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Accelerating quantification of oligo therapeutics with robust microfluidic.BZ200624
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Accelerating quantification of oligo therapeutics with robust microfluidic.BZ200624
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Segment:0 .
Hello, everyone, and Thank you for attending today's webinar. Accelerating quantification of therapeutics with robust microfluidic methods I'm your host. Taylor and Francis senior editor Tristan fry, and I'll be your host for today's event. Before I introduce you to our speaker, I'd like to quickly cover a few housekeeping items. You've joined the presentation and will be listening via your computer's audio by default. If you prefer to connect to a different speaker, you can adjust your audio settings by clicking the arrow next to the microphone icon at the bottom of the Zoom window.
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So we are delighted to have John Chappell presenting for us today. John is the senior global scientist support manager at gyros protein technologies, where he is responsible for training the field application team, as well as implementing customer collaborations and scientific innovations. John has 25 years of experience in the contract research industry supporting both pre-clinical and clinical drug development.
He is particularly interested in validation requirements and ensuring the data generated will be acceptable to regulatory authorities. Thank you for joining us today, John. I'll now hand over to you to start today's presentation. Good afternoon or the time zone where you're currently at. So as Tristan mentioned, I'm Jon Chappell. I'm senior scientific support for gyros protein technology, and I've been at gyros protein technology for six years approximately.
And then prior to that, I worked for many global crows for over 20 years developing and validating immunoassay. As was mentioned, and is actually back in the mid 90s where I actually did an oligonucleotide assay research project to develop a hybridization using an immunoassay type format. So I've actually got a long history doing oligonucleotide analysis, so I was very excited to try and get oligonucleotide assays to work on the gyro lab platform.
So today I really want to summarize some work that we've done both internally, but also we're also collaborating with external collaborators to also continue the work in oligonucleotides. And the oligonucleotide work really started from a customer request and we wanted to optimize and get the best out of our platform. And also we feel that the gyro lab platform suits the analysis of oligonucleotides with its many advantages.
So today I want to cover a little bit about oligo nucleotides. I'm sure most of the audience will know about oligonucleotide therapeutics, and I'll give examples of this and that. It's very much a growing area, and then I'll give you an overview of microfluidic oligo assays and also how they compare to traditional hybridization Elisa assays.
Then I want to go through the use of an offline incubation and then an and then the assay is using automated detection on the platform, which is one format that can be used. And the second assay format is to look at fully automating the platform using our mixing, which I think gives the greatest benefits. But obviously both approaches have certain advantages. So I'll go through that and then I'm going to go through some nuclease options as well.
The use of nucleases can be very helpful, particularly in reducing metabolite interference. So that's really a consideration when developing hybridization assays. So the oligonucleotide area is very much a rapid grown of area. And actually it started originally back in the 80s and 90s. And then it went through a bit of a boom and then it stopped and then it's very much rapidly growing now.
I mean, even last year we had four approved oligonucleotide therapies in 23, and overall there's been total 20 total oligonucleotide therapeutics, of which half of those are antisense oligos. And this is the main area that we're going to discuss today. But there are other kind of areas for oligos as well. And obviously in the post COVID vaccine kind of environment, there's a lot more interest in developing oligonucleotides, particularly with the alternative delivery mechanisms.
And in general, it's very much a vast and growing area. As you see here. There's various approved AstraZeneca, Ionis, Novo Nordisk, et cetera, have all recently had therapeutics approved. So in terms of the types of oligonucleotides. So as I mentioned today, I'm going to be talking about antisense oligonucleotides, which are targeted messenger RNA sequences, and they're normally approximately like 20 bases.
And there's been a lot. This is probably the most common. And the first area that people have been working on. But there are also siRNA or small interfering RNA. Again, they can prevent the production of a disease causing genes and many of these are in clinical trials and we also have microRNA and other forms include aptamers, which have a much larger sequence and have a three dimensional can act like a protein even by having binding et cetera.
So they're much larger. And things like CRISPR, which is a very exciting area of oligonucleotide and growth. And, and obviously there's other things like genome editing. But today really I'm going to concentrate on the antisense which basically this project is concentrated on. So antisense oligonucleotides are small, synthetic, single, stranded nucleic acid molecules, and they're designed to bind complementary RNA sequences.
So they're meant to effectively in most cases, stop the production of a disease producing protein. There are various delivery systems. So they can be conjugated to targeting ligands. I've heard a lot about recently about antibody oligo conjugates, for example, as well as other things that can maybe target it to the liver, for example, to deliver the oligo to the liver and but also can be encapsulated in nanoparticles like lipid nanoparticles, for example, to improve the delivery because one of the main things with antisense over its history is really to try and get it to its target organ or to the site of action.
And the main thing to do that is to really to increase its half life and to really do that they've modified the antisense chemistry. So on the left here in terms of the chemical sequence, you can see the normal nucleotide sequence. This kind of the first generation was the phosphorothioate, where you had a sulfur molecule being substituted into the backbone and then you had the second generation, two or more and two more and then the third generation PMO.
But all these modifications are designed to increase nuclease resistance and to prevent it being metabolized at following dosing, et cetera, and to ensure that it gets to its target organ or to where it's going to act, basically. So the important considerations with oligonucleotide is mean, obviously sensitivity. They're dosed like a small molecule, so they are typically administered at low doses.
They obviously lack of interference and metabolite cross-reactivity. So obviously metabolites where you may get n minus 1, n minus 2, for example, may also be still be biologically active and matrix effects can be a big thing. Obviously for pharmacokinetic analysis, you normally collect the blood serum, for example. But there's a lot of interest in tissue distribution.
So where the actual oligo distributes in the body. So we I've done a lot of work on tissue depending on the oligo or membrane like lung tumor, liver, et cetera, et cetera. And that obviously when you're measuring in tissue that you have to homogenize and have sample prep procedures to actually measure and extract from the tissue. And also, I've done a lot of work with cerebrospinal fluid as well.
So it depended obviously on the mechanism of action, obviously, and of which tissues and which organs you're going to look at. So the first is really to look at a dual hybridization assay. So as I mentioned, an antisense is approximately 20 bases. So when we look at a dual hybridization assay, we treat it as if it's an immunoassay. So we look for a complementary capture and detection probes.
So we're using Watson hit a watson-crick nucleotide combination, so complementary a to C to G. So effectively we take the parent sequence and we design a complementary probe for that normally about half the length for capture and then also again half the length for detection. And in this case, you can see we're using a biotinylated for capture and a fluorescent label for detection.
So basically you run it like an immunoassay and then you wash away unreacted probes after the hybridization. One of the potential disadvantages of this assay format is that if you do get metabolites 3 prime and 5 prime metabolites, they can still cross-react in the assay and this cross-reactivity can actually be quite high. So this is really is not necessarily selected for parent.
So that kind of introduces kind of the immunoassay format. And now we're going to move into the Jairo lab platform, the microfluidics. So this gives a schematic of the basic principles of the Jairo lab. So we have a microfluidic structure at the bottom of a CD or compact disk, and we have an affinity column at the bottom of that microstructure.
And that has a strict streptavidin bead and you need a biotinylated antibody for capture and you need a fluorescent Alexa label, a 647 normally for detection and it's microfluidics. Everything is moved throughout the CD by a combination of capillary action and centrifugal force. The instrument itself will actually spin the CD. And this allows the fluids to travel throughout the microstructure.
One of the big advantage of the lab is that all the samples are loaded onto the CD and then when it spins, they all move across the microscope structures at the same time. So you almost get simultaneous processing of samples on the CD, which has a lot of advantages. And because it's microfluidics and because of the reaction times as part of the assay, it normally takes approximately one hour to complete a whole CD.
And the CD normally runs an equivalent amount of samples to an Elisa plate, depending on the assay volume on the CD. And actually, on the right hand side, you can see the lab Explorer, which is our one system which can process one CD at a time. But also there is a five CI/CD system, the ixpand, which can process up to five CDs.
So when it comes to the two formats. So we had a semi-automated and fully automated. So the semi-automated assay uses our standard CDs or compact disks. And this basically, as I mentioned, has a micro-structure with an affinity column at the bottom, but also has a common reagent channel. So reagents like the capture and the detection reagent get added to the CD in blocks of 8, around the CD, and then we have individual sample inlets for the samples.
So the samples are individually loaded around the CD. And as I mentioned earlier, sort of movement through these. The CD is achieved by a mixture of capillary action and centrifugal force. The hydrophobic barriers kind of help to control the liquid flow. And then we ramp up the centrifugal, the spinning of the CD to move the liquid through the hydrophobic barrier. The advantage of the semi-automated format is it allows us to change the assay volume.
So we have the smallest CD is the 20 nanoliter CD, so that has 20 nanoliters of sample being introduced into the sample inlet. And then the biggest CD is the 4,000 nanoliters. And then we have CDs at 200 and 1,000 nanoliters so by actually shifting with a different CeeDee, you can shift the analytical range. So if you want a very sensitive assay, you want to look at a very high volume CD.
So 4,000 nanoliters and then for example, if you want a very kind of low sensitivity, when you're measuring high concentrations, you can actually measure that, use the 20 nanoliter. So it gives a lot of flexibility and actually you can take the same assay and develop it and then use it on different CDs to adjust the analytical range, which a few people are doing. And it can be a way of avoiding diluting samples, for example.
But obviously, it means you have to validate your assay on multiple CDs. And then for the fully automated CD, this is actually the mix in CD. So this is slightly different microchannel to the normal CD. It has a mixing chamber in the middle. So this mixing chamber allows the addition of 20 nanoliters of sample and you can add up to four steps in the mixing chamber.
It was actually designed for anti-drug antibody assays where for a fully automating acid dissociation procedures. But this CD is very useful where you want to increase contact time and to pre incubate things. So for example, we've had people that want to measure free targets, so they want to dissociate and also dissociate drug, for example. But also it's good for the oligonucleotide acids, which I'm talking today, allows you to add the sample and then the complementary probes sequentially into the mixing chamber.
You can actually change the timing of an incubation within the mixing chamber. So it gives you a lot of flexibility. And obviously the advantage of that is you can really increase sensitivity. But obviously the more incubation time you use, the longer the assay becomes. So it's like a trade off depending on what you need for your assay.
And as I said, what the mixing CD does allow you is to fully automate the whole steps in the assay. So I'll start off with the offline incubation. So this actually started off as a master's project, which I mentioned at the very end of the presentation. So we had a master's student working in our lab in Uppsala and we're very keen as a company to do scientific research and to try and develop new potential ways of measuring things and new sort of technical advances.
So we had a student actually start working on the assays as part of a project. So we took a commercial sequence which is based on amondys 45 or casimersen, and this is actually a PMO, so a third generation oligonucleotide chemistry. And we actually had the sequence artificially manufactured. So it wasn't a case of buying the drug.
We actually had it synthetically synthesized and then we had complementary probes actually manufactured as well, a biotinylated and a Lexa floor sequence as well. We actually also not really going to talk about it today. That much, but we also looked at digoxigenin as an indirect detection. And that is a very popular method for detection of oligonucleotides.
So that is actually an option in lab format. But I'm not actually going to really speak about that much today. But just to let everybody know that is an option. And actually for the offline incubation, we started with the 1,000 nanoliter the 1,000. So that's kind of towards a more sensitive assay, not the most sensitive CD. And also we looked at different buffers, so rexhep and rexhep.
So the gyro lab has a variety of different buffers with different properties. They actually really do help the microfluidics and the transport of the liquids through the CD. And we spent many years developing these buffers and they're very much optimized to use on our platform. And very I always recommend, that people look at our buffers and we had our capture and detection probes were in rexhep.
Rexhep actually contains a higher degree of detergent, so which can be very useful in this case. And what we did in this case, we actually pre-incubated the capture detection and the analyte offline for one hour. And then once the reaction is hybridized, it can then be transferred to the CD automatically. And as explained really we pre incubate the components offline.
We measure the analyte capture and detection and then incubate. This case we incubate at room temperature for one hour. But this is where this assay format gives you a lot of flexibility because you can depending on the temperature of the hybridization, you can do room temperature or you can do 37 or other temperatures. So this gives a lot of flexibility. And then the microplate is loaded onto the gyro lab and then the gyro lab will actually do the whole assay through the CD.
And approximately this offline incubation takes approximately two hours and the automated is for capture and and detection only. So this is an example data. So this was actually using a 50% matrix. One thing that the giro lab is really good at actually is using quite low dilutions and sample 50% matrix or even 100% matrix can be run through the CD.
So that can particularly when you try and trying to get very low sensitivity. Can that be an example of an advantage. And in this case, we had an assay with an analytical range of 1 picomolar to 10,000 picomolar. In general, giro lab technology gives you approximately four logs of analytical range, and this shows you the data of three operators in three separate runs.
And one thing about the giro lab technology, it's very reproducible and it's very robust. So this is consistent data that we see. And then also we had discussions that we needed to one of our customers. We talking to had very, very high concentrations of analyte, particularly in different matrices, in things like tissues, for example.
So we wanted to make sure that we had good dilution or linearity and we could achieve a neat serum at $50,000. And we've got really good sort of linearity. And this was run over 3 operators of 3 over 3 runs. And as you can see, we got very, very precise and linear dilution into the analytical range. And then kind of precision and accuracy.
I mean, in this case, actually, we tested the look at five picomolar. This is on the 1,000 nanoliter. So potentially we could go a lot lower by using the 4,000 CeeDee where we have increased volume. And then we looked at QCs, five concentrations with the upper limit at 8,000. And we have a good precision and accuracy and a very reproducible performance on the platform.
And one thing that was quite important in this case was to look at the incubation time. So we looked at 37 versus room temperature, and we found that there really no difference in incubating between room temperature and 37 degrees. And what this shows actually is in this case, the assay could be run at and hybridized at room temperature, but with the awful lot. As I mentioned, already using the offline incubation, it would allow us to use higher incubation temperatures if needed, depending on what we need, how the reagents hybridize.
So I want to move now on to the fully automated assay. So as I mentioned, this uses the lab mixing CD. And the advantage of this is that you add your samples, your diluted samples to the plate, you put it in the instrument. And then it will run the assay from start to finish. And as I mentioned, it uses the mixing CD. And in this case, the mixing chamber allows for incubation, as I mentioned, and it allows for the sequential or combined addition of reagents.
And we first we looked at the dual hybridization assay where we added to the mixing chamber and give further details of how the assay was set up in the next slides. But just an overview, again, it's very similar to the dual hybridization in terms of the sorry, the offline incubation, exactly the same assay, exactly the same capture and detection and the same buffers.
And then this shows you the assay format. So we start off with the sample. And the sample is added into the CD and then adds to the mixing chamber. And next in we add the capture reagent. And this is added, as I mentioned, by centrifugal force and capillary action. And it gets added to the mixing chamber. And this is mixed for one hour.
And then you can actually change. We looked at incubations from five minutes all the way up to 30 minutes. And basically so you can actually optimize these parts. So in the mixing chamber, you'll see now that the analyte has now has now hybridized with the capture probe to half of its sequence. And then we add the detection probe, which is the Alexa flood detect.
And then this is then again, you can change the incubation time. And it's added like the other reagent through the reagent inlet. And then that gets added to the mixing chamber. And then in this case, as you can see now have got both the capture and detection probe bound to the analyte. You could actually premix the capture and detection together and add them together.
So that's another potential assay format. But for this assay this assay format work the best. And then basically the hybridized mixture will then be introduced over the column, over the strep. So all the reactions taking place in the mixing chamber, then it's by it's a stronger centrifugal force will move the whole mixture and then it passes over the column.
And then it becomes the biotinylated probe, which was used for capture, becomes immobilized on the streptavidin bead. And then the instrument will automatically detect the amount of fluorescence using laser induced fluorescence using the Alexa 647. The advantage of using laser induced fluorescence, it gives good sensitivity but also gives a broad dynamic range.
So when we look at the incubation times, we can actually as I mentioned, we actually incubated for 30 minutes, but we looked at different reaction times. So can actually shorten the incubation. So five minute, 10 minutes and actually by shortening the incubation, you can actually lower the signal to background. So it's very much for the end user to try and optimize the best conditions for the assay that they're running.
And then the fully automated platform. We actually we ended up with a lower IQ than the offline, so we ended up with an IQ in serum of 20 of two picomolar. So it was slightly more sensitive. You can probably tell by the curve shape it's slightly straighter at the bottom end, so you can see it's slightly more sensitive. Maybe that's even though it's a lower volume CD, we've ended up with higher sensitivity.
So which is quite interesting. And this was in 50% matrix and as I mentioned, 30 minute incubations we ended up with an assay range of 0.64 to 10,000 picomolar and good interest curves and good precision and accuracy in general. So when we compare the offline versus fully automated, you can see actually in reality, the actual total time is very similar.
So by using the mix CD, we end up with approximately 2.5 hour runtime and also with the pre-incubation offline, we also end up with approximately two hours. So actually in terms they're very similar, but obviously the advantage of the automated is that the instruments doing all the pipetting, so there's less chance for analytical error when actually doing the automated method.
Sorry and then just as I mentioned, we'll also going to discuss the nuclease cutting assay or the NSCA. And this assay format uses a single probe. And this probe in this case has a complete, completely complementary has a biotin at one end and an Alexa flaw at the other end. So obviously the dual hybridization had two complementary probes to the dual hybrid and the NSCA has one complementary probe.
And what you do in this case is that you once hybridized. You, then you can introduce a nuclease and the nuclease will chew up any single stranded DNA or RNA. So this allows the method to be more selective. So if you do get any n minus 1, n minus 2 or even larger metabolites, the nuclease will chew the parts of the complementary probe, which isn't completely hybridized.
So it has the advantage of being more selective. There are disadvantages of the nuclease cutting assay as well. I know that there can be batch to batch variability with nucleases for example. And also the dynamic range may not be as good as the dual hybridization assay, but it obviously is more selective. And in a lab format we actually tested both S1 and micrococcal nuclease and we actually also tested mung bean and other nucleases in the project.
But I'm just going to show some data from the S1 and the micrococcal nuclease. And then I'm going to show you the assay format. So in the mixing CD, so so we start up by adding the capture probe. And that gets added to the CD and the mixing chamber. And then we add the sample. Again, we mix in for one minute and that's built into the method.
And then it incubates for one hour. And then it will hybridize in the mixing chamber. And then that then you run the complexes across the column. And then in this case, as you see here on the right hand side, you'll see you have the hybridized probes, but also you have the probes that are unreacted. And then we can add a nuclease across the column.
And the nuclease will pass through the column and then chew up the single stranded unreacted probe. And actually, when it comes to the nuclease assay formats, we looked at both adding them to the nuclease, adding it to the mixing chamber all across the column, and you have the flexibility to do either. And for the micrococcal assay, we found that it was better to add it across the column.
But again, it depends on the enzyme. We also we looked at measuring using multiple nuclease washes. That's one of the advantages of the Jairus format. You can because it's the flow through technology. You can actually look at adding the nuclease across the column multiple times to potentially help wash away the unreacted and metabolites. Sorry and then so then just looking at the S1 nuclease data compared to the micrococcal nuclease data, the S1 nuclease gave slightly more sensitivity.
But one of the issues with S1 nuclease is it tends to work at quite low pH and it's more selective for more selective than the micrococcal for single stranded. It's probably the main nuclease that people are using in industry for cutting assays. So we but we as part of the project, we looked at multiple nucleases. And as I mentioned, you can add the nuclease either across the column or across the mixing chamber.
And as I mentioned, there is further optimization. There was complexity with both the S1 and the micrococcal, but they do work and but further optimization is actually needed. And we have a few customers out there that we're working with that are working on cutting assays on the platform. So as I mentioned, in terms of metabolites. So we actually, as part of the project, we had n minus 1, n minus 2 and n minus 3 metabolites made with both the 3 prime and 5 prime n And we can see there's some even with the nuclease and this is the micrococcal there's some cross-reactivity at the 3 prime end and then a minimal cross-reactivity at the 5 prime end and there's reflective of the activity of the enzyme.
And then when we looked at the dual hybridization assay, we found quite a lot of cross-reactivity at the n minus 1 end, around 40% cross-reactivity. And as I mentioned, we also we are able in the jarrah lab format to incorporate nuclease washes. So we actually looked at a nuclease wash nuclease wash across the column after the dual hybridization and we actually managed to reduce the cross-reactivity at the n minus 1 that we see here to around 20% just by adding a nuclease wash.
We were also concerned at potentially splitting up the small gap between the complementary probes, but it seemed to work in the assays that we ran basically. So in conclusion, the lab platform can be used for oligonucleotide analysis. And it obviously something I haven't mentioned a lot today is that the lab uses extremely small sample volumes, so it can give you a very robust assay using minimal sample volume, but also will reduce reagent use as well.
And we have the option of fully automating the assay and letting the instrument do all the work, or you do an offline incubation and then run that onto the instrument and both options are available and possible for the same assay. The offline incubation gives you the greater flexibility because it allows you to use different CDs but also different incubation temperature. And that gives you a potential advantages depending on the assay range that you need to achieve and the sensitivity.
And then finally. So here are some references. So we've actually published on and presented data at multiple conferences, including the and these are the references in the industry for that we consulted when developing hybridization assays. And I would like to Thank some of my colleagues for Frida. Frida was the master student that started off doing this project and did some really good and exciting and very successful project as a master's thesis.
And then she then became a joined our team in Uppsala. So she's now part of the company after a master's thesis, said Uppsala. In Uppsala. We have a long history of supporting students locally. And this is a great way to get scientific innovation. And then also I'd like to Thank Joris, who's our field application scientist who works with the French speaking areas and Southern Europe, and Joris continued this project as kind of training, but also some application and market support to actually generate more data and continued the project on.
So I'd like to Thank both Frida and Joris for their help in the work and the data that was presented today. And finally, I would like to Thank you all for listening and would be very happy to answer any questions on this subject. Brilliant well, Thank you very much for your presentation, John.
If you do have any questions for our expert, then please continue to submit them using the Q&A button at the bottom of your screen. We've received a number of questions so far, so we'll start addressing some of these on air now. The first question we have is have you compared quantitation results between giro lab hybridization assay and stem loop. No, no, we're not had the obviously this started off with as an in-house project and we also continued to collaborate with a number of customers to actually help them set up oligonucleotides in their company.
But we've not done any direct comparison with any other techniques, including Eliza. Actually we've gone straight for developing the assay on our platform because we feel there's probably a lot of advantages for doing so. Fantastic And then the next question is, so we've heard that oligonucleotides can cause carryover due to the oligo sticking to the microchannel in the CED.
Can you comment on this. Yeah, that's actually a really good question, and something something I was actually concerned about because I have a long history measuring oligonucleotides and I know from working in groups that do both small and large molecule that my mass spectrometry colleagues used to hate doing oligonucleotides because they knew that it stuck to everything. The chromatography as well as the ion trap in the mass spec and people doing oligonucleotide analysis by LCMS generally would dedicate a system to measuring oligos.
So I was actually really concerned actually that we might get some stickiness of the oligo one to the needles and secondly to the column. But actually we found that as long as people use the Brexit buffers, we generally we're not seeing any sticking of the oligo, at least the ones we tested in house anyway. We've not seen any sticking of the materials to actually the CD and we have a feeling that people in the past may have tried giro lab for oligos and not used the appropriate buffers.
So may have got some analyte sticking into the column, but it has not been our experience or something that we've seen. But we're obviously happy to work with it, but it's probably about buffering. It's important that you have the right buffers being used. And then what is the max volume that can be added to the mixing chamber. Mixing CDs.
Yeah so it's really 200 nanoliters. So it's. So you add 2 to 200 nanoliters and you can add 4 200 nanoliters steps. So we have had some customers ask to add the sample twice, for example, to increase the volume. But in general, we would recommend that, 200 nanoliters and then you can have up to four steps. So you can, for acid dissociation, for example, for ADR, we add sample acid and then neutralizing.
But we also have had a fourth step, for example, adding the target and anti target antibody. So you have a lot of flexibility in that fourth step and you can change the timing and the incubation times for all the steps in the mixing CD. Excellent and then have you developed ASO or ASO assays? And did the antibody or nanobody interfere with the capture and detection probes? So I'm trying to understand the question.
So is that anti oligo antibody assays? So AB ASO or ASO assays? I assume that's antibody assays and Nab assay. Yeah Yes Yeah yeah Yeah yeah yeah. I mean something this mean auntie. We've not really looked at immunogenicity assays on the GI lab for oligos. I know there has been some reported antibodies being produced clinically, so I know there has been some interest.
I mean, ADA can be developed on the platform, so there's no reason why not. It's not something we've necessarily tested at this stage, basically. And sorry, the assay was referring to nanobody. OK So this is like a conjugate basically antibody, a nanobody conjugate. We not specifically, but one of our partners is interested in this area, so that's something we may be looking at in the future.
That's all I can say at this stage. Fantastic Thank you. And what are some major differences in processing DNA versus RNA oligonucleotides using gyro's platforms. I wouldn't say there's any specific difference. It's just about having the complementary probes. So I've done in the past, I've done both RNA and DNA probes. So it's both are possible. Fantastic and can you comment on the best method for tissue homogenization?
So best buffer to use or something like that. Well, that's a complicated case. I mean, we didn't specifically do tissue work as part of this project, but we have had customers doing tissue work. And this actually favors the offline incubation assay because you can build in the homogenization and the treatment of the tissue as part of the offline incubation. I mean, personally, I had a lot of experience using proteinase k for using as a protein digestion of tissues.
That's something I've used in the past. But I mean, in terms of homogenization, this is a whole huge area and there's much more information in the literature than I have on the top of my head on the best homogenization methods, basically. Fantastic and so why should we consider the giro lab over traditional Elisa hybridization? Hybridization assays.
Obviously dynamic range, but it depends on the sample volume as well. So there are advantages of larger dynamic range. Also fully, as I kind of say, it's fully automating the method. So it can take some of the labor part out and also make the data lot more robust. I know from my own experience that hybridization acids can be quite tricky sometimes and they are quite labor intensive and you can get analysts to analyst variability by manually doing assays.
So this gives you the advantage of fully automating. So as I said there, it depends. It depends sometimes the giro lab will give you a superior assay and sometimes you can probably do it by Elisa. So it depends on the needs of your project. But we like to give people the option, particularly people that have giro labs in their lab or want to achieve a lab. It just is something else that they can use and measure on the platform.
So that's the important given them people a lot of flexibility to use the platform for multiple different assay types. And what next steps do you have planned for developing oligo assays? I mean, this is an ongoing project. We still have a few things to do in house, but the main thing I'm particularly looking at higher volume CDs, for example, of the offline incubation to see if we can really get sensitivity of less than 1 picomolar would be good.
But from some of the conversations we've had with sponsors, it seems more the large dynamic range and minimizing sample dilutions may be more important than necessarily sensitivity, because I think we've already got the sensitivity to support most pharmacokinetic sort of analysis of oligos and probably the other area they want to look at more is tissue work, to be honest, because that's one main thing people do analyze is the tissue distribution of the oligo.
And the oligo levels in tissues can vary can be much higher concentrations and you may need lower sensitivity. So it gives us a lot of flexibility. Fantastic and what's the minimum copy of oligos that your method is able to detect. But I mean, this is not PCR, so we're really mass units. So it's really one picomolar of the oligo. So it's a concentration.
It's not copies basically that we're measuring, which is more reflective of PK rather than PCR because you want to you're measuring concentration over time. So OK. And then since your shell is around 1 picomolar, does that mean times 10 to the 11 copy. I would need to. I'd need to my mathematical brain at this after just presented.
I've probably try and get back to this question. I'll look I'll rather than answer it now. Fantastic And then has any tissue work been performed using the giro lab platform. Yeah as I mentioned in house we haven't done any in-house, but we've had a couple of customers that are using it for tissue analysis. And as I mentioned, it's an important consideration for oligonucleotide assays.
So Yeah, we will continue to support and from what we've seen so far, there has been no problem with any sort of homogeneity issues or any once you homogenized it, any issue causing blockage of the microfluidics, that's something else that someone was concerned about. So as long as you do the appropriate treatment of the tissue, we extract and and we haven't seen any issues with flow through of that element through the microfluidics.
OK, fantastic. And then I think final two questions we've got. What are the volume requirements to perform oligo assays on the platform and then use gyros on purpose as more likely from the audience. Yeah OK. Yeah so. So in terms of volume, the depends on the CD that you use, but it's anywhere from 6 to 12 microliters of diluted sample in the micro in added to the plate and then that's taken in across to the CD and that's a diluted sample.
So it depends on your so if you have a 1 and a 2, you're going to need less. You definitely need less than 5 microliters pretty much for any assay that you run on the platform, basically. Excellent and then the final question then is, what is the main advantage of the offline incubation assay format since you're not fully automating the assay? Yeah, the main advantage, as I tried to mention during the presentation, really is flexibility.
It allows flexibility in allowing for different incubation temperatures. So the actual instrument itself will run the hybridization in the mixing at room temperature. So by doing offline, you can do 37 degrees on. I know from my experience I've had one assay where we're heating up to 50 odd degrees to melt and to basically to allow the hybridization. So it gave us that flexibility.
People that are developing tissue assays, they're already going through laborious extraction of the tissues. So it kind of fits well with the offline incubation. You're still automating the assay. It's just can it's probably works better with the offline. And as I mentioned, you can use different CDs to actually kind of change your analytical range. If you want a very sensitive assay, you can use the 4,000 nanoliter CD.
And if you want to get low sensitivity, you can use the 20 nanoliter and everything else in between. Fantastic And then there's just one final question. Do you need to extract the DNA or from the sample or just lyse the tissue. It depends on the tissue. But I mean, potentially you could. Lyse, as long as we haven't got any particulate matter.
As long as it's in solution, it's potential. You could measure it. I'm thinking of things like cell cell culture, supernatants and things, for example, which is a very common kind of matrix that we see going through our platform, particularly in our bioprocess area. So pretty much, Yes, either is an option basically. And sorry, another one just come through. Are you going to develop a multiplex assay?
No plans at this stage, but they're obviously in the jar lab format that, to actually do a multiplex is difficult because we use a single laser. There is, there are potential in the expand to gyro plex where we can on the same sample potentially you could run it on five separate assays simultaneously or consecutively. So that's what we call gyro plex.
But multiplex is something we would love, but at this stage we'd need a big technology development to do so. Fantastic Well, Thank you very much, John, for answering our audience questions today. Before we wrap up, do you have any last comments that you'd like to add. No Thank you, everybody, for your time. If you've got any questions, please don't hesitate to contact us.
Happy to support potential customer collaborations if you want to. If you're interested in measuring oligonucleotides and we'd be happy to collaborate with you. And we find this is an exciting area and I think it's an area which actually does suit our platform. So I really want to help support people out there develop oligonucleotides. Fantastic well, I'd like to once again Thank our wonderful presenter, John, as well as you, our listeners, for your time and questions.
Don't forget to visit us at bioanalysis hyphen zone.com/webinars for more webinars. Thank you again for attending.
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Hello, everyone, and Thank you for attending today's webinar. Accelerating quantification of therapeutics with robust microfluidic methods I'm your host. Taylor and Francis senior editor Tristan fry, and I'll be your host for today's event. Before I introduce you to our speaker, I'd like to quickly cover a few housekeeping items. You've joined the presentation and will be listening via your computer's audio by default. If you prefer to connect to a different speaker, you can adjust your audio settings by clicking the arrow next to the microphone icon at the bottom of the Zoom window.
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So we are delighted to have John Chappell presenting for us today. John is the senior global scientist support manager at gyros protein technologies, where he is responsible for training the field application team, as well as implementing customer collaborations and scientific innovations. John has 25 years of experience in the contract research industry supporting both pre-clinical and clinical drug development.
He is particularly interested in validation requirements and ensuring the data generated will be acceptable to regulatory authorities. Thank you for joining us today, John. I'll now hand over to you to start today's presentation. Good afternoon or the time zone where you're currently at. So as Tristan mentioned, I'm Jon Chappell. I'm senior scientific support for gyros protein technology, and I've been at gyros protein technology for six years approximately.
And then prior to that, I worked for many global crows for over 20 years developing and validating immunoassay. As was mentioned, and is actually back in the mid 90s where I actually did an oligonucleotide assay research project to develop a hybridization using an immunoassay type format. So I've actually got a long history doing oligonucleotide analysis, so I was very excited to try and get oligonucleotide assays to work on the gyro lab platform.
So today I really want to summarize some work that we've done both internally, but also we're also collaborating with external collaborators to also continue the work in oligonucleotides. And the oligonucleotide work really started from a customer request and we wanted to optimize and get the best out of our platform. And also we feel that the gyro lab platform suits the analysis of oligonucleotides with its many advantages.
So today I want to cover a little bit about oligo nucleotides. I'm sure most of the audience will know about oligonucleotide therapeutics, and I'll give examples of this and that. It's very much a growing area, and then I'll give you an overview of microfluidic oligo assays and also how they compare to traditional hybridization Elisa assays.
Then I want to go through the use of an offline incubation and then an and then the assay is using automated detection on the platform, which is one format that can be used. And the second assay format is to look at fully automating the platform using our mixing, which I think gives the greatest benefits. But obviously both approaches have certain advantages. So I'll go through that and then I'm going to go through some nuclease options as well.
The use of nucleases can be very helpful, particularly in reducing metabolite interference. So that's really a consideration when developing hybridization assays. So the oligonucleotide area is very much a rapid grown of area. And actually it started originally back in the 80s and 90s. And then it went through a bit of a boom and then it stopped and then it's very much rapidly growing now.
I mean, even last year we had four approved oligonucleotide therapies in 23, and overall there's been total 20 total oligonucleotide therapeutics, of which half of those are antisense oligos. And this is the main area that we're going to discuss today. But there are other kind of areas for oligos as well. And obviously in the post COVID vaccine kind of environment, there's a lot more interest in developing oligonucleotides, particularly with the alternative delivery mechanisms.
And in general, it's very much a vast and growing area. As you see here. There's various approved AstraZeneca, Ionis, Novo Nordisk, et cetera, have all recently had therapeutics approved. So in terms of the types of oligonucleotides. So as I mentioned today, I'm going to be talking about antisense oligonucleotides, which are targeted messenger RNA sequences, and they're normally approximately like 20 bases.
And there's been a lot. This is probably the most common. And the first area that people have been working on. But there are also siRNA or small interfering RNA. Again, they can prevent the production of a disease causing genes and many of these are in clinical trials and we also have microRNA and other forms include aptamers, which have a much larger sequence and have a three dimensional can act like a protein even by having binding et cetera.
So they're much larger. And things like CRISPR, which is a very exciting area of oligonucleotide and growth. And, and obviously there's other things like genome editing. But today really I'm going to concentrate on the antisense which basically this project is concentrated on. So antisense oligonucleotides are small, synthetic, single, stranded nucleic acid molecules, and they're designed to bind complementary RNA sequences.
So they're meant to effectively in most cases, stop the production of a disease producing protein. There are various delivery systems. So they can be conjugated to targeting ligands. I've heard a lot about recently about antibody oligo conjugates, for example, as well as other things that can maybe target it to the liver, for example, to deliver the oligo to the liver and but also can be encapsulated in nanoparticles like lipid nanoparticles, for example, to improve the delivery because one of the main things with antisense over its history is really to try and get it to its target organ or to the site of action.
And the main thing to do that is to really to increase its half life and to really do that they've modified the antisense chemistry. So on the left here in terms of the chemical sequence, you can see the normal nucleotide sequence. This kind of the first generation was the phosphorothioate, where you had a sulfur molecule being substituted into the backbone and then you had the second generation, two or more and two more and then the third generation PMO.
But all these modifications are designed to increase nuclease resistance and to prevent it being metabolized at following dosing, et cetera, and to ensure that it gets to its target organ or to where it's going to act, basically. So the important considerations with oligonucleotide is mean, obviously sensitivity. They're dosed like a small molecule, so they are typically administered at low doses.
They obviously lack of interference and metabolite cross-reactivity. So obviously metabolites where you may get n minus 1, n minus 2, for example, may also be still be biologically active and matrix effects can be a big thing. Obviously for pharmacokinetic analysis, you normally collect the blood serum, for example. But there's a lot of interest in tissue distribution.
So where the actual oligo distributes in the body. So we I've done a lot of work on tissue depending on the oligo or membrane like lung tumor, liver, et cetera, et cetera. And that obviously when you're measuring in tissue that you have to homogenize and have sample prep procedures to actually measure and extract from the tissue. And also, I've done a lot of work with cerebrospinal fluid as well.
So it depended obviously on the mechanism of action, obviously, and of which tissues and which organs you're going to look at. So the first is really to look at a dual hybridization assay. So as I mentioned, an antisense is approximately 20 bases. So when we look at a dual hybridization assay, we treat it as if it's an immunoassay. So we look for a complementary capture and detection probes.
So we're using Watson hit a watson-crick nucleotide combination, so complementary a to C to G. So effectively we take the parent sequence and we design a complementary probe for that normally about half the length for capture and then also again half the length for detection. And in this case, you can see we're using a biotinylated for capture and a fluorescent label for detection.
So basically you run it like an immunoassay and then you wash away unreacted probes after the hybridization. One of the potential disadvantages of this assay format is that if you do get metabolites 3 prime and 5 prime metabolites, they can still cross-react in the assay and this cross-reactivity can actually be quite high. So this is really is not necessarily selected for parent.
So that kind of introduces kind of the immunoassay format. And now we're going to move into the Jairo lab platform, the microfluidics. So this gives a schematic of the basic principles of the Jairo lab. So we have a microfluidic structure at the bottom of a CD or compact disk, and we have an affinity column at the bottom of that microstructure.
And that has a strict streptavidin bead and you need a biotinylated antibody for capture and you need a fluorescent Alexa label, a 647 normally for detection and it's microfluidics. Everything is moved throughout the CD by a combination of capillary action and centrifugal force. The instrument itself will actually spin the CD. And this allows the fluids to travel throughout the microstructure.
One of the big advantage of the lab is that all the samples are loaded onto the CD and then when it spins, they all move across the microscope structures at the same time. So you almost get simultaneous processing of samples on the CD, which has a lot of advantages. And because it's microfluidics and because of the reaction times as part of the assay, it normally takes approximately one hour to complete a whole CD.
And the CD normally runs an equivalent amount of samples to an Elisa plate, depending on the assay volume on the CD. And actually, on the right hand side, you can see the lab Explorer, which is our one system which can process one CD at a time. But also there is a five CI/CD system, the ixpand, which can process up to five CDs.
So when it comes to the two formats. So we had a semi-automated and fully automated. So the semi-automated assay uses our standard CDs or compact disks. And this basically, as I mentioned, has a micro-structure with an affinity column at the bottom, but also has a common reagent channel. So reagents like the capture and the detection reagent get added to the CD in blocks of 8, around the CD, and then we have individual sample inlets for the samples.
So the samples are individually loaded around the CD. And as I mentioned earlier, sort of movement through these. The CD is achieved by a mixture of capillary action and centrifugal force. The hydrophobic barriers kind of help to control the liquid flow. And then we ramp up the centrifugal, the spinning of the CD to move the liquid through the hydrophobic barrier. The advantage of the semi-automated format is it allows us to change the assay volume.
So we have the smallest CD is the 20 nanoliter CD, so that has 20 nanoliters of sample being introduced into the sample inlet. And then the biggest CD is the 4,000 nanoliters. And then we have CDs at 200 and 1,000 nanoliters so by actually shifting with a different CeeDee, you can shift the analytical range. So if you want a very sensitive assay, you want to look at a very high volume CD.
So 4,000 nanoliters and then for example, if you want a very kind of low sensitivity, when you're measuring high concentrations, you can actually measure that, use the 20 nanoliter. So it gives a lot of flexibility and actually you can take the same assay and develop it and then use it on different CDs to adjust the analytical range, which a few people are doing. And it can be a way of avoiding diluting samples, for example.
But obviously, it means you have to validate your assay on multiple CDs. And then for the fully automated CD, this is actually the mix in CD. So this is slightly different microchannel to the normal CD. It has a mixing chamber in the middle. So this mixing chamber allows the addition of 20 nanoliters of sample and you can add up to four steps in the mixing chamber.
It was actually designed for anti-drug antibody assays where for a fully automating acid dissociation procedures. But this CD is very useful where you want to increase contact time and to pre incubate things. So for example, we've had people that want to measure free targets, so they want to dissociate and also dissociate drug, for example. But also it's good for the oligonucleotide acids, which I'm talking today, allows you to add the sample and then the complementary probes sequentially into the mixing chamber.
You can actually change the timing of an incubation within the mixing chamber. So it gives you a lot of flexibility. And obviously the advantage of that is you can really increase sensitivity. But obviously the more incubation time you use, the longer the assay becomes. So it's like a trade off depending on what you need for your assay.
And as I said, what the mixing CD does allow you is to fully automate the whole steps in the assay. So I'll start off with the offline incubation. So this actually started off as a master's project, which I mentioned at the very end of the presentation. So we had a master's student working in our lab in Uppsala and we're very keen as a company to do scientific research and to try and develop new potential ways of measuring things and new sort of technical advances.
So we had a student actually start working on the assays as part of a project. So we took a commercial sequence which is based on amondys 45 or casimersen, and this is actually a PMO, so a third generation oligonucleotide chemistry. And we actually had the sequence artificially manufactured. So it wasn't a case of buying the drug.
We actually had it synthetically synthesized and then we had complementary probes actually manufactured as well, a biotinylated and a Lexa floor sequence as well. We actually also not really going to talk about it today. That much, but we also looked at digoxigenin as an indirect detection. And that is a very popular method for detection of oligonucleotides.
So that is actually an option in lab format. But I'm not actually going to really speak about that much today. But just to let everybody know that is an option. And actually for the offline incubation, we started with the 1,000 nanoliter the 1,000. So that's kind of towards a more sensitive assay, not the most sensitive CD. And also we looked at different buffers, so rexhep and rexhep.
So the gyro lab has a variety of different buffers with different properties. They actually really do help the microfluidics and the transport of the liquids through the CD. And we spent many years developing these buffers and they're very much optimized to use on our platform. And very I always recommend, that people look at our buffers and we had our capture and detection probes were in rexhep.
Rexhep actually contains a higher degree of detergent, so which can be very useful in this case. And what we did in this case, we actually pre-incubated the capture detection and the analyte offline for one hour. And then once the reaction is hybridized, it can then be transferred to the CD automatically. And as explained really we pre incubate the components offline.
We measure the analyte capture and detection and then incubate. This case we incubate at room temperature for one hour. But this is where this assay format gives you a lot of flexibility because you can depending on the temperature of the hybridization, you can do room temperature or you can do 37 or other temperatures. So this gives a lot of flexibility. And then the microplate is loaded onto the gyro lab and then the gyro lab will actually do the whole assay through the CD.
And approximately this offline incubation takes approximately two hours and the automated is for capture and and detection only. So this is an example data. So this was actually using a 50% matrix. One thing that the giro lab is really good at actually is using quite low dilutions and sample 50% matrix or even 100% matrix can be run through the CD.
So that can particularly when you try and trying to get very low sensitivity. Can that be an example of an advantage. And in this case, we had an assay with an analytical range of 1 picomolar to 10,000 picomolar. In general, giro lab technology gives you approximately four logs of analytical range, and this shows you the data of three operators in three separate runs.
And one thing about the giro lab technology, it's very reproducible and it's very robust. So this is consistent data that we see. And then also we had discussions that we needed to one of our customers. We talking to had very, very high concentrations of analyte, particularly in different matrices, in things like tissues, for example.
So we wanted to make sure that we had good dilution or linearity and we could achieve a neat serum at $50,000. And we've got really good sort of linearity. And this was run over 3 operators of 3 over 3 runs. And as you can see, we got very, very precise and linear dilution into the analytical range. And then kind of precision and accuracy.
I mean, in this case, actually, we tested the look at five picomolar. This is on the 1,000 nanoliter. So potentially we could go a lot lower by using the 4,000 CeeDee where we have increased volume. And then we looked at QCs, five concentrations with the upper limit at 8,000. And we have a good precision and accuracy and a very reproducible performance on the platform.
And one thing that was quite important in this case was to look at the incubation time. So we looked at 37 versus room temperature, and we found that there really no difference in incubating between room temperature and 37 degrees. And what this shows actually is in this case, the assay could be run at and hybridized at room temperature, but with the awful lot. As I mentioned, already using the offline incubation, it would allow us to use higher incubation temperatures if needed, depending on what we need, how the reagents hybridize.
So I want to move now on to the fully automated assay. So as I mentioned, this uses the lab mixing CD. And the advantage of this is that you add your samples, your diluted samples to the plate, you put it in the instrument. And then it will run the assay from start to finish. And as I mentioned, it uses the mixing CD. And in this case, the mixing chamber allows for incubation, as I mentioned, and it allows for the sequential or combined addition of reagents.
And we first we looked at the dual hybridization assay where we added to the mixing chamber and give further details of how the assay was set up in the next slides. But just an overview, again, it's very similar to the dual hybridization in terms of the sorry, the offline incubation, exactly the same assay, exactly the same capture and detection and the same buffers.
And then this shows you the assay format. So we start off with the sample. And the sample is added into the CD and then adds to the mixing chamber. And next in we add the capture reagent. And this is added, as I mentioned, by centrifugal force and capillary action. And it gets added to the mixing chamber. And this is mixed for one hour.
And then you can actually change. We looked at incubations from five minutes all the way up to 30 minutes. And basically so you can actually optimize these parts. So in the mixing chamber, you'll see now that the analyte has now has now hybridized with the capture probe to half of its sequence. And then we add the detection probe, which is the Alexa flood detect.
And then this is then again, you can change the incubation time. And it's added like the other reagent through the reagent inlet. And then that gets added to the mixing chamber. And then in this case, as you can see now have got both the capture and detection probe bound to the analyte. You could actually premix the capture and detection together and add them together.
So that's another potential assay format. But for this assay this assay format work the best. And then basically the hybridized mixture will then be introduced over the column, over the strep. So all the reactions taking place in the mixing chamber, then it's by it's a stronger centrifugal force will move the whole mixture and then it passes over the column.
And then it becomes the biotinylated probe, which was used for capture, becomes immobilized on the streptavidin bead. And then the instrument will automatically detect the amount of fluorescence using laser induced fluorescence using the Alexa 647. The advantage of using laser induced fluorescence, it gives good sensitivity but also gives a broad dynamic range.
So when we look at the incubation times, we can actually as I mentioned, we actually incubated for 30 minutes, but we looked at different reaction times. So can actually shorten the incubation. So five minute, 10 minutes and actually by shortening the incubation, you can actually lower the signal to background. So it's very much for the end user to try and optimize the best conditions for the assay that they're running.
And then the fully automated platform. We actually we ended up with a lower IQ than the offline, so we ended up with an IQ in serum of 20 of two picomolar. So it was slightly more sensitive. You can probably tell by the curve shape it's slightly straighter at the bottom end, so you can see it's slightly more sensitive. Maybe that's even though it's a lower volume CD, we've ended up with higher sensitivity.
So which is quite interesting. And this was in 50% matrix and as I mentioned, 30 minute incubations we ended up with an assay range of 0.64 to 10,000 picomolar and good interest curves and good precision and accuracy in general. So when we compare the offline versus fully automated, you can see actually in reality, the actual total time is very similar.
So by using the mix CD, we end up with approximately 2.5 hour runtime and also with the pre-incubation offline, we also end up with approximately two hours. So actually in terms they're very similar, but obviously the advantage of the automated is that the instruments doing all the pipetting, so there's less chance for analytical error when actually doing the automated method.
Sorry and then just as I mentioned, we'll also going to discuss the nuclease cutting assay or the NSCA. And this assay format uses a single probe. And this probe in this case has a complete, completely complementary has a biotin at one end and an Alexa flaw at the other end. So obviously the dual hybridization had two complementary probes to the dual hybrid and the NSCA has one complementary probe.
And what you do in this case is that you once hybridized. You, then you can introduce a nuclease and the nuclease will chew up any single stranded DNA or RNA. So this allows the method to be more selective. So if you do get any n minus 1, n minus 2 or even larger metabolites, the nuclease will chew the parts of the complementary probe, which isn't completely hybridized.
So it has the advantage of being more selective. There are disadvantages of the nuclease cutting assay as well. I know that there can be batch to batch variability with nucleases for example. And also the dynamic range may not be as good as the dual hybridization assay, but it obviously is more selective. And in a lab format we actually tested both S1 and micrococcal nuclease and we actually also tested mung bean and other nucleases in the project.
But I'm just going to show some data from the S1 and the micrococcal nuclease. And then I'm going to show you the assay format. So in the mixing CD, so so we start up by adding the capture probe. And that gets added to the CD and the mixing chamber. And then we add the sample. Again, we mix in for one minute and that's built into the method.
And then it incubates for one hour. And then it will hybridize in the mixing chamber. And then that then you run the complexes across the column. And then in this case, as you see here on the right hand side, you'll see you have the hybridized probes, but also you have the probes that are unreacted. And then we can add a nuclease across the column.
And the nuclease will pass through the column and then chew up the single stranded unreacted probe. And actually, when it comes to the nuclease assay formats, we looked at both adding them to the nuclease, adding it to the mixing chamber all across the column, and you have the flexibility to do either. And for the micrococcal assay, we found that it was better to add it across the column.
But again, it depends on the enzyme. We also we looked at measuring using multiple nuclease washes. That's one of the advantages of the Jairus format. You can because it's the flow through technology. You can actually look at adding the nuclease across the column multiple times to potentially help wash away the unreacted and metabolites. Sorry and then so then just looking at the S1 nuclease data compared to the micrococcal nuclease data, the S1 nuclease gave slightly more sensitivity.
But one of the issues with S1 nuclease is it tends to work at quite low pH and it's more selective for more selective than the micrococcal for single stranded. It's probably the main nuclease that people are using in industry for cutting assays. So we but we as part of the project, we looked at multiple nucleases. And as I mentioned, you can add the nuclease either across the column or across the mixing chamber.
And as I mentioned, there is further optimization. There was complexity with both the S1 and the micrococcal, but they do work and but further optimization is actually needed. And we have a few customers out there that we're working with that are working on cutting assays on the platform. So as I mentioned, in terms of metabolites. So we actually, as part of the project, we had n minus 1, n minus 2 and n minus 3 metabolites made with both the 3 prime and 5 prime n And we can see there's some even with the nuclease and this is the micrococcal there's some cross-reactivity at the 3 prime end and then a minimal cross-reactivity at the 5 prime end and there's reflective of the activity of the enzyme.
And then when we looked at the dual hybridization assay, we found quite a lot of cross-reactivity at the n minus 1 end, around 40% cross-reactivity. And as I mentioned, we also we are able in the jarrah lab format to incorporate nuclease washes. So we actually looked at a nuclease wash nuclease wash across the column after the dual hybridization and we actually managed to reduce the cross-reactivity at the n minus 1 that we see here to around 20% just by adding a nuclease wash.
We were also concerned at potentially splitting up the small gap between the complementary probes, but it seemed to work in the assays that we ran basically. So in conclusion, the lab platform can be used for oligonucleotide analysis. And it obviously something I haven't mentioned a lot today is that the lab uses extremely small sample volumes, so it can give you a very robust assay using minimal sample volume, but also will reduce reagent use as well.
And we have the option of fully automating the assay and letting the instrument do all the work, or you do an offline incubation and then run that onto the instrument and both options are available and possible for the same assay. The offline incubation gives you the greater flexibility because it allows you to use different CDs but also different incubation temperature. And that gives you a potential advantages depending on the assay range that you need to achieve and the sensitivity.
And then finally. So here are some references. So we've actually published on and presented data at multiple conferences, including the and these are the references in the industry for that we consulted when developing hybridization assays. And I would like to Thank some of my colleagues for Frida. Frida was the master student that started off doing this project and did some really good and exciting and very successful project as a master's thesis.
And then she then became a joined our team in Uppsala. So she's now part of the company after a master's thesis, said Uppsala. In Uppsala. We have a long history of supporting students locally. And this is a great way to get scientific innovation. And then also I'd like to Thank Joris, who's our field application scientist who works with the French speaking areas and Southern Europe, and Joris continued this project as kind of training, but also some application and market support to actually generate more data and continued the project on.
So I'd like to Thank both Frida and Joris for their help in the work and the data that was presented today. And finally, I would like to Thank you all for listening and would be very happy to answer any questions on this subject. Brilliant well, Thank you very much for your presentation, John.
If you do have any questions for our expert, then please continue to submit them using the Q&A button at the bottom of your screen. We've received a number of questions so far, so we'll start addressing some of these on air now. The first question we have is have you compared quantitation results between giro lab hybridization assay and stem loop. No, no, we're not had the obviously this started off with as an in-house project and we also continued to collaborate with a number of customers to actually help them set up oligonucleotides in their company.
But we've not done any direct comparison with any other techniques, including Eliza. Actually we've gone straight for developing the assay on our platform because we feel there's probably a lot of advantages for doing so. Fantastic And then the next question is, so we've heard that oligonucleotides can cause carryover due to the oligo sticking to the microchannel in the CED.
Can you comment on this. Yeah, that's actually a really good question, and something something I was actually concerned about because I have a long history measuring oligonucleotides and I know from working in groups that do both small and large molecule that my mass spectrometry colleagues used to hate doing oligonucleotides because they knew that it stuck to everything. The chromatography as well as the ion trap in the mass spec and people doing oligonucleotide analysis by LCMS generally would dedicate a system to measuring oligos.
So I was actually really concerned actually that we might get some stickiness of the oligo one to the needles and secondly to the column. But actually we found that as long as people use the Brexit buffers, we generally we're not seeing any sticking of the oligo, at least the ones we tested in house anyway. We've not seen any sticking of the materials to actually the CD and we have a feeling that people in the past may have tried giro lab for oligos and not used the appropriate buffers.
So may have got some analyte sticking into the column, but it has not been our experience or something that we've seen. But we're obviously happy to work with it, but it's probably about buffering. It's important that you have the right buffers being used. And then what is the max volume that can be added to the mixing chamber. Mixing CDs.
Yeah so it's really 200 nanoliters. So it's. So you add 2 to 200 nanoliters and you can add 4 200 nanoliters steps. So we have had some customers ask to add the sample twice, for example, to increase the volume. But in general, we would recommend that, 200 nanoliters and then you can have up to four steps. So you can, for acid dissociation, for example, for ADR, we add sample acid and then neutralizing.
But we also have had a fourth step, for example, adding the target and anti target antibody. So you have a lot of flexibility in that fourth step and you can change the timing and the incubation times for all the steps in the mixing CD. Excellent and then have you developed ASO or ASO assays? And did the antibody or nanobody interfere with the capture and detection probes? So I'm trying to understand the question.
So is that anti oligo antibody assays? So AB ASO or ASO assays? I assume that's antibody assays and Nab assay. Yeah Yes Yeah yeah Yeah yeah yeah. I mean something this mean auntie. We've not really looked at immunogenicity assays on the GI lab for oligos. I know there has been some reported antibodies being produced clinically, so I know there has been some interest.
I mean, ADA can be developed on the platform, so there's no reason why not. It's not something we've necessarily tested at this stage, basically. And sorry, the assay was referring to nanobody. OK So this is like a conjugate basically antibody, a nanobody conjugate. We not specifically, but one of our partners is interested in this area, so that's something we may be looking at in the future.
That's all I can say at this stage. Fantastic Thank you. And what are some major differences in processing DNA versus RNA oligonucleotides using gyro's platforms. I wouldn't say there's any specific difference. It's just about having the complementary probes. So I've done in the past, I've done both RNA and DNA probes. So it's both are possible. Fantastic and can you comment on the best method for tissue homogenization?
So best buffer to use or something like that. Well, that's a complicated case. I mean, we didn't specifically do tissue work as part of this project, but we have had customers doing tissue work. And this actually favors the offline incubation assay because you can build in the homogenization and the treatment of the tissue as part of the offline incubation. I mean, personally, I had a lot of experience using proteinase k for using as a protein digestion of tissues.
That's something I've used in the past. But I mean, in terms of homogenization, this is a whole huge area and there's much more information in the literature than I have on the top of my head on the best homogenization methods, basically. Fantastic and so why should we consider the giro lab over traditional Elisa hybridization? Hybridization assays.
Obviously dynamic range, but it depends on the sample volume as well. So there are advantages of larger dynamic range. Also fully, as I kind of say, it's fully automating the method. So it can take some of the labor part out and also make the data lot more robust. I know from my own experience that hybridization acids can be quite tricky sometimes and they are quite labor intensive and you can get analysts to analyst variability by manually doing assays.
So this gives you the advantage of fully automating. So as I said there, it depends. It depends sometimes the giro lab will give you a superior assay and sometimes you can probably do it by Elisa. So it depends on the needs of your project. But we like to give people the option, particularly people that have giro labs in their lab or want to achieve a lab. It just is something else that they can use and measure on the platform.
So that's the important given them people a lot of flexibility to use the platform for multiple different assay types. And what next steps do you have planned for developing oligo assays? I mean, this is an ongoing project. We still have a few things to do in house, but the main thing I'm particularly looking at higher volume CDs, for example, of the offline incubation to see if we can really get sensitivity of less than 1 picomolar would be good.
But from some of the conversations we've had with sponsors, it seems more the large dynamic range and minimizing sample dilutions may be more important than necessarily sensitivity, because I think we've already got the sensitivity to support most pharmacokinetic sort of analysis of oligos and probably the other area they want to look at more is tissue work, to be honest, because that's one main thing people do analyze is the tissue distribution of the oligo.
And the oligo levels in tissues can vary can be much higher concentrations and you may need lower sensitivity. So it gives us a lot of flexibility. Fantastic and what's the minimum copy of oligos that your method is able to detect. But I mean, this is not PCR, so we're really mass units. So it's really one picomolar of the oligo. So it's a concentration.
It's not copies basically that we're measuring, which is more reflective of PK rather than PCR because you want to you're measuring concentration over time. So OK. And then since your shell is around 1 picomolar, does that mean times 10 to the 11 copy. I would need to. I'd need to my mathematical brain at this after just presented.
I've probably try and get back to this question. I'll look I'll rather than answer it now. Fantastic And then has any tissue work been performed using the giro lab platform. Yeah as I mentioned in house we haven't done any in-house, but we've had a couple of customers that are using it for tissue analysis. And as I mentioned, it's an important consideration for oligonucleotide assays.
So Yeah, we will continue to support and from what we've seen so far, there has been no problem with any sort of homogeneity issues or any once you homogenized it, any issue causing blockage of the microfluidics, that's something else that someone was concerned about. So as long as you do the appropriate treatment of the tissue, we extract and and we haven't seen any issues with flow through of that element through the microfluidics.
OK, fantastic. And then I think final two questions we've got. What are the volume requirements to perform oligo assays on the platform and then use gyros on purpose as more likely from the audience. Yeah OK. Yeah so. So in terms of volume, the depends on the CD that you use, but it's anywhere from 6 to 12 microliters of diluted sample in the micro in added to the plate and then that's taken in across to the CD and that's a diluted sample.
So it depends on your so if you have a 1 and a 2, you're going to need less. You definitely need less than 5 microliters pretty much for any assay that you run on the platform, basically. Excellent and then the final question then is, what is the main advantage of the offline incubation assay format since you're not fully automating the assay? Yeah, the main advantage, as I tried to mention during the presentation, really is flexibility.
It allows flexibility in allowing for different incubation temperatures. So the actual instrument itself will run the hybridization in the mixing at room temperature. So by doing offline, you can do 37 degrees on. I know from my experience I've had one assay where we're heating up to 50 odd degrees to melt and to basically to allow the hybridization. So it gave us that flexibility.
People that are developing tissue assays, they're already going through laborious extraction of the tissues. So it kind of fits well with the offline incubation. You're still automating the assay. It's just can it's probably works better with the offline. And as I mentioned, you can use different CDs to actually kind of change your analytical range. If you want a very sensitive assay, you can use the 4,000 nanoliter CD.
And if you want to get low sensitivity, you can use the 20 nanoliter and everything else in between. Fantastic And then there's just one final question. Do you need to extract the DNA or from the sample or just lyse the tissue. It depends on the tissue. But I mean, potentially you could. Lyse, as long as we haven't got any particulate matter.
As long as it's in solution, it's potential. You could measure it. I'm thinking of things like cell cell culture, supernatants and things, for example, which is a very common kind of matrix that we see going through our platform, particularly in our bioprocess area. So pretty much, Yes, either is an option basically. And sorry, another one just come through. Are you going to develop a multiplex assay?
No plans at this stage, but they're obviously in the jar lab format that, to actually do a multiplex is difficult because we use a single laser. There is, there are potential in the expand to gyro plex where we can on the same sample potentially you could run it on five separate assays simultaneously or consecutively. So that's what we call gyro plex.
But multiplex is something we would love, but at this stage we'd need a big technology development to do so. Fantastic Well, Thank you very much, John, for answering our audience questions today. Before we wrap up, do you have any last comments that you'd like to add. No Thank you, everybody, for your time. If you've got any questions, please don't hesitate to contact us.
Happy to support potential customer collaborations if you want to. If you're interested in measuring oligonucleotides and we'd be happy to collaborate with you. And we find this is an exciting area and I think it's an area which actually does suit our platform. So I really want to help support people out there develop oligonucleotides. Fantastic well, I'd like to once again Thank our wonderful presenter, John, as well as you, our listeners, for your time and questions.
Don't forget to visit us at bioanalysis hyphen zone.com/webinars for more webinars. Thank you again for attending.
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