Podcast Transcript

David Brühlmann [00:00:47]:
Picture a clone that looks flawless in a 96-deep-well plate, then disappoints the moment it hits a production bioreactor. It’s a costly, familiar problem in biologics development. Today’s guest built a company to solve it. Kent Rapp, co-founder and CEO of Biolinco, uses DNA barcoding to track a clone’s identity from early screening all the way through scale-up. Let’s find out how it works. Kent, welcome. It’s good to have you on today.

Kent Rapp [00:02:24]:
It’s great to see you too, David.

David Brühlmann [00:02:26]:
Absolutely. To start us out, share something that you believe about bioprocess development that most people disagree with.

Kent Rapp [00:02:34]:
I think I probably have a lot of thoughts about bioprocess development that might be slightly controversial, but I think a big one would be that biotech loves to automate their problems instead of solve them.

I work mostly in the cell line development space, and I’ve been working in that space for a while now. I’ve seen that they want to improve their throughput a lot of times and just screen more cells whenever they have a challenge. The answer is always, “Screen more cells. We can find the one.”

This is missing a lot of the context and the connection between the cells and what the actual goal is. They are trying to find a cell that lives up to its potential in these manufacturing environments that are larger scale, but they’re actually only screening at really small scales. So, their answer historically has just been to screen more cells at the small scale. You’ll find the answer eventually.

But what they really need to be doing is measuring the cells at the larger scales, in the context of where it’s important, and putting that in the context of the entire bioprocess development pipeline, from vector design all the way through large-scale performance.

David Brühlmann [00:03:46]:
You’re making a really good point, Kent. You need to understand the problem you’re solving and then solve the problem first, and then automate. And sometimes, I had a chat actually with an automation specialist, and he said, “You know, sometimes the solution is not even to automate because the solution is much, much simpler.“

Kent Rapp [00:04:04]:
Yes, yes, for sure, for sure. And I mean, don’t get me wrong. I mean, sometimes automation definitely is the solution. You just have to go into it knowing that that’s what you’re looking for, rather than using it as a crutch to fix a problem that it won’t actually solve.

David Brühlmann [00:04:20]:
I’m excited to talk about cell line development today, but let’s start out with yourself. Draw us into your story. What drew you into science? What was that spark? What were some interesting pit stops along your way, now leading to your role as a company founder?

Kent Rapp [00:04:37]:
I was always kind of interested in science and did chemical engineering in undergrad. I had a brief stint where I interned at Regeneron, kind of right as it was hitting its major growth phase, and that gave me the bug for biotech. So, I’ve always been interested in the bioprocess scene ever since then.

And then I had a fellowship where I was able to live and work in Germany for a year. As part of that, I was working at a startup company, and that’s what really gave me the bug for startups. So, I decided to go to grad school to better understand the bioprocessing scene, the biomanufacturing scene, and also to potentially start a company.

As I looked around at all these biotech startups, it was completely saturated with PhDs. Everyone had one. So, I figured this has got to be the right path for me. I can’t go wrong.

And sure enough, I joined a biomanufacturing lab when I was in grad school at Johns Hopkins. And after seeing a lot of the challenges that the biomanufacturing industry faces year after year after year, and working on research and proposals to address these challenges, I was talking to a friend, and we came up with the idea for a way to improve cell line development using DNA barcoding.

So, there happened to be an expert at Johns Hopkins who specialized in DNA barcoding and lineage tracing, and he was the perfect collaborator for us to marry this idea of DNA barcoding and lineage tracing with biomanufacturing and really get it into the commercial space.

And when you think about it, cell line development is actually a great place for this technology because you require a single cell and a single cell line, and that’s what lineage tracing specializes in. It specializes in identifying single cells, their lineage, their daughter cells, and understanding their performance in the context of everything else going on around them. It was a perfect application from our perspective.

And then I was very fortunate to be in Maryland, which has a lot of support for startups and commercial ideas. So, we were able to get some external funding from the state of Maryland, from some local venture capital groups that were interested in commercializing technology, and Johns Hopkins itself to actually implement our idea and build on the science and develop a proof-of-concept data package that we could then take to potential customers and show off how this worked.

Once we got there, we have been able to just recently launch the company. We launched two months ago, got our first customer as well, our first customer contract. And we are now growing and expanding and building on the platform.

David Brühlmann [00:07:30]:
Well, this is great. You get your first customer within two months of founding a company. That’s awesome. Let’s start with the elephant in the room. What, or where, does the conventional approach to clone screening actually fall short today? What is the problem?

Kent Rapp [00:07:47]:
That is a great question. So, I think that the conventional approach tends to fall short because it is missing a lot of the context, both that comes before and after in cell line development.

Before, you are often screening your cells and looking for a lead molecule that you want to move forward. And sometimes companies are using different cell lines, different vectors to do this screen than they’re actually planning on using for manufacturing. So, by the time they move into the cell line development space, they are putting it in a different cell line, different vector, and, surprise, it performs differently.

And you get these unexpected results, and it becomes a challenge for the cell line development team and downstream manufacturing to continue developing it the way that was expected initially.

Some companies have mitigated this by actually aligning on what cell line and vectors they’re using, but they’ll often still screen many different vector designs to find an optimal one. And they still have to make a decision about which to move forward with. From the early vector screening stage to the cell line development stage, there’s still a gap there.

And then from the cell line development to the later biomanufacturing stage, there’s another gap because cell line development is often screening in high throughput in a 96-well plate or a microbioreactor or something else. And then the actual bioreactor environment that is happening in these bioprocesses is in many, many orders of magnitude larger volumes, with gas control, fed-batch culture, sparging. It’s much more controlled and involved than a static 96-well plate or even a shaking 96-well deep-well culture.

The cell line development team is in this tricky position of having to optimize with some vector and molecule that they’ve been handed that hasn’t necessarily been optimized for what they’re trying to do, while also trying to predict how it will perform at larger scales without actually being able to measure confidently at those larger scales.

David Brühlmann [00:10:00]:
This is a huge challenge. And when you’re developing a cell line, you want to screen as many cells as you can, and also try to analyze not only productivity but other parameters, such as product quality, as early as you can. And then, hopefully, what you’re seeing at small scale is predictive enough to get similar results at larger scale.

So, where does your technology now come into play with all these challenges and complexities a cell line development scientist is facing?

Kent Rapp [00:10:34]:
At Biolinco, we have kind of reimagined what cell line development could look like. And instead of trying to just brute-force screen more cells, we rethought how to get better measurements out of the experiments that we already have to do.

We are able to label every single cell with a unique DNA barcode, and that allows us to grow all of the cells together in a pool and understand their individual characteristics by linking them to a DNA barcode.

What that allows us to ultimately do is grow a mixed pool of cells in a bioreactor and then measure the actual manufacturability of the molecule we’re interested in, in a bioreactor environment, rather than having to screen the cells individually in a 96-well plate or 1,000 different bioreactors if we want to screen 1,000 cells.

By linking all of this data with a barcode, we have converted all of our measurement challenges into sequencing measurements instead of offline, more complicated, or difficult serial measurements that you have to do on individual cultures.

And sequencing costs have come down dramatically, and you can get much higher throughput. So, as a result of all of this, we can improve your throughput. And we can also give information earlier by screening at a pool stage for the individual clonal performance. And we can do it at a manufacturing scale, so in a bioreactor environment that’s true to the conditions you’ll eventually have, with all of your feeds, pH control, shear forces, et cetera.

David Brühlmann [00:12:19]:
So, once you have your pool, instead of screening and analyzing at small scale, you take all these cells you have and go directly into a benchtop bioreactor to analyze the entire pool. Is that correct?

Kent Rapp [00:12:35]:
Yes. So, we can analyze all of the clones, all the cell lines in the pool, in that bioreactor because they’re uniquely labeled and are distinguishable by the DNA barcode.

David Brühlmann [00:12:47]:
And how do you detect or measure the barcode? How does that work?

Kent Rapp [00:12:52]:
We have had to develop a lot of assays that convert the standard manufacturing and cell line development measurements that are of interest into sequencing variations instead.

For growth, that’s probably the easiest one to explain. What you can do is you can measure your pool growth and cell density over time in a fed-batch culture, just as you would for any other cell line. And then you can also take a sample every single day from that culture and sequence it.

By sequencing the DNA barcodes in that set, you now have measurements of the fraction of the population that each cell line makes up of the larger pool. We can then just combine those two measurements—the actual pool cell density and the fractional representation of each cell line or barcode in that pool—to get the individual cell density of the cell line that we’re interested in.

David Brühlmann [00:13:53]:
And how do you account for population drifts or changes? Because as you move forward in a run, certain cells will grow faster than others, and so the distribution will change.

Kent Rapp [00:14:04]:
Yeah. So, that is the biological heterogeneity that we love and we’re interested in, and what ends up actually making a great versus an okay cell line through the cell line development process.

We are able to track all of these things with extremely high sensitivity. We can detect cells as rare as 1 in 100,000. With standard cell line development methods that use plate-based assays, this would translate to about 1,000 96-well plates, which is about 100 times more than I think most companies are currently screening at.

We have incredible sensitivity because of the sensitivity of DNA barcoding and the high-throughput sequencing that has been iterated on over the past several decades. And the population drift is not actually as problematic as you would think, because when you can measure with such incredible detection levels, you can measure really rare events and get those really rare, high-performing clones.

David Brühlmann [00:15:03]:
And once you have identified your high-performing clone, what is the next step? Do you go back to your pool? What happens then?

Kent Rapp [00:15:12]:
Obviously, you can’t have your totally pooled culture and use that for manufacturing. You still have to get your monoclonal cell line.

We’ve also integrated a selectable marker into the cells that is dependent on the DNA barcode, and we can use a CRISPR activation-based method to specifically target the DNA barcode, activate our selectable marker, and then select out just the cell line that we want with really high sensitivity.

We’ve been able to see roughly 99.8% specificity for the cell line that we want, 70% recovery efficiency of the cell line, and are able to retrieve these clones with really high accuracy from the mixed pool.

David Brühlmann [00:15:58]:
And how does the workflow look like once you have extracted your pool? Do you still do cloning and the subsequent steps, or can you skip that entirely?

Kent Rapp [00:16:09]:
That’s something that we’re currently working on. So, I know that the industry would love to move away from having to do monoclonal validation and everything, especially with new transposase technologies. And we are also in that boat.

We have a DNA barcode that allows you to validate that all cells that you’re using are from the same cell line and the same clone. So, we do expect that we could sort out, for example, 100 cells that all are of the same cell line and use that small subpool for large-scale manufacturing.

That would help with outgrowth speeds and the outgrowth efficiency for your actual single-cell cloning step because you’re no longer putting a cell in a well by itself. You’re able to put a cell with all of its friends and then help it grow.

But as of now, with FDA regulations, and because we’re still an early-stage startup, we’re building this out. We are relying on the tried-and-true kind of method of single-cell cloning of the high-performing clone that we want, and then making sure that we’re using all of the tested and proven monoclonality verifications. So, visual inspection, et cetera.

David Brühlmann [00:17:21]:
How does this overall speed of the cell line development using your technology compare to the traditional one?

Kent Rapp [00:17:31]:
We get that question a lot. Time is of the essence when you’re trying to move into a clinical regulatory filing, an IND filing, and we believe we can speed up the process by overlapping a lot of your lead candidate selection and vector design with the cell line development process.

Right now, they have to be done sequentially. So, first you choose your vector, you make sure you do a quick pool study to make sure that you’re getting reasonable product purity and titers, and then you’ll take that vector or cell pool and do single-cell cloning on it to move into your next stage.

We don’t have to do these intermediate tests with the technology that we’ve developed. We can take your initial screen, your initial pool, and then get all of the data that you could possibly want from it. And immediately identify which clone is going to be the one that you want in terms of productivity, titer, growth. And we’re expanding our assays to include some critical quality attributes now, like product purity.

And once you have that information, you can simply pull out the one that you want using our CRISPRa selectable marker, and then use that for large-scale manufacturing. You can skip all of the intermediate tests.

David Brühlmann [00:18:46]:
What is the impact of this DNA barcode, which will stay in the cell, obviously? Is this relevant at all in terms of any safety aspects later on? Or is this regulatory relevant?

Kent Rapp [00:19:03]:
Yeah, the regulatory element is actually—there isn’t a huge issue there. The barcode is not transcribed. It is not present in the cell. It is purely in the DNA just for purposes of labeling and tracking.

And when you’re inserting an entire transgene that’s expressing a new protein, has all these other features on it and vector elements, the barcode is a very, very vanishingly small fraction of the entire construct that is being inserted into the cell.

So, there aren’t major regulatory implications on the actual barcode front. It’s just a helpful tool that helps you get more out of your single-cell cloning process.

David Brühlmann [00:19:46]:
That’s where we’ll pause for now. Kent Rapp has already given us plenty to chew on, and there’s more ahead, from characterization to lessons learned as an entrepreneur. Stick around for Part 2.

If you are enjoying the conversation, please leave a review on Apple Podcasts or your favorite platform. It generally helps others find the show. Thanks so much for tuning in, and I’ll see you next time.