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Duration: 17 Min

Rapid and Robust Analytical Solutions for Identifying Clones in Cell Line Development Processes leveraging the ValitaTiter and Valita Aggregation Pure Assay

This talk was presented as a part of the Bioprocess Quality, Data and Analytical Solutions webinar with BIO-EXPO. Hear from Eligio Iannetti, PhD, Sr. Business Development Manager, Cell Health at Beckman Coulter Life Sciences about ValitaTiter and Valita Aggregation Pure assays. These simple, high-throughput plate-based assays are designed for antibody titer and protein aggregation quantification and can seamlessly integrate into automated workflows.

Victoria takes the audience through:

Transcript

Our next presenter is from Beckman Coulter Life Sciences, also part of Dan and her he is a legio, you know, the business development manager for cell health and the Vellita cell portfolio in Europe. Allegio is going to introduce us to the innovative Vellita tighter and the newly launched Vellita aggregation assays. These simple high throughput plate based assays are designed for the antibody tighter and protein aggregation quantification seamlessly integrating into automated workflows. Welcome Allegio.

Thank you very much, Barry. Thank you very much for joining in for connecting. It's a pleasure to be here. As you can see, we have a little poll as well.

And we're really curious to know and to hear from you guys about what's your typical method for protein aggregation measurement because this is a new technology we have recently launched. So say that we can start really. I was already introduced. So again, you can go to anytime that's my name, Allegio and netty and I'll go straight into the presentation.

So today I'm presenting two solution, but it's a time valid aggregation. Those have been developed with a number of analytical application and one of those has been the cell line development workflow within the monochrome, the large and monochrome antibody process. So the monochrome antibody process, as you know, go all the way from discovery to where the focus is really finding the best molecule, going through the cell line development where the focus is really finding the best clone through process development and scale up and manufacturing. And just to zoom in a little bit more on the cell and development process, we start as an input with lead molecules that were discovered previously and we end up with the best clones. The best clones can be variably around 10, 20, depending really from the specific workflow, how it was designed.

But there are definitely a number of homologies among the different workflow, different companies and then overall there are strong similarities. So we start from a large number of clones over the course of the last, let's say 10 years, probably this is, it calmed down a little bit, but it can easily be up to more than 1000 of clones that are initially screened to identify the best 10, the best 20. So we go from higher volume, sorry, smaller volume with many, many clones to bigger volume, so up to 5, 10 ml with just a few clones. It is critical to have a rapid and robust analytical solution to identify those best clones.

Some of those are obviously tighter in aggregation. Within this process, there are still a number of challenges and those are mostly, let's say in part due to the manual process that is still happening a lot. So the manual process takes really a number of challenges in relation to the time, in relation to the fact that it's more complex obviously to manage all those parallel workflows and these come with obviously time, there is a significant amount of time that is spent, but also there are other problematics that come up like the risk of contamination, the inconsistency, the possibility of bringing not the best clones forward, because the data somehow sometimes are not as centralized as you would like.

So at Beckman, we have also a really solid and strong automation capability and team, as you probably know, and here there are some of the cell line development applications that have been already automated by the Beckman team. So all the way from the genomics, really at the beginning of the process where we have the vecton of construction and the transient first and stable after transfection, monochrome analysis, cell culture, molecule analysis, cell analysis, analytics and data. And today as mentioned, we're going to touch on two specifically, more specifically, so tighter quantification and protein aggregation quantification. So those two are fully available and they are available in multiple plates. These are coated with a Liophilus probe and as was already briefly introduced, they really allow rapid and throughput quantification assays.

So they share some similarities, those two solutions. So first of all, the accuracy and precision, these can be based on the specific workflow on the hardware or the number of factors, it can be below 3% of variation and below 5% accuracy bias. Simplicity, it's a key key point, simplicity to measure. So just add, mix and read is that simple and simple to analyze because it is a relative quantification assay. So you simply need to interpolate your sample with the data you've generated. Rapidity, as we said already, so you can measure really up 96 samples in less than 50 minutes. Again, this is dependent a little bit from your workflow, from your source sample and so on, but it can be achieved in 15 and less, less than that. Trueput, over under 50 samples in less than an hour, super easy to automate. The fact that is add, mix, read, make it really, really simple to automate. There are no shaking between, there is no centrifugation involved, so everything gets just easier to be automated. Also the capital expenditure, it's an interesting point because you need just a print reader. Now the technology behind and Valita, Titer and Vali-Dagration is fluorescence polarization.

The concept is quite simple. When a molecule, it's free, it rotates in solution with a certain speed. When this molecule, our probing is case bind to a target, its molecular weight is going to increase. So if we take the example of human Hg, our Titer probe is about 10 kilodaltons, so it rotates at a certain speed when our probe is free and bound in solution. When you can negate, it binds to the Hg to the target, that is typically a human Hg around 150 kilodaltons.

Obviously the complex will become of 160 kilodaltons. There is a big shift in molecular weight that will influence a shifting molecular rotation and therefore in fluorescence polarization. We can detect that with a plate reader and I'm going to touch on it on the next slide. So talking about the workflow, we start really from the plates. There is no additional reagents, nothing that you need to add on top. You just have the plates that are ready to be used. The problem has been dried down, lay of the lies at the bottom of the wall.

You add your own media, catch on media, or commercial buffer. We can obviously support you and discuss what is the best approach based on a number of factors like pH and so on. And then you add your sample, really simply, 60 microliters of sample in the 96-well plate and 40 microliters of sample in the 334-well plate. You mix and you incubate at room temperature. So there is no special temperature condition.

There is no shaking. Really simple. You add mix, you incubate at room temperature, different incubation time for the different products. So for the lower detection range of titers, you have five minutes. For the higher detection range, you have 15 minutes.

And for the aggregations, or protein aggregation, or quantification, you also have five minutes. As a last step, you read with a plate reader. So here, obviously, we have a molecular device's plate reader.

We work really closely, obviously, with Moldeve. And a full 96-well plate can take less than two minutes to be read. So basically, in less than 15 minutes, as I said before, you can read all your 96 samples.

And now, in these slides, you really got a breakdown of where the time is going. As said, all what you need, it's really the plate and the plate reader. The plate reader needs to have a fluorescent polarisation detector, and also the right excitation and emission filters.

So the tighter probe has a red spectra, and then, sorry, there's a green spectra, and the aggregation probe has a red spectra. But we can really support you closely. So we're happy to discuss all the details with you. Look at your own plate reader.

If you have one, also advise you in purchasing a new one, and providing really a full solution. And that's what we do really. We come into your lab and we support you closely to stop everything from the plate reader to the standard curve, to the workflow, to the pipette thing, really. We take great pride in supporting really closely all this process. And here, I'm going to jump a little bit in detail around Valita Titer.

And to start with, I would like just to get your attention to this really concrete, really quantitative benefit of using Valita Titer. You can screen your best clones, as we said, in less than 15 minutes. So this is something that other solutions struggle to achieve because, you know, in average, you can easily go over one hour. And so we're really proud of this incredibly, incredible speed that we are bringing really to your labs. But besides the speed and the ease of use, there is another big, big benefit. There are boosts of our data, the performance. So here you can see a data comparison between Valita Titer and HPLC protea.

So one of obviously one of the gold standard between Valita Titer and BLI and between Valita Titer and the LISA. So you can see 20 samples that have been quantified by our industry partners. And you can see on the top graph, the alignment, the trend analysis, and the bottom graph, you can see the correlation.

So all of those samples have a correlation of 0.9. And that's really what we, that's our goal. We come to your lab, we set up everything, and we build a comprehensive data set where we are demonstrating that our solution is really aligning with your current method. So we really create the data set for you that once we have gone, you are fully ready to integrate the solution because everything has been set up, everything has been qualified.

And, you know, you get really, I think, some nice confidence to proceed and use your solution, our solution. And more, you know, in detail, looking at the accuracy bias and precision, as said before, also this can go below 3% in precision. So, you know, looking at different replicas and less than 5% of accuracy bias. So when quantified pre, pre-quantified known samples. I have to say that the bar graph are there, are quite invisible.

That's just because they're really, really tight and really small. And that's, that's really what, what you can achieve. So besides unprecedented speed, throughput and ease of use, this is also an extremely reproducible and robust assay. Just a couple of slides more about protein aggregation. So also in this case, really quantitative output, you can quantify your data in less than 15 minutes. We have again two industry partners, dataset, more than 40 samples have been quantified, at the same time with Valita aggregation and with HPLCSEC, again, the gold standard for this kind of analysis. On the right, instead, we have a few more samples where we have grouped us in the different aggregation range. So between 0 to percent, between 2 and 5% and above 10%. At the moment, we can achieve 0.5% of percent of aggregation.

So that's really what we have been demonstrated so far. So we can, we can be really sensitively in quantifying the protein aggregation. We can, as an output, we give you percent of our aggregation, monomer versus all the rest, basically. And this graph again, around the precision and accuracy.

So similarly to Valita data, we have less than 3.5% for precision and less than 5% on, on accuracy bias. Okay. So as a last slide, really on the automation side of things, as I say, this is an extremely easy to automate. Together with the automation colleague, we have fully implemented protocols so that if you have a Biomek from Beckman as a liquid handling, you're ready to go, you have the protocol, we can provide you, then you can quantify your data and your protein aggregation with a, with a, with a Play 3, that obviously can be fully integrated. And, and you can achieve more than 40, 450 samples in less than one hour. So that's really the, the throughput that you can reach. And that's it for me. Obviously, I'm really happy to take any question.

Thanks, Ligio. Impressive. Really, really neat stuff. Do I have to remove cells from the sample, or do I have to purify the protein before running Valita Titer?

That's a really good, really good point. So I'm happy to ask this question because that's what one of the biggest benefit. Valita Titer is super robust to cell presence. So we've demonstrated up to 50 million cells per ml. But honestly, we can go even higher.

More recent data showed really that they can be, they can be pushed even higher. So there is no need for any centrifugation and if you're traction, you go straight from culture to the plates. So that, that simplified things quite a lot because as I mentioned before, there is no additional step, especially from an automation point of view. That's really, we believe that's a great advantage. That's what also they've told us. So yeah, you're straight from culture to, to play to our place and to results.

Okay. A couple of questions about reagents. And I'm going to combine them into one question. So how many reagents does Valita Titer require? And is, is the reagent specific to the antibody and does it react to the HCP?

Yeah, yeah, that's another really good question. So there are no reagents. That's a beauty of it. You get the plate and that is ready to go. The flow of the probe, which is constituted by the fluorophore together with the binding peptide, it is dried down, lyophilized. So you add your sample, our probe, get in suspension and that's it. So there is really no proprietary buffer, no additional reagent.

That's simplified everything because, you know, there are many of those key and protocol where you have solution A, solution B, C, D and, and it gets complex. You know, you have to bring everything to room temperature. Here you just take the plates out, you add your sample and you go into the plate reader basically because you can incubate into the plate reader.

So that simplifies things, things quite a lot. And no, you don't need any specific, you know, reagent because everything is ready. And this solution, Titer is really specifically designed to quantify IGG. So you, you know, the target needs to be, well, web work obviously mostly with IGG. But the point is you need to have an FC domain, so a component obviously of the FC domain. But any molecule having an FC component can be quantified by our solution.

And yeah, this is HCP and not the protein absolutely do not make much of an significant effect. We've been looking at this really for years now. So it's really robust. It's based on protein G, so really gold standard in the industry. So yeah, yeah, really simple from Maria Gent point of view. Terry.

Well, thank you, Aligio. Thank you for being with us today. That is Aligio Yannetti. He's from Beckman Coulter Life Sciences.