the big ones. Yeah, IP 10 has been popular recently as well. Just a lot of markers. So we can do a lot of different things, which is great. 

So we’ll go into a little bit more about the MSD assays and the kind of data you can get out of it in a bit. So something interesting about how MSD is becoming the preferred platform. What are the main advantages of using MSD over, say, a conventional colorimetric sandwich ELISA? Sure. So, yeah, MSD is very similar to an ELISA, the technique is pretty much the same. It’s a plate-based assay. You add some stuff, some magic happens in the plate and you get an answer, to put it simply. It’s a very simplified view for a complicated technique. But it is just more sensitive than ELISA. So we can detect smaller amounts of whatever these analytes may be. It has a larger range, so if you have a disease that has a very, very tiny amount of something that could cause something to go wrong, we can detect that with MSD, not always by ELISA. It requires less sample volume than ELISA, which is nice. So in an ELISA you probably need, you know, a hundred microliters of serum to run one ELISA. We can do MSD with like 20 microliters, and we can get information on more than one item. With MSD we can multiplex, whereas with ELISA, you have to do one ELISA per whatever you’re looking for. With MSD, we can look at up to 10 different analytes per well, which is nice. So we can do 10 for one, basically. So it’s just faster. We get more information and it uses less volume. 

And that’s a big thing, right? Because in a clinical setting you can get a decent amount of blood from a human, whereas if you’re looking at a preclinical study, an animal model, depending on what the model is, sample volumes do become restricted, especially in life when you’re just sampling. So what about the kinds of samples that you get out of an MSD assay? Sure. Yeah. So we support all the animal work that we do here. So that’s both small and large animal. Small animal could mean anything from mice and rats basically. We also have rabbits, which we are just starting to do some of their blood analysis, that’s new for us. And then we have NHPs. So depending on the animal model, it really makes a difference of what we’re looking at and how many time points we can actually collect from these animals. So a mouse or a bone study, we might just have a predose and a necropsy terminal dose. We might have one time point, one six hours after dosing or something. And usually that’s it. We might have three total for the mouse. But for NHP studies, we might have a whole time course, a whole PK time course of, you know, 1, 2, 4, 8, 16 hours later and then the next month and the next month and the next month, going on for six months, 12 months, whatever it may be. Wow. So it can be a lot. Yeah, it can be a lot. And sometimes clients want their data right away after the six-hour time point, we want the information. Other times it’s, you know, we can wait after the first month and give us a whole plate full of data, but either way is fine with us. What’s nice about these assays is that we can do a partial plate, we can run 15 samples and get them the data they need immediately, within that same day or the next day. So I think that’s super helpful for them when they’re trying to do multiple studies at once or they’re trying to do a dose response, or they’re trying to figure out what dose is important to get what they want to do in. But it’s quick, so it’s nice that we can do it quickly and get some kind of answer. 

So I’m curious, when you say quick, what are we talking about? Are we talking about a full day? A couple of hours? A half day? What is quick kind of translate into? Probably about a half day. It really depends on the plate, on exactly how many items they’re looking for. But most of the time, you know, you add your samples, you incubate it for an hour or two, you add the detection antibody another hour or two, and then you kind of just wash the plate a bunch of times, which takes some time. So yeah, it’s usually about four to six hours to do a plate. And on that plate, so same day, I mean, it’s technically the same day you could get the results. Yep. So usually we do it the next day or depending on what the time point is, we can do it on fresh samples or frozen samples. So it’s really up to whatever they’re looking for when they’re looking for it. So I want to dig a little bit deeper on the fresh and frozen, because when you were talking about batching, so I immediately jumped to the fact that it’s the plasma serum samples, whatever you’re analyzing has to be frozen. So how do you guys freeze it? Is it minus 20, minus 80? And have you seen any difference in sample integrity and experiment performance between fresh and frozen samples? Yeah, so some of the analytes that we do are very picky. Whereas it does make a difference. So we try to keep track of how many freeze thaws we do. We try not to do freeze thaws if we don’t have to. So we try to plan it in advance with the client and saying, you know, when do you want your data? If you have all these time points the first week, do you want it after the first week? We’ll run all those together. We don’t want to wait. Sometimes we can repeat a sample, that’s fine. But again, if we can reduce the amount of freeze thaws, that’s better for us. Yeah. Just because sometimes it does make a difference, sometimes it doesn’t. Some of these analytes are very sturdy, you know, whether you freeze them, they’re still going to be hanging out in your plasma. But some do start to break down so your results could not be quite as accurate as you would want them to be. Right, right. Because it really depends on that specific study and what you’re looking for. I’m guessing you have run enough samples that you have a good idea which are the picky ones and which ones are the sturdy ones. So, sturdy analytes. Yeah. Even when it comes down to processing. So some of the processing just to get to serum or plasma, some of it has very, very particular instructions depending on the analyte that we’re looking at. So even that can make a difference. Got it. Got it. So I’m curious, when you mentioned small and large animal models, right? And if you kind of just broadly classify them as say, rodents as representative of small animals, and say NHPs or rabbits as large animal models right? 

Now, what’s the split? And then because the reason I ask this is with small animals, they have their pros and cons, like, you know, you’d mentioned, and large animals also have the pros and cons. So what do you, where do you, where are the clients, you know, where are they moving towards? Do they prefer small animals or do they jump right into more expensive large animal studies? So I’m kind of biased because I’m also a small animal study director at Biomere. So I do some of the small animal stuff. Um, so I see a lot more of the small animal. But speaking for the lab itself, we see it’s about 50/50 for the work that we do in the lab. 

I think, you know, client, it depends on the client. So some clients have never tested their compounds before in an animal. Um, so they’re going to put it in a mouse first because mice are cheaper and they’re easier to work with. Whereas the NHPs are very expensive and, you know, they need a lot more handling, a lot more care, a lot more support. Right. So they and they might only have a small amount of compounds to start with, so whatever their drug may be. So they want to use a small amount in a small animal just to see what happens. But they do, you know, after that, even most people do move on to larger animals just to see what happens there. 

Whether there’s some kind of reaction, some kind of toxic reaction or another problem that may happen. Um, because again, the larger you get, the closer you are going to be to what it might happen in humans. And that’s what we want to see. So, yeah. And I was, I was thinking about, you know, one idea that popped into my head is I think sometimes with, you know, drugs targeting different neuro, neurodegenerative or neurological disorders, I wonder if it makes sense sometimes to jump straight to a large animal because, you know, mouse brain versus human brain, there’s like a million papers out there that talk about the differences and the, you know, the poor translational value. 

Whereas I think it’s well established that NHP is just, they’re much better because in terms of the CNS anatomy and, you know, physiology and how everything works. So sometimes I wonder if that plays a part depending on what the drug is targeting. Yeah. And I’ve been here almost nine years now and I feel like even in the past few years, even with the FDA approving certain drugs, that the rules are changing. I feel like, you know, I don’t know all of the, all the regulations and rules because that’s not my role, but we definitely see, you know, people are using either a humanized mouse model instead of going from rat to whatever, Guinea pig to all the steps. Um, or they’re going straight, they’re skipping a couple steps to go to NHPs and first and not having to do some of the mouse work or some rodent work. And I think that it’s making a difference. 

It’s getting these drugs, you know, into the clinic faster. Hopefully that’s the goal. Um, so I know I call it skipping a step, but it’s not really skipping if we’re trying to make it more efficient and make it better for the patients that are waiting for some kind of treatment. Right. And I mean, if there is an option, as you said, to use as a humanized mouse model or just go straight to a more translational model, why bother? I think in the, in the old days, probably people would put it directly in a mouse and kind of see, see what happens. But I think I do believe the drug development community is getting smarter and a lot more thoughtful on study design and they want to make sure that, you know, they go in with the right model right off the bat and don’t waste time and money because everything adds up at the end, you know? Right. And yeah. And speaking of time and money, you know, one of the fascinating aspects of MSD is that multiplexing capability. And then you touched upon it a little bit earlier, but I want to dig a little bit deeper because I think that’s such a critical advantage because I mean, if you can get, you know, multiple data points from 20 microliters of sample and get a whole like panel and get an array of information, why not? As opposed to, you know, single analyze sandwich, which is limiting. 

So more about how, you know, are your clients are thinking about singleplex versus multiplex because I think pros and cons in both. Yeah. So MSD has been really, really helpful with us. Um, they are really trying to even get any ELISAs that we have onto the MSD format, so we can even take one ELISA and put it onto our MSD plates, and it’s just, we get much better data. Um, so that’s been helpful. But I think just getting more out of that one sample has been really the big push. And clients now, you know, they know that now we have MSD, we’ve only had it for two and a half years now, and, and even over the past two years it’s been, you know, we started off with, you know, maybe 30 that year, and then the second year we did like 200 different plates. Wow. So that’s a huge difference. 

This year, the same we were on track to do another 200. So really that means that we are doing MSD every other day in the lab, so running something for someone, and that’s huge. And we’re, you know, we’re not a giant company, but you know, we’re big enough to get that enough information out to the clients. Um, so that’s been helpful. But I think really getting them as much data as they can from that sample has been huge for us. 

And do you see, you know, this study design where it’s kind of a combo or combination of multiplex and singleplex where you kind of, the analogy I think of is you’re casting the net as wide as possible in the first pass, and once you know how many fish you can catch, then you can go in with a proper fishing line and get the, get the fish of interest. So is that the, do you see a lot of these kind of approaches to start the study design? Yeah, I mean, I guess, you know, it also depends how much money they’re willing to throw at it. But oftentimes someone, they’ll come and they’ll say, oh, you know what, we don’t know. We’ll just look at all the cytokines. 

I’m like, well, do you realize how many, all the cytokines, that means a lot. That’s like hundreds. So let’s, we can’t do that. Right? Let’s pick some. Right. Um, but MSD does have some set panels, which we’ve been trying to offer. Um, they’re, you know, 10, 8, 6, 8, 10 different, um, cytokines or analyzed that we look at that I think helped people narrow it down a little bit. And then from there they might be like, you know what, I’m only interested in whatever this specific response, this specific t-cell response or whatever it may be. Can you look at these two or these four? And then, yeah, the answer is yes, great. 

Let’s  do that instead of running, you know, 20 at once. I think makes a lot of sense because you want to find out which are the most informative markers because, you know, when I’ve seen different MSD data, a lot of times, you know, when you see those graphs from MSD, they’ll see no change in, right. I don’t know most of the markers, but then there’s always those informative ones that are, you know, fluctuating in response to, you know, whatever drug you’ve dosed or just baseline fluctuation as an inflammation, inflammatory response or whatever. And those are the ones you really want to zero in on and analyze a lot more. 

I was going to say, I think that people are trying to be more cognizant of the time points they’re picking as well to make sure they’re capturing the data that they want to actually capture. Um, you know, so whether it’s, I mean, the, the most popular one we do is a six hour time point because it, it seems to be with, you know, all these LNPs that we’ve been doing since covid, um, six hours after you get dosed, there’s some kind of immune reaction, right? And then it usually goes away within 24 hours. And that’s fine that, you know, we see that in people with the covid vaccine. It’s just, it just is how these, these treatments are. Um, but now that we know that, now that everyone does it, hey, we’re doing a six hour time point. Okay, great. We know what to look for, we’re going to see something go higher six hours and then go back down. 

Um, and that’s been the case for most of these drugs that are working or that they want to continue to move on through the clinic. Well, that’s a hugely important point, right? Because if you don’t get your time points right, you’re going to miss an entire event. Yeah. Right. If you like, for example, you mentioned six hours is, um, it seems to a good point, but if you say you wanted a 12 hour, you probably missed, you missed that window. So curious, just a curiosity question. And all the assays that you’ve run, what’s the earliest time point that you’ve, that you’ve been able to test after dosing. Five minutes? No way. Oh goodness. 

Five minutes. So you dose and then immediately collect blood. Yep. And have you see, did in that particular study, did you see a change or did you identify something unique if something cool? Yeah, so they, uh, they were lucky they actually did the five minutes because they did, I think they did 5, 15, 30 and then one hour, two, four hours, something like that. And they did see within that first hour things went wild and then after that it was fine, which is good to know, you know? 

Yeah. Some kind of cytokine storm is going on for sure. But it is good to know because you have a patient sitting there and you give them something, Hey, so why don’t you sit there for 30 minutes and then everything’s fine. You know, it is good to know. And I think that was the case, you know, even with the, with the COVID vaccine and things like that. And, and as you mentioned, LNPs being one of the favorite drug delivery vehicles, so that’s probably going to, I wouldn’t be surprised if that triggered a really rapid, you know, transient response that people need to be aware of. So it doesn’t necessarily cause long-term effects, but short term it makes it fee, it makes, you know, it causes an, has an issue.

You mentioned, so MSD, you know, if you check out their, their, you know, portfolio and you check out their entire product line, they just are constantly adding panel after panel. And I think they’re super thoughtful about how they put their panels together. So, um, in the studies that you’ve been running, do you primarily use just the off the shelf available panels or, um, do you see requests coming in for, Hey, let’s customize this, or, I don’t want, I, I want one, two, and three in this panel, but I, I, and then I want to add, you know, A, B, and C. Do you see interesting requests like that?

Yeah, customization is huge. And that’s also why we love MSD because we can, we can say, Hey, we want this panel, but instead of this one thing, can we have this instead? And then we can make a custom plate, and they send it to us and we just run it and it’s lovely. Um, wow. You know, they work with us to say what they have validated, what they haven’t validated, um, yeah. You know, our, our support person is always willing to work with me and I could say something that they haven’t tried. I’m like, well, how do we try it? Which buffer do we use? Like what, what dilution should I do? And you know, they’re, they’re very responsive and getting us the answers we need to get our clients the data that they need. Um, but it’s a lot of customization. So, and, and that’s great because we can do up to 10 per, well, sometimes clients only want six or they want, you know, they want to use a certain panel, but add two more and if the panel is six, we can add on four more, so why not? Right. Um, get the data that they need. Yeah.

That’s amazing. And do you anticipate, you know, an increased demand for custom panels? Because I think once people have their, you know, whatever, based on the studies they’ve done to date, they probably have some good ideas which markers are the most informative. So do you an anticipate an increased demand for customization?

I think so, and I think now that people, you know, they hear the word MSD all the time, I feel like it’s become way more popular. So it’s either MSD or Luminex, which we don’t have Luminex, but we have MSD, so we can do most of the same things. Um, and it’s definitely becoming more popular. And now that we can customize everything, I think it’s going to just, just fly from there just to get as much as we can out of the samples.

I think makes a lot of sense because you want to find out which are the most informative markers because, you know, when I’ve seen different MSD data, a lot of times, you know, when you see those graphs from MSD, they’ll see no change in, right. I don’t know most of the markers, but then there’s always those informative ones that are, you know, fluctuating in response to, you know, whatever drug you’ve dosed or just baseline fluctuation as an inflammation, inflammatory response or whatever. And those are the ones you really want to zero in on and analyze a lot more.

No, I definitely see that as a huge advantage. My understanding is that your lab has developed solid expertise in MSD assays. This is extremely helpful because I believe you can support in vivo studies. I’ve read some papers where they validate a lot of MSD and LUMINEX for clinical samples and trials. Using this in preclinical studies is very helpful because you know which markers to look for.

Have you seen any unexpected markers? We’ve discussed cytokines and the classic inflammation markers, but do you have any examples of unique markers that surprised you?

I think it sometimes depends on the model they’re using. They are trying to combine everything now. We might have some panels for diabetes or something else, and I’ll realize we can just add insulin to a panel of cytokines, which isn’t something I would have considered doing at the same time. But we can, and that’s great for them. I think that’s the main example I can think of off the top of my head; we’ve had a lot of requests for insulin lately. I would’ve never thought of that one. We also frequently add CK, which is a muscle damage marker, to panels. Adding it in addition to cytokines when you’re not dosing into the muscle is interesting to see.

I have a logistical question. You mentioned customization is on the rise. The advantage of an off-the-shelf product is that you place an order, the plates arrive in a day or so, and you can run your assay. How long does it take to get custom plates from MSD? Does the time depend on the level of customization needed?

It’s just as fast as ordering a regular plate because we do it in-house. They help us design it and match what we want on the plate. When the plate arrives, we perform the first step of coating it with the desired antibodies. We keep track of which antibodies are used and then we can run the entire plate. It’s fast because they have the antibodies ready to go; they line them up and send them out. We can then pick and choose and link the antibodies to the plate ourselves.

Do the off-the-shelf plates also come pre-spotted with the antibody?

It depends. Most of the time, we add them ourselves. They have antibodies, a linker, the buffer, and everything we need ready to go. Our first step is to coat the plate with the antibody we are using, and then we add our samples.

I assume they have validated and tested the antibodies, because the data from a sandwich assay is only as good as the antibody. Do they have a huge menu of validated antibodies?

Yes, they have many different antibodies that they have validated for use in different plates. They have figured out which buffers and antibodies to use and which antibodies can be placed next to each other on the same plate. They’ve done a lot of work so we can just ask for what we need, and they can help us.

That adds so much flexibility. That’s good to know because I was envisioning pre-spotted plates. If they can just mix and match the antibodies in a kit and ship them, that makes things much easier. I think that’s a huge benefit of using MSD; you control the entire experiment from start to finish.

This has been really helpful. Anything else you’d like to add about the MSD capabilities? Use it more, give us more work. It’s great to hear about BIO’s expanding portfolio. I am convinced your lab has this assay locked down and running very smoothly. It seems like you’re running more and more assays, especially to support in vivo studies in both small and large animals. I also love the concept of customization. It allows people to get as much data as possible at a reasonable price point that hopefully won’t break the bank.

And to our listeners, if you are interested in learning more, email bd@bio.com and check out the Biomere website at biomere.com for more information. Thank you, Mandy, for joining me today. I definitely learned a lot and wish you and the lab good luck for the year.

Thank you so much.

Summary:

In a Q&A session, Anjli Venkateswaran and Amanda Clark discussed in vitro assays that support animal studies at Biomere, a preclinical Contract Research Organization (CRO) in Worcester, Massachusetts. Amanda Clark, the senior program director of laboratory sciences at Biomere, received both her bachelor’s and doctoral degrees from Boston University. Biomere, which is a preclinical CRO, has locations in Worcester, Massachusetts; Richmond, California; and multiple sites in China. The company’s services include discovery, AME, and both GLP and non-GLP toxicology services.

Biomere’s lab supports in vivo animal studies by running plate-based assays after animals have been dosed. These assays help researchers understand what is happening after dosing, by looking for inflammation markers, cytokines, and drug interactions. The lab uses plate-based assays such as ELISA and MSD. According to Amanda Clark, the trend has been to use more MSD assays because they are faster and provide more information.

MSD assays have several advantages over conventional colorimetric sandwich ELISAs. These advantages include:

• Higher sensitivity, allowing for the detection of smaller amounts of analytes.
• A larger range, which can detect very small amounts of a substance that could cause a problem.
• The ability to use less sample volume; for example, an MSD assay can be run with about 20 microliters of serum, whereas an ELISA may require 100 microliters.
• Multiplexing capabilities, meaning an MSD assay can look at up to 10 different analytes per well, while an ELISA is limited to one analyte per assay.

The Biomere lab supports a range of animal studies, including those with small animals (such as mice and rats), rabbits, and NHPs (Non-Human Primates). The lab sees a roughly 50/50 split between small and large animal studies. Small animals are often used first because they are less expensive and easier to work with, especially when a client has a small amount of a compound to test. However, many clients eventually move to larger animals to observe for toxic reactions or other issues, as larger animals are more similar to humans. In recent years, there has been a trend for some studies to skip certain steps, such as going directly from a humanized mouse model to NHPs, to get drugs to the clinic faster.

The quick turnaround time for MSD assays is another benefit. A typical plate can be completed in about four to six hours, allowing for results to be available on the same day or the next day. The lab can run partial plates with as few as 15 samples to provide clients with immediate data. The samples can be either fresh or frozen, though some analytes are “picky” and their integrity can be affected by freeze-thaw cycles. The lab tries to plan in advance with clients to reduce the number of freeze-thaws whenever possible.

The demand for multiplexing and customization of MSD panels is increasing. MSD offers pre-set panels of cytokines and other analytes, but the lab also receives many requests for customized panels. The ability to create custom plates is a significant advantage of MSD. The process is fast, as the lab works with MSD to design the panel, and MSD provides the necessary antibodies, which the Biomere lab then uses to coat the plates before running the samples. The customization allows clients to combine markers, such as adding insulin or the muscle damage marker CK to a cytokine panel.

A six-hour time point after dosing is a common choice for many studies, particularly those involving LNPs (Lipid Nanoparticles), as it often captures an immune reaction that resolves within 24 hours. However, some studies have tested time points as early as five minutes post-dosing, which revealed a rapid, transient “cytokine storm” that subsided within an hour.

For more information about Biomere’s services, people can visit their website at www.biomere.com