We dive deep into gene therapy during this conversation with Dr. Cliff Froelich. He covers what vectors his team works with, what methods and technologies they use to monitor quality and safety, and he reminds us why it all matters. As always, you’ll get a bit of fun too!
Viral vectors are a cornerstone of gene therapy and many employ experts in the viral vector services space to help design and produce their specialty vectors. These service providers are experts at making sure you get the vector you want with a titer and purity you need for your application.
We’re joined in this episode by Dr. Cliff Froelich, Head of Analytical Development for a viral vector services provider. Cliff and his team work with AAV, lentivirus, and other vectors to support multiple, and simultaneous, client projects. Specifically, we dive into how they use various analytical and molecular methods to monitor and assess identity, strength, purity, impurities, potency, efficiency, empty/full ratios, safety, and more. As you might expect, it’s not a one-method-does-it-all approach or solution. Yes, digital PCR is in the mix here, and Cliff does a great job of outlining where it shines relative to the other methods they use regularly in their GMP practice.
In our career corner portion, you’ll hear about Cliff’s circuitous career path, which includes stints in the poultry industry and time as a clinical dietitian. Through it all, and into his current role, Cliff brings a passion and genuine interest for the science and its potential to affect lives.
Visit the Absolute Gene-ius pageto learn more about the guests, the hosts, and the Applied Biosystems QuantStudio Absolute Q Digital PCR System.
Jordan Ruggieri 00:00
Why did the viral vector go to therapy? It had a tough time dealing with all its host attachment issues. Why was the viral vector always the center of attention in the lab? Because it had a knack for delivering genes in the most viral way possible.
Christina Bouwens 00:15
Hmmm.
Jordan Ruggieri 00:18
There's got to be something in there.
Christina Bouwens 00:31
Welcome to Absolute Gene-ius, a podcast series from Thermo Fisher Scientific. I'm Christina Bouwens.
Jordan Ruggieri 00:36
And I'm Jordan Ruggieri. And today we are thrilled to welcome another Gene-ius to the show, Dr. Clifford Froelich.
Christina Bouwens 00:43
Cliff is the head of a viral vector services lab in Plainville, Massachusetts where he uses the Absolute Q and dPCR for a variety of amazing applications. Cliff's career in biochemistry stretches back nearly 15 years, and it's a pleasure to share more about his work, past and present, with you today.
Jordan Ruggieri 00:59
Christina, do you want to hear a viral vector scientist pun?
Christina Bouwens 01:02
Absolutely.
Jordan Ruggieri 01:03
Why did the viral vector scientist always bring a map to the lab?
Christina Bouwens 01:07
Why?
Jordan Ruggieri 01:08
Because navigating through gene delivery pathways is more confusing than a maze.
Christina Bouwens 01:18
Well, I'm super excited to have you here Cliff. I've been looking forward to this all week. It's been my favorite conversation, I've been I've been talking you up.
Cliff Froelich, PhD 01:25
That's awesome. It's great to find a needle in a haystack.
Jordan Ruggieri 01:30
Cliff, maybe we can hop right in. Can you give a little bit of background on yourself and what you do?
Cliff Froelich, PhD 01:37
Yeah, so I'm Clifford Froelich. I'm the senior manager and head of analytical development at the viral vector services location in Plainville, Massachusetts. Our role there is to provide services to our clients to make various kinds of viruses for gene therapy. And so my role is to assist our bioprocess sciences group with providing the data that describes what they've been working so hard to do. And so without that data, it's just very expensive water and so the data paints that landscape, it tells the story. And we utilize that to assess whether we were successful, and then also optimize things and improve things. Really, ultimately, in the end, the goal is to transfer the, all the intellectual property into the GMP side of the site and make drugs to help people.
Christina Bouwens 02:37
And I know like, we're super focused here about digital PCR, but you see a lot of technologies in the work that you do. Can you talk about some of the different technologies that that you see and encounter on a daily basis in the work that you do, And kind of the, the work that the viral vector lab sees?
Cliff Froelich, PhD 02:53
Yeah, I mean, so obviously, you know, just start with it, it's about the, essentially the five key attributes that need to be tested for any drug product that's going out. So you know, whether it's a safety test, whether it's identity, strength, purities, impurities, all of those things, and then obviously, potency, efficiencies are all key. And within that, you know, obviously, the PCR realm, whether you're talking about Q, dd, d, all kinds of start to feed into strength, into identity, we can assess some impurities utilizing that technology. But also where we come from is understanding empties versus full viruses. So not all viruses are immediately stocked with the genome, they can often be empty. There is mixed philosophy in the in the field of whether those empties are important or not, when we are dosing a patient, whether it's to clear the immune system so that the full ones can actually make it to the target tissue. So as we're going through, we want to understand, you know, what, what's the, what's the quality? And when I say quality, it's a big 'Q' quality. How good is it for me because it is going to the patient. Because it could be my mother, it could be my brother, my sister. It must be the highest standard. And so the more that we know about it, the better. And we really don't want to limit ourselves to the technology.
Jordan Ruggieri 04:23
Cliff, could you actually even go a step back and talk about like, what is a viral vector lab? And how do you, what's actually the ultimate goal that you're trying to achieve?
Cliff Froelich, PhD 04:34
Maybe we need to take a step back even further and just look at the word. Obviously, you know, viral is going to be a virus. So typically, we are producing, manufacturing an AAV which is more of our bread and butter at the Plainville site. That's going to be an adeno associated virus. There are adenoviruses. So we all may be familiar with the J&J vaccine for COVID. That was an adenovirus. Then you could be looking at a lentivirus, or we could be even looking at a herpes simplex virus. All of these are non-replicating, they are referred to as ‘suicide vectors’ because they cannot make a copy themselves on their own. And they're glorified FedEx trucks to deliver a genome. And so that delivery truck in the science world is known as a vector. For these viruses that we produce they're either carrying a single stranded DNA, which is AAV, double stranded if it's adenovirus, and RNA payload if it's a lentivirus. But all of your coding something in order to target a gene that is maybe not correct. Yeah, maybe it just got mutated through environmental reasons. Maybe it just got, you know, transferred through homologous recombination from parents, etc. And for whatever reason, it's not functioning. And what our goal is, is to be able to use this FedEx truck, and put the correct gene sequence in place so that we can improve lives. There's a myriad of ways to apply this. That's where we are utilizing these viruses as vectors, to carry these genomic sequences to a person.
Christina Bouwens 06:29
I love that. And I think that's one of the, that's honestly the reason that drives me every day to you know, do what I do. And that was what drove me to continue following cool technologies and cool tools was the ability to, you know, someday something that I do is going to impact real people. So I guess that kind of brings me to my next question is when we're looking at these FedEx vehicles, bringing these cool deliveries to really unique destinations. Um, what about digital PCR makes it really useful right here, right now, to help you in the work that you're doing?
Cliff Froelich, PhD 07:02
I've done this for, I'm going to date myself, probably a little over two decades now. And I actually remember being in undergrad when PCR was invented. And to have seen the evolution of it is absolutely phenomenal. You know, going from standard curves of the qPCR to now absolute counts, absolute measurement, absolute determination is extremely tight. And actually coming from a quality background, where percent CVs, you know, where you're measuring variance, and you have to mathematically fit to a logarithmic data set on a qPCR, it immediately takes your percent CVs that might be low, at 2 or 4%, and bumps it to at least one magnitude more, 20 or 40%. I think the other bonus for the Absolute Q is the addition of a robotic arm and the and the plate hotels. When you're in my realm where I've got hundreds or thousands of samples coming in because we've got all these clients, we're screening all these conditions, etc. We can just go crazy with a robot and set these up and have them sitting in a hotel and let them run overnight. And so what it does do, it improves the ergonomics. It improves even the pipetting accuracy on that. And then we're actually utilizing the members of the team for what we actually pay them for which is their brainpower not because they're a human robot. We instead have them go ahead and get the data that we need to accelerate our clients’ roles, to accelerate their intellectual property, to get the drug to the patient.
Jordan Ruggieri 08:53
Continuing on this digital PCR train of thought, when you are evaluating a new piece of technology, let's say it's the Absolute Q, or really any type of new technology, what are your expectations? What do you look for? And how do you actually get that new piece of technology into your lab?
Cliff Froelich, PhD 09:12
I think it takes multiple approaches. I'm a little bit more systematic. And so you know, usually I kind of want almost a URS, what are my user specific requirements? For me, I have to keep in mind that whatever I'm going to develop or build in my lab needs to translate into a GMP ready lab and so then it needs to meet all the validation criteria, the IOQ's. You know, the whole shebang as well as the qualification of the method. Then I usually have a demo. You know, obviously, it's first and foremost, maybe a lunch and learn, or some seminar. But then if we can actually be able to have a loaner in the lab for, you know, a couple of weeks to a month. But you know, it really, honestly, Jordan, it really varies across the board. In all cases, I bring my team in, because I want a diverse input. I needed a diverse understanding of what people's opinions are, and then I can build a business case. And that's my role, then, is to clear the roads for that, get the funding for that, and then we move forward.
Jordan Ruggieri 10:24
You know, diving into even more on the, the why you're, you know, you're looking into this new technology. So I mean, look, you're in a regulated environment, right? You're working with customers that, that are trying to develop things that will end up in, you know, a subject or a person, right. How do you identify that you need a new technology? I'm assuming things aren't just going around and blowing up in the lab, right. So how do you actually sit there and say, "Hmm, I actually need something new?"
Cliff Froelich, PhD 10:54
You know, obviously, we have to be up to date in the science, in the journal articles, etc. The FDA could demand it. So that's the second part. But then on top of that, as part of something that I require of my team, and I think it's best practice, is we trend all of our data. At least within my shop, we we’re going to have our own site reference standards running through most of our assays all the way through, no matter which client we are testing for, so that I can track and trace how well the assays performing. And it's, you know, that way it's not a client-specific performance question, it's across the board. You know, is it is an analyst, or is it the instrument. And it's, you know, let's say I've purchased an ELISA kit that's commercial and I'm just always seeing it, you know, where it bounces around by 25%. Well, that, that's a lot. That's an obscene amount. In some cases, I've seen ELISA kits where they may have an invalid rate of, of 25, 30%. By letting the data drive it, listening, going to conferences, staying educated, and listening to even the regulatory group who you know, I mean, FDA, the EU, will listen, talk, collaborate, and assess, “Alright, is this good enough or not?”
Christina Bouwens 12:21
Awesome. I had a question. Because I think I heard kind of two different themes going. There's the first, which is a very, like when we think about gene therapy, and all the variety of diseases that it can really, all the variety of disease research that it can impact. It feels very custom, right. There's lots of different things that it can impact. And when we think about dPCR, we all have to think about novel biomarkers that we can track. But then I heard you also talk about some of the different quality and safety aspects that you know you're using to look at in your lab. So, what types of markers are you most often looking for? Are they the more standard like res DNA, viral titer quantitation markers? Or are you looking at more of these custom novel markers where you need like, you don't want to be creating these novel standards all the time? What do you see more commonly come to your lab?
Cliff Froelich, PhD 13:12
When we're talking about the gene of interest itself, that needs to be, should be in the in the virus. Usually, we will make something special for that gene of interest. A lot of times those genes have been engineered. You know, some of them so you know, for instance, again AAV. Usually the capsid can typically only hold a virus, or a viral genome no bigger than 4.7 kilobases. That's it, that's its limit. And so some genes are bigger than that and so they're often engineered. In other cases, maybe it's a smaller gene, but they tack on different promoters, different introns, other things like that. And so what we usually do, and it's kind of twofold, we do need to make it specific to the gene of interest, because we need to demonstrate, per the FDA, specificity. So if I've got two clients that are producing factor A, I need to be able to show the difference between those two in the facility at the same time. But at the same time, I also need to be able to create amplicons of the genome across a unique portion of the genome or of the viral genome that is not inherently found in you or me. And the reason for that is one of our quasi-functional assays, quasi-potency assays, is a tissue culture infectious dose 50, TCID50. All right, so we, here we take the virus that's been purified, and we're just checking to make sure it's infectious. And what that titer, that infectious titer might look like. So we'll add it to HeLa cells. We'll infect it, we'll culture it, we'll grow them up, we'll go ahead and harvest it. And then what we do is look for, you know, look, look for that material. But we need to be able to discriminate that from the HeLa genomic indigenous genome. And so if I can make a span that goes from the gene of interest over into an SV40 domain, or into the ITR, or into, you know, the promoter that I mentioned before, then that's going to be unique. And so that what that does is it provides, it works twofold. One for strength, the other for the attribute of more functionality or potency. But then to, Christina to your, I guess the other dimension, which would be more of the impurities, like the host cell DNA, you know, we could, at least in early phase, we can probably get away and when I say early phase, it may be phase one, phase two of a clinical trial. We probably get away with, you know, just generally looking for ALU repeats. You know, and these are repeats are found in human cells, and he would in human genes. And what it does is it helps us quantity, “Alright, how much of the HEK293 genome co-purified with the virus with it that's an impurity, how much is still there?” But I don't know what's in that basket. I only know that it weighs 20 grams. And so you could go general. And that's good enough for phase one and two. Now the caveat is that we're also expected to have very short contaminating DNA segments, no greater than 200 bases. And so a lot of times we'll utilize like, capillary electrophoresis, and things like that, to understand whether we've got, do we have a full-length gene? Or is it just a truncated part, which doesn't really matter? You know, that's, that's where our concern would be is that is that a trouble? So you know, there's a lot of factors and a lot of ways that this all impacts it and utilizing a dPCR approach like Absolute Q can assess the plasmid, it can assess, you know, the residuals, the indigenous genome from the HEK293's, and anything else that we may be concerned about as before we ever inject that product into a person.
Christina Bouwens 17:27
I love hearing you talk about all this synergies really, and how thorough the entire process is. I think that's what I really love the most about this conversation because I think so much we hear and we think about, you know, how is this one technology used to, you know, to tell this story to put this puzzle together and really, it is truly a synergy of all these different tools in a toolbox coming together. Um, because this story has to be tight, this ship is run extremely thorough and nothing is released without us being extremely sure that you know, that these products are ready to go and I think that is like the most incredible part of this entire story is just you know, how many touch points and how many, you know, extremely intelligent people get together to make sure that the right thing is ready at the right time. So I just, I love this.
Christina Bouwens 18:16
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Jordan Ruggieri 18:36
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Christina Bouwens 19:00
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Jordan Ruggieri 19:10
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Christina Bouwens 19:27
And now back to our conversation.
Jordan Ruggieri 19:33
Awesome. All right. So Cliff, welcome to what we are calling the career corner of the of the podcast. Can you actually, I mean, just give us insight. How did you even get into this viral vector space? What was your educational background and maybe even some of your career pathway to get there?
Cliff Froelich, PhD 19:52
I think it's probably pretty similar for many people, a very circuitous route. I've always been a dork. I've always loved science, ever since I was a kid asking for a chemistry set from my mom and growing little, little micro crystals and whatnot. Because, well, I like to blow stuff up, test things and, and cook. Cooking is everybody's chemistry owl. So, and I say that because my background is, I have, I got my bachelor's degree in both nutrition and food science from Texas A&M University. I dug nutrition, and I chose nutrition, because I realized that maybe the first medicine is what you eat. I knew I wanted more education so I stayed at Texas A&M, and I got a master's in nutrition doing microbiology in a poultry science department and I raised 400 chickens. And what I was looking for was the rate limiting amino acid in the nutritional feed of growing chicks. Because in the poultry industry if you can optimize the diet, you can have the biggest, fattest bird that sold by pound in the shortest amount of time. But when I walked into nutrition, I wanted to work in a hospital and wanted to be a clinician at whatever. And so I ended up doing that, I'm actually a registered dietitian. I went after my master’s and passing my boards, after doing my clinical rotations, and worked at the University of Texas Medical Branch in Galveston, Texas as a registered dietitian. And I oversaw the burn unit there. Orthopedic surgery and neurosurgery were under me. Medical ICU was also under me. And then I periodically had to oversee the nutritional support for the Texas state prison system that was attached to the hospital. So working with those inmates who have, who are at various levels of their lives. Some are pregnant. Some are at the end of their lives. They all have the same challenges we all do, they're just in a different home. I found that I was able to do my work in about half a day and I found myself sitting in the library reading Nature and Science every day, because I really missed that science. I really missed nerding out over all of that, despite all helping patients or working with people and treating kids with diabetes and all that jazz, I missed it. So after a year of work, he has clinical dietitian. I was like, “I need a PhD.” I've got to go on for my PhD. I was accepted to a few places but I ended up opting to go to Louisiana State University. Specifically, the medical center up in North Louisiana in Shreveport, Louisiana. I joined the Department of Biochemistry and Molecular Biology and there I was under the tutelage of Eric Furst. Eric Furst was one of the first postdocs of Sir Alan Ferscht who literally wrote the book on enzyme kinetics. He was knighted for writing this book. And so I was, I feel like I learned enzyme kinetics classically from essentially ‘The Man.” But I was doing both single turnover and multiple turnover kinetics using stop flow fluorescence spectroscopy, surface plasmon resonance, and we started to dabble with the X ray crystallography, because here we were, we were busy understanding the function of an enzyme. How does it work? It's a machine, you know, it's doing something, it's turning something over. In our case, it was tyrosine-tRNA synthetase. So it was busy taking tyrosine hydrolyzing ATP and connecting it to tRNA so that you could create protein in your body one amino acid at a time. So we were trying to understand how that enzyme could work. Whether we could switch the chirality and engineer our own proteins from L to D amino acids, and really just really just trying to dig into what can we do. How can we break the system? So that's what we did, and I absolutely had a blast. I really wanted to expand more on the structural side. Um, so I made a beeline as a postdoc to St. Jude Children's Research Hospital. I studied under Eric Enemark who is a preeminent X-ray crystallographer. We worked on DNA helicases. Because if I could understand how to stop DNA replication, then we can stop cancer. Because every cell that's dividing cancer, all it is dividing cells, they all have to have a genome. But if they don't have a genome then they're not functional cells, so if we can stop that, then we you know, if we just stop the helicase, then we're good to go. So we were, we were working on that. And so, I was I was able to be the very first individual to purify and determine the structure of a eukaryotic DNA helicase with single stranded DNA bound to it. And so, I was able to determine this down to 1.19 angstrom. I could see the hole in the in the benzene, benzene ring of the phenylalanine of the proteins because I had such tight resolution. I could rotate it, I could see how the DNA is all binding to it. And, and it was, it was a seminal paper. It was published in eLife. It was one of the very first papers when eLife first came out. And so it was it was really fun. It was an absolute blast. But then I needed a real job and St. Jude's got a GMP facility there on site. And so I found out about a position that was available, I applied for it, and I got it, within the analytical development QC group. We made monoclonal therapeutic antibodies, monoclonal antibodies, we had cell therapies, we had lentiviruses. So I was one of 20 people on a team to cure 11 little boys of bubble boy disease. We had kids who were flying in on private chats, because they had to be ultra clean, and six months, nine months later, they were getting on commercial flights. They're outside, they're living in normal life. Again, this is why we do what we do, right? And so so I had a great opportunity there and St. Jude is also home of a couple of WHO directors. And so we were working in in in BSL-3 suites working on viral genome stocks, or viral cell stocks to essentially anticipate maybe the next pandemic. But I moved on from there to other, to other companies to grow. And so while I was at St. Jude, after about six months at the GMP facility, I was hired and as the manager of QC. Everybody seemed to trend to come to me and talk to me and ask questions and stuff. So I cut my teeth and learned to lead and to guide and to mentor there. And so since then, I've been some form of leadership director, senior director, now to Thermo Fisher, scientific VVS, as senior, you know, as senior manager of the, of the analytical development here. So, that's a long story, but there you are.
Jordan Ruggieri 28:01
I don't even know where to go from there. It was that was just, just inspiring.
Cliff Froelich, PhD 28:06
In all reality, it's, I didn't do it alone. It took a lot of really cool and really smart people. You know, and that's the thing is I get to hang out with some really smart people. And, and usually, most of those individuals are so smart that when they think, their brainwaves kind of come and slap me in the forehead. But you know, it's being able to ask the questions, not being afraid to ask the questions. I've gotten to a point where nothing embarrasses me.
Jordan Ruggieri 28:36
Is that, is that the advice you'd give? You know, ask questions? Is that kind of the advice you give to anyone?
Cliff Froelich, PhD 28:42
Yeah, my advice is just don't be afraid. Nothing. Nothing scares me anymore. I'm always embarrassing myself. If I had children, they would be constantly embarrassed. But uh, but, you know, it's just asking the questions, and some of them may seem really obvious, but no one asked it.
Jordan Ruggieri 29:03
We ask everybody actually comes on the podcast, it's a perfect segue, actually, was there something in your mind that stands out as your most embarrassing moment in the lab? And then on the other side, something that you're the most proud of?
Cliff Froelich, PhD 29:20
I think that any drug product that I get out I'm the most proud of. You know, the fact that I've done what I've done so far is pretty cool. But that's, I want more, I'm greedy. Um, you know, I want to I want to save more people. Embarrassing, you know, there was that time I dropped the bottle of TEMED and it smelled like rotten fish everywhere. Um, in the lab, it was absolutely horrible. So you know, again, I'm dating myself, I poured my own SDS page shells and we had TEMED there, as a, you know, a polymerizing. Anyway, yeah, there is that, you know, obviously being elbow deep doing the necropsies in chickens while you've got pizza sitting on the side because we're on a farm and it wasn't a true laboratory, and we're, we're doing that during our masters. You know, you know, and, and when you're doing X-ray crystallography, obviously, you need to do this, usually on sites, although St. Jude, we were very blessed because we could remote into the synchrotron and control the goniometers, and all the robotics and we could actually collect all of our crystals, crystal data, essentially from a closet in my pajamas at three in the morning. But, you know, oftentimes it's better to go on site. And you know, if you go to a particle accelerator ring, like a synchrotron, they're very large. They usually, you know, ours was Argone, and they they've got tricycles everywhere. So like at three in the morning, you might be having races to going around in circles literally because it's synchrotron but on a big, massive tricycle. You know, we all are kids at heart, so we just have a blast.
Jordan Ruggieri 31:08
I don't have any other questions. Christina, anything on your end?
Christina Bouwens 31:11
No that is it for me. I just wanted to thank you so much Cliff. This has been fantastic. Such a, such a fun, a fun session and a really, really great, great time talking to you. It's been excellent.
Cliff Froelich, PhD 31:22
Well, thank you. It's been an honor to have been asked to do this. It's a pleasure.
Christina Bouwens 31:28
That was Dr. Cliff Froelich, head of analytical development at Thermo Fisher's viral vector services lab located in Plainville, Massachusetts. Thank you so much for joining us for today's episode of Absolute Gene-ius. It was produced by Sarah Briganti, Matt Ferris, and Matthew Stock. With more great science around the corner in future episodes, stay curious and we'll see you next time.
Christina Bouwens 31:48
Jordan, I have a good, I have good joke for you.
Jordan Ruggieri 31:51
I'm always down for a good joke.
Christina Bouwens 31:52
Considering we just heard a lot about chickens. This is a very popular one in my house right now. Because I have a five-year-old and a two-year-old. Why did the chicken cross the road?
Jordan Ruggieri 32:01
Why did the chicken cross the road?
Christina Bouwens 32:13
Kleck-caw!
It's a classic