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Segment:0 .

MARTHA POWELL: Hello, everyone. And thank you for attending today's webinar-- Incorporation of Microsampling Techniques in Bioanalytical Assays, DBS and Capillary Microsampling. I'm Martha Powell, a digital editor here at Future Science Group. And I'll be your host for today's event. Before I introduce you to our speakers, I'd like to quickly go over a few housekeeping items.
MARTHA POWELL: Firstly, all the widgets are resizable and movable. So feel free to move them around and get the most out of your desktop space. You can expand your slide area to maximize it to a full screen by clicking the arrows in the top right-hand corner. A copy of today's slide deck will be available on our on-demand console. And our feedback survey is located in the resource list. We encourage you to download any resources or links that you might find useful.
MARTHA POWELL: Please let us know your thoughts on today's webinar by tweeting us @BioanalysisZone, using the hashtag #bzwebinars. Or simply share today's webinar with your friends and colleagues using the share widget on your screen. Some networks may cause slides to advance a bit more slowly than others. So logging off your VPN is recommended. If your slides are behind, pushing F5 on your keyboard will refresh the page.
MARTHA POWELL: If you have any other technical problems, you can find additional answers to some common technical issues located in the help widget at the bottom of your screen. Finally, an on-demand version of today's webinar will be available approximately an hour after the webinar and can be accessed using the same audience link that was sent to you earlier. So we're absolutely delighted to have Xiaonan Tang and Yang Lu from Frontage Laboratories presenting for us today.
MARTHA POWELL: Xiaonan joined Frontage Laboratories in 2008 and is responsible for small molecule bioanalysis under the GLP environment. Xiaonan has extensive experiences in method development and validation. He has supported over 300 nonclinical and clinical studies as a principal investigator. Yang Lu jointed Frontage Labs in 2015. Yang's team is responsible for developing and validating LC-MS/MS based methods for small molecular drug quantitation and supporting bioanal-- oh, sorry, and supporting bioanalytical assays in preclinical and clinical studies.
MARTHA POWELL: So we invite the audience to submit any questions you may have for our presenters throughout the webinar using the Q&A widget to the right-hand side of your screen. We'll pose your questions to Yang and Xiaonan in a live Q&A session after the presentation and then follow up any outstanding questions offline. So thank you again for joining us today. And I'll now hand it over to our presenters to start today's presentation.
MARTHA POWELL:
XIAONAN TANG: Thank you, Martha, for the introduction. My name is Xiaonan. It is my great pleasure to give this presentation on BioAZone. Today, I'd like to talk to you about the incorporation of Dried Blood Spots, or DBS, in bioanalytical assay. My talk is made up of three parts-- the overview of DBS sampling, the LC-MS/MS MD and MV for DBS samples. And then I will talk about the application of the DBS bioanalytical assays on regulated bridging studies, where both plasma and DBS samples are collected and analyzed.
XIAONAN TANG: I hope after the talk you are convinced that using DBS microsampling technique, we can generate reliable PK data as well as when plasma samples are used. So Dried Blood Spot testing is a form of biosampling where blood samples are blotted and dried on filter paper. The dry samples can easily be shipped to any analytical lab for analysis.
XIAONAN TANG: The concept of DBS was first introduced in Scotland in 1963 as a method to screen for metabolic diseases. And since then, the method has been widely used for screening purposes all over the world. However, the technique has not been widely utilized in the pharmaceutical industry until 10 years ago, thanks to the advancement in mass spec technology in terms of detection sensitivity. So what is the advantages of DBS sampling?
XIAONAN TANG: The largest advantages of DBS sampling is that it greatly reduces blood samples volume needed. As you can see from the picture, only 20% of the sample volume is needed for each sample. And because of this, for PK studies, for example, when mice, or rats, animals are used, the number of rodents needed can be reduced by up to 75%. And because of this, serial PK profiling becomes possible when DBS technique is used while when plasma sample is collected, only composite profiling is possible.
XIAONAN TANG: And serial PK profiling can generate a much more reliable and consistent PK profile because all the time points are collected from the same animals. With the composite profilings, the animals are bled alternately and at different time points. And the variability between animals can be very large, especially for PO dosage. For clinical studies, DBS sampling is also more patient friendly, especially for pediatric studies or studies with critically ill patients.
XIAONAN TANG: The handling of DBS samples is also a lot simpler than traditional blood collection procedures. There's no centrifugation needed to harvest plasma, no freezing needed. You don't need to transfer the plasma into the second container, no need to aliquot. DBS also is safe. All pathogens, including HIVs and hepatitis Bs, becomes inactivate on contact with the filter paper.
XIAONAN TANG: So the personnel in a lab is safe handling these samples. It is lower cost, not just because less rodents, less animals, and less compound are needed. The shipping is also easier because it's not considered biohazardous anymore. You don't need a specialized courier to ship those DBS samples, as you would with plasma samples. Storage, most of the cases DBS samples can be stored at room temperature, so no need for freezer space.
XIAONAN TANG: Next, I'm going to talk about some of the challenges with DBS when a bioassay is needed. The first challenge would be, obviously, the sensitivity because the small sample size can be used during extraction. Usually a 2-millimeter punch would have a little bit less than 3 microliter of blood samples. And that's all you can use to develop a method. So that's one of the limitations.
XIAONAN TANG: Sample quality is another concern. Spot-to-spot variation can be a big problem. I've seen some really terrible DBS samples. And hematocrit is another concern. With different hematocrit levels, the spot size can become bigger or smaller and thus affect the quantitation. Compound stability is the third challenge. In general, compounds are stable on DBS.
XIAONAN TANG: And that is why DBS samples can be stored at room temperature. However, if you happen to work with some unstable compound, it is not as easy as plasma samples if you want to apply some inhibitors or stabilizers.
MARTHA POWELL: That's great. Thank you, Xiaonan. So we've now got a quick poll. What is your biggest method development challenge when working with blood samples? So I'm just going to give you a few seconds. You should be able to answer this. And then we'll take a look at the results. Just a few seconds left to answer that.
MARTHA POWELL: Great. So I can see, most people think that the small sample size is the biggest challenge. So thank you very much for answering that. And I'll now hand it back to Xiaonan.
XIAONAN TANG: So this slide is the standardized procedures for the DBS sample collection and handling. I will not go into much details of this. There's plenty of informations on the internet. In this slide, I listed the main parameters that needs to be assessed during a DBS bioanalytical method validation. So I categorized these parameters into three main groups. The first groups I believe all the audience are very familiar with.
XIAONAN TANG: These are parameters that's needed for both plasma method as well as DBS method. The second group-- including dilution integrity, matrix effect, selectivity, and recovery-- these parameters need to be evaluated for both plasma and DBS method. However, the study designed to perform these evaluations are very different for DBS methods. The third category is including spot size, sampling location, evaluation, and hematocrit evaluations.
XIAONAN TANG: These are the key and unique parameters to DBS sample assays. So only DBS method, you need to evaluate these parameters. And I'm going to cover more details later. So how do we go about and develop a DBS-LC/MS/MS method? The upfront part, including the sample tuning, parameters, optimization, mass spec, and including the gradient optimizations-- those are all just exactly the same as traditional plasma methods.
XIAONAN TANG: The difference lies in how we prepare a standard and QCs and how we extract the samples. The DBS is a special matrix, unlike liquid samples. You cannot vortex them. You cannot dilute them. And homogeneity cannot be ensured. So we have to make sure that, when we prepare DBS as standard and QCs, we make sure the samples are homogeneous.
XIAONAN TANG: So I listed a couple of key points that we need to pay more attention during the preparation of standard and QCs. So the procedure is like this. We spike blank blood with the compound of interest. And we mix it well. We apply a certain volume of the whole blood onto the filter paper. And we get a spot, and then we let it dry.
XIAONAN TANG: It becomes DBS. One thing I need to emphasize here is that when we spike the blood with the compound, we want to make sure that the spiking solution should be less than 5% of the total volume. The reason is, if the spiking solution is too much, it's going to change the viscosity of the blood and therefore change how it diffuses on a paper. And it'll affect the quantitation with the assay samples.
XIAONAN TANG: This slide shows you typical extraction procedures for DBS samples. It's very straightforward. First, take a punch from the center of the DBS card-- usually a 3 millimeter or a 6 millimeter in diameter. And we put this disk in a tube, and we add extraction solutions containing internal standard. Sonicate vortex extract the samples out of the disk and into the liquid phase.
XIAONAN TANG: And then the samples can be either reconstituted and inject. Or if sensitivity is a concern, it can be further concentrated by using liquid-liquid extraction or solid phase extraction. So following the previously discussed rules and method, we developed two methods. Let's call it compound A in both rat DBS and dog DBS.
XIAONAN TANG: In this slide, I show you the procedures for these two methods, and they are identical. And this procedure is similar to the typical procedures I just showed you. There's one extra step, which is centrifuge for two minutes. We add this step just to get rid of some small particles in a liquid. This is a typical chromatic round for LLOQ, with a concentration of 5 nanogram per mil.
XIAONAN TANG: As you can see, even with a small sample volume, the sensitivity is quite good, and the signal to noise is also very, very good. Starting from this slide, I will show you some of the selected validation results for the rat DBS method. The dog methods are similar. So this slide shows the linearity of the calibration standards. Among nine runs, each run has two set of curves.
XIAONAN TANG: The linearity are very good, based on the R-square values. And the percent bias are generally less than 2%, which is very good. The acceptance criteria is actually plus/minus 15. Not only that, the percent CV is also very tight. They're all single digit. This slide shows you the result from the three intra-runs. So all four levels of QCs passed. There's no single failed QCs.
XIAONAN TANG: Accuracy and positions are good. In this slide, I show you the recovery of the compound A from rat DBS. Normally, it would be done at low and high levels. But in this case, we also covered AQL concentrations. So the recoveries are consistently high among all levels and among all replicates. And this is the key for a successful validated method.
XIAONAN TANG: So I would closely monitor this recovery during method development phase and making sure that the recoveries are consistent and they are high. Matrix effect is close to 100% for both low and high QC levels. This is expected for all DBS methods or all micro sampling methods, simply because, really, there is not much matrix added during the extraction.
XIAONAN TANG: Dillution integrity-- so as I said before, DBS samples cannot be diluted using a blank matrix because they are solid. So the way we did this is we extracted AQL DBS samples, and then we diluted it with the extraction solutions containing same levels of internal standard concentration. And the results are very good. The effect of spot size-- so the effect of spot size is one of the evaluations done to evaluate the homogeneity of the spot.
XIAONAN TANG: So to do this experiment, we prepare a DBS spot using two different blood volumes, 25 microliter versus 50 microliter. The idea is, when larger volume of blood is used, due to the resistance on the paper, the thickness of the spot at the center tends to be thicker, and therefore, it will affect the quantitation. So we have to make sure that's not the case. And for this method, we have shown that percent bias between the two a difference spot size-- there's no obvious difference.
XIAONAN TANG: So in case the nurse or the personnel from the animal facilities-- they accidentally added too much blood or too little blood, you will know that that would not affect your quantitation results. The effect of sampling locations-- this is another test for homogeneity test. So same spot size is used.
XIAONAN TANG: However, the punching location is different. So we are comparing the punch from the center versus the punch at the edge of the spot, and we compare the extract samples between this, too. The idea is we want to evaluate if the blood is distributed evenly along the radius of the spot. In this case, there's no difference between the two. So the passing result of this test comes in handy when-- especially helpful when you have limited number of spot per sample.
XIAONAN TANG: Usually, we get three to four spot per sample for us to do reassays and ISRs. But in case you only receive one, you can punch three disks of one spot without touching each other, and you can use one of them as assay and the other one as reassay and ISR, et cetera. We also evaluated extensively short-term stability at all kinds of extreme temperatures, minus 20, 37 degrees with or without a dessicant pouch.
XIAONAN TANG: So this evaluation is used to mimic the shipping process. Because when we ship DBS samples, usually, no refrigeration is needed, and no dry ice is added. So the DBS samples are actually the same as the ambient temperature. It can be really hot or really cold. So we want to make sure that, during the shipping process, the concentrations won't change, and the drug is still stable.
XIAONAN TANG: Finally, the hematocrit test, where DBS prepared using blood at low and high humidity levels are measured and compared against their nominal concentrations. According to my experience, the DBS with low hematocrit levels tends to be more problematic. Just as in this case, as you can see, QC samples with 30% hematocrit blood actually has a larger deviation from the nominal concentration.
XIAONAN TANG: When it's less viscous, it tends to spread bigger on a paper, and the disk of the same punch would actually have less blood volumes. Well, hematocrit effect have been recently highlighted as an analytical concern for DBS. But personally, I think hematocrit impact on quantitative analysis of DBS samples are minimal for preclinical study, especially for TK studies, because only healthy animals are used.
XIAONAN TANG: And same for the majority of the clinical studies, so only for those studies with a population in some severe disease situations-- for example, patients with renal impairment or oncology patients, where we expect the hematocrit levels would be abnormal. Then it's going to be a concern, and then we can evaluate how we handle that.
XIAONAN TANG: So with these two successfully validated methods, we apply them on two bridging GLP studies, one for rat and one for dog. Each study has a little over 200 samples, and we'd take 10% of all the samples and perform the intra-sample reassay. The acceptance criteria is-- the reassayed values should be within 20% of the original values, which is within the two red bars.
XIAONAN TANG: As you can see, for the rat studies, 100% of the samples passed. And for the dog studies, 81% of the samples passed. So both of them have exceeded the acceptance criteria of 2/3 of all the ISR samples, indicating that not only the method is repeatable but also the spot-to-spot variability is also minimized because the ISR evaluation was done using a separate spot as the original.
XIAONAN TANG: So now let's look at the data. Because this is bridging studies and we have collected both DBS and plasma concentrations-- so we measured both using plasma method and DBS method. The blue lines are the concentrations from DBS, and red lines are from plasma. As you can see, they are quite different. The DBS are a lot lower. Does that mean we failed to recreate the PK profiles?
XIAONAN TANG: No, because DBS-- we are measuring the concentrations in blood and now plasma. So how do we compare these two? Let's introduce the concept of blood plasma partitioning, the K value, which is the ratio between the concentration in blood versus the concentration in plasma. And in this case, compound A in rat blood, the K is 0.7. So let's apply that ratio to the DBS concentration. After correction, the two curves are a lot closer.
XIAONAN TANG: By average, the difference is close to 10%. As we know, the biological method has a plus/minus 15% acceptance criteria. So I would say that we successfully recreated the PK profiles from the plasma using a DBS bioanalytical assay. Now, let's look at the dog study. So the dog study-- the two curves are very close to begin with, without the correction factor.
XIAONAN TANG: If we accept that 0.7 factors to the blue line, it's going to be too high. But luckily, when we measured the K values for compound A in dog blood, we found that the K value is very close to 1. It is 0.931. And when we apply that, the two curves become overlapped. So the agreement is amazingly good. So by this slide, we can conclude that we are able to recreate a TK profile from plasma samples by using both rat and dog DBS bioanalytical assays.
XIAONAN TANG: So this slide shows you how we get these partitioning parameters for both dog and rat. Due to the time limitation, I'm going to skip this slide. If you're interested, you can download the PDF file and look into it. One thing I want to point out is, to get these parameters, we did the test at the concentration of 500 nanogram per mil, which is a lot less than most of the assay samples.
XIAONAN TANG: So the numbers could be a little bit off if it's concentration-dependent. We don't know yet. So in previous slides, I only showed the mean values from samples collected at the same time points and from the same group. In this slide, I plugged all 200 individual samples with their concentrations in plasma and a concentration in DBS, x-axis being plasma concentration and y-axis being the DBS concentration.
XIAONAN TANG: And the correlation between the two are pretty linear. The slope of the fit is 0.64, which is 89% of the K value that we determined at 500 nanogram per mil. But if you look at the other concentrations, they are at 50 or even 100 times higher than that. So again, if it is concentration-dependent, we might get slightly different, values and it's only going to make the agreement better. So to summarize, DBS is a good alternative to traditional blood collections, and they can be well incorporated with bioanalytical methods and studies as well.
XIAONAN TANG: During the method development and validation, we want to make sure that those parameters that are unique to DBS should be assessed. And this is mandatory according to the newly released FDA validation guidance. I'm going to cover that later. And I have used a case study to demonstrate that bioanalytical data using DBS samples and DBS bioanalytical assay can generate reliable and repeatable TK profiles, as well as the TK profile generated using traditional plasma method.
XIAONAN TANG: Of course, we have to admit that there are some natural challenges associated with DBS or blood samples where the impact of hematocrit is always a challenge and spot homogeneity is going to be a problem as well. So my last slide, I would like to talk about some regulatory perspectives on DBS. As with anything involving drug development, DBS requires regulators' approval.
XIAONAN TANG: I could be wrong. But as far as I know, they haven't issued definitive rulings on how they will handle new drug applications that uses this technique. I do know that they encourage comparative studies with plasma and other established technologies, just like the bridging studies I've shown you. I cited a few statements from their perspectives on microsampling.
XIAONAN TANG: I'm sorry about the typo in the footnote by Dr. Viswanathan. It was published a few years ago. I recommend you to read it if you're interested in learning the perspectives on DBS technique or microsampling technique from a regulator's point of view. With that said, FDA did share rules on how validation of DBS bioanalytical methods should be performed. So in the Bioanalytical Method Validation Guidance for Industry issued May this year, they did list how DBS assays should be performed.
XIAONAN TANG: So the last two bullet points are the citations from this literature. The first one basically listed all the parameters that needs to be assessed during the full validation. It actually also included the repeatability, which is the ISR. And they also recommend correlative studies with traditional sampling should be conducted during drug development, which is exactly what we have done in that bridging test.
XIAONAN TANG: So one thing worth noting is that, in the 2013 draft of this guidance, there's one sentence, and I quote, "Although the dried blood spot methodology has been successful in individual cases, the method has not yet been widely accepted." So it's not very encouraging. But in this final version, they removed this sentence, which, to me, is a good sign to whoever is interested in developing new programs using DBS.
XIAONAN TANG: So that wraps up my talk, and I will hand it over to Dr. Yang Lu. And he will talk to you about capillary microsampling. Thank you.
YANG LU: OK. Hello, everyone. My name's Yang from Frontage. So today, I'm going to talk about also microsampling, but from a different perspective. It's using microscale volume of samples by using the capillary technique. So the topic of my talk, it will be, A "Tube" to A "Drop," Capillary Microsampling Technique and its Advantages in Today's Bioanalytical Research.
YANG LU: So as Xiaonan just mentioned, we have every reason to apply new techniques to reduce the volume of sample we need for bioanalysis. So to do bioanalysis, the ultimate goal is to determine the drug concentration in biological matrices. And usually, plasma concentration is the most important parameter we look at to support PK modeling, PK/TK analysis, and things like that.
YANG LU: So for the analysis lab, so we usually use LC-MS to support this kind of work. But the difference is the sample volume. Because we do need a certain volume of biological samples to support this sample extraction, which will be analyzed later on. As Xiaonan just mentioned, the DBS can apply a different or alternative way to reduce the sample volume to be used.
YANG LU: But there is-- or there could be-- a concern that using a dried blood spot is actually changing the biological matrices from the usually tested positive samples, especially for some cross-termination or cross-comparison studies that the previous data were acquired using plasma sample. Sometimes scientists are a little concerned to use different matrices. So the question comes to is there a way to simply just reduce the volume of plasma sample that we need to use?
YANG LU: For information, usually for a preclinical study, the sample we need to use for bioanalysis will be about 25 to 50 microliter. So the question comes to why do we need to reduce the sample volume that is used for bioanalysis? That is because, first of all, as I said, 25 or 50 microliters of plasma needs to be used. It means we need to take about 100 or even more of blood per time point from the animal at each time.
YANG LU: That's because we can only acquire about half of the blood volume to acquire the plasmas. And to get a full PK profile from the animal, from an animal study, we need to have different time points. Usually, time points is listed like this. This is just like a dummy example, probably from predose all the way to 24-hour. So the sample volume needs to be collected will be over a mil or 1 milliliter of whole blood.
YANG LU: And that will be very challenging, especially when using small animals. For example, a mouse that weighed about 30 grams, the total blood volume is only like 1.2 to 1.5 milliliter. It will be very hard to collect enough volume from a single animal. So this approach is kind of difficult. And to achieve that, a typical study design for a tox study is to have satellite group or subgroups in each dosing level.
YANG LU: This just shows a very general example. Even in the same dosing group, we need to have three or four subgroups. Each group will have five to 10 animals to achieve enough volume we need to acquire at each time point for bioanalysis. And that just raised some concerns. And it can be the limitation for this conventional sample collection or sample design.
YANG LU: The first concern is, of course, the ethical concern, that we need to increase a lot of animal number we use in each study. There are also a scientific concern, which can impact the result. First of all, it's an individual variance, just like Xiaonan mentioned. Especially for small rodents, the individual variance could be very high, especially using [? field ?] dosing. Another one is animal physiology, that if we keep using the same-- especially at the first several time points, the sample needs to be collected very frequently from the same animals.
YANG LU: And a quick reduce of blood volume in each animal could cause a change or impact on the animal physiology, which can affect the ultimate PK result also. Another possibility is unexpected death, that if some of the animals died during the sample collection because of losing a huge amount of blood, that can impact the total statistic calculation when we do PK modeling or PK profiling. There are also accuracy concerns and efficiency concerns due to the sample collection procedures using it the conventional way.
YANG LU:
MARTHA POWELL: That's great. Thank you, Yang. So we have another poll question for you now. What drove your interest in using microsampling techniques? I'll just give you a few seconds to answer that. Just a more seconds. That's great. Thank you.
MARTHA POWELL: So as we can see, efficiency is the top scorer. But it's quite split between both efficiency and ethical concern, which is interesting. So I'll just hand you back to Yang now.
YANG LU: OK. Thank you, everyone, for your opinion. So actually, that comes back to the very general question, is there a way that we can use less volume of sample? So actually just [INAUDIBLE] is from two to a drop. If we can finish the sample collection using just a drop of blood, that will be fantastic. So actually there is a way. It's a little bit different from what Xiaonan mentioned. But this is an alternative way to using less sample volume to support not only bioanalysis, but also we're sharing the blood samples with other scientists, like a biochemist or hematologist.
YANG LU: So this technique I want to talk about is capillary microsampling technique. So the capillary microsampling is pretty much using a capillary to collect blood sample and separate the plasma we need for bioanalysis. The first step will be at each time point, there will be punch-- the tail vein on the animal-- I'll just use a mouse as an example-- punch a drop of blood in tail vein, and using a capillary, usually a 32-microliter capillary, to collect the blood.
YANG LU: And we can seal the capillary by using-- excuse me-- by using scientific wax to seal both ends of the capillary and then using a specifically designed rotor to centrifuge the capillary-- this step you can see in the second picture-- to separate the plasma from red blood cells. And then the next step will be transferring the collected plasma sample into a secondary capillary, too, which has a set volume-- 4 microliter, 10 microliter.
YANG LU: There are different kinds. So in this case, let me use the previous example. If we just follow the same study design for a 24-hour tox study, a seven time point study only need to use 224 microliters of whole blood. So that's totally bearable for a small animal like a mouse or even bigger, like a rat. That means we can collect all the time points from one single animal.
YANG LU: So now the question comes to our bioanalysis part. How can we apply the sample collected in capillary tube to our bioanalysis research? So actually the first step, different from the conventional sample collection way-- the first step we need to think about is how can we wash out the sample from the capillary tubes?
YANG LU: So actually there are many things we need to consider. The first of all is when we wash out the samples, or wash out of the compounds from capillaries, we need to make sure the compounds washed out are homogeneous. So selection of the washout buffer or washout matrix is critical. There are many options we can use. We can use just a buffer solution.
YANG LU: Or we can use some organic solvent. Sometimes we use matrix. Sometimes we use surrogate matrix, et cetera. So this is the thing we need to closely monitor when we do method development and validation. And I will talk about that a little bit in one of my later slides. But this is the most different step from the method when we use conventional sample.
YANG LU: So also, try to wash them out, we need to apply some other techniques. Like, we need to vortex the tubes, or sometimes we need to centrifuge them. Sometimes sonication is needed. Again, so this will be closely monitored during method development. So how can we fit this capillary microsampling technique into bioanalysis, which is required by GLP regulations?
YANG LU: First of all, there are additional requirements or additional parameters we need to test during method development and validation. For example, the recovery is one thing we need to keep an eye on. The conventional way, we just need to measure the recovery or extraction efficiency during the extraction procedures. But when we are using a capillary microsampling technique, we need to also test the recovery rate or efficiency of the washing out step.
YANG LU: So pretty much the recovery we tested is a combined recovery ratio of both washout and extraction. Also, when we test the stability of the sample, we're not only testing the stability of the drug in matrix, for example, plasma, we also need to test the stability of the compound in washout buffer, no matter what solution we're using to wash it out. We also need to test some potential absorption issue, whether there's any unspecific binding to the capillary or not.
YANG LU: Of course, we need to measure the impact of different capillary size that result of our validation. So finally, Frontage has been working with our collaborators on different methods and different projects using capillary microsamplings. At this moment, we have developed more than 10 methods using capillary microsampling, supporting over 40 projects. They are all GLP regulated. And we have tested over 3,000 samples.
YANG LU: Again, they are all tox studies. So not huge amount of sample, because they are relatively small. So to prove that the capillary microsampling technique is feasible for bioanalysis work, I just show the ISR result for one of our studies. And you can see most of the study-- most of the sample-- are very reproducible. That just supported that the capillary microsampling technique is well-adapted in today's bioanalysis work and can provide a very reliable data.
YANG LU: So there are some challenges when we're trying to develop a microsampling incorporated bioanalysis method. The first concern-- or the first challenge-- as Xiaonan mentioned in the Dried Blood Spot method, is the sensitivity. So even though we're using like a 10-microliter capillary tube, we have to wash it out using some reagent, no matter it's a matrix or it's a buffer.
YANG LU: Just for example, let's say we use one 90 microliter of the reagent that resulted to 200 microliter in total of the washout sample. Another way we can say this-- these samples are 20-fold diluted. So if we're using 20 microliter of the washout sample for extraction, that means the actual plasma sample we're using is only 2 microliter. And that's all we can use, because we also need to consider potential reassay and ISR down the road.
YANG LU: So that gives us the challenge-- how can we make our method more sensitive? So that just comes to the bioanalysis part. We need to apply new extraction procedure-- maybe STE or LOE to concentrate the sample. Sometimes, we need to use a higher sensitive instrument. And we need to fine tune the method to get a better peak, as good as possible, and to increase the signal-to-noise ratio to have a good peak for our sensitivity requirement.
YANG LU: Another challenge is, as I mentioned, the washout procedure. So it's not as easy as the picture shows to try to make it washed out. Actually, we need to put some efforts during this method development part to make sure that our sample can be washed out efficiently. Also, it needs to be washed out consistently. Because we don't want to have different recovery from different samples.
YANG LU: Also, we saved a lot of blood or biomatrices. But there comes a price. That is the sample handling is much more challenging than the usual, conventional way to collect the samples. And that can cause potential contamination. So the accuracy of the sample transfer is a key during the sample collection procedure at the animal facility.
YANG LU: Because we need to transfer whatever collected in the first or the primary capillary to the second route capillary tube. And that brings the challenge that we need to make sure the second recovery tube is completely filled. Because if you don't fill the second capillary tube, you don't know exactly how much volume you have in there. And that will cause a big problem when we do the PK profiling.
YANG LU: And also, this contamination issue comes from the transfer staff also. There is a chance that either the primary or the secondary capillary got contaminated. There is another concern about sample loss. There are two faults. The first fault of sample loss is sometimes when we collect the plasma in the primary capillary tube, we don't know whether that's enough for a 10-microliter tube.
YANG LU: And during the transfer, if a scientist realizes, oh, there's not enough volume to fill the secondary tube, then this sample is pretty much useless. And the only thing we can do is to discard it. And the second point for the sample loss is that during the sample transfer from the first to the second capillary tube, there's the chance that it can drop the tube and cost the loss of sample.
YANG LU: Also, there is potential to over-dilute the sample. Because when we receive the samples at the bioanalysis site, we wash out the sample using the reagent. And if the volume we add is too much, that means we may see a lot of [? BQL ?] sample. And that can cause the over-dilution. And that comes to the very last end on this page is there's no backup sample using this technique.
YANG LU: If you over-dilute the sample, there's no way you can turn it back. So that's why Frontage developed an alternative one-capillary approach to try to avoid and try to prevent these potential issues. So this is a general procedure, how we collected the sample and how do we use that to analyze the compounds. The first couple of slides show-- the first couple of pictures show how do we treat the capillary tube before even we collect a sample.
YANG LU: The primary-- or the only-- capillary tube was prescored at a certain lens or certain volume. And then it will use the same way as the regular capillary tube sample collection, and using the centrifuge to separate the plasma from red blood cells. But actually, the difference comes here is there's no transfer from the primary to the secondary capillary tube.
YANG LU: Instead, we would just snap the primary tube and just put that in the containers. So in this picture, I show that the snapping of the primary tubes is by hand. But actually, when we actually practice that, we use a pair of tweezers. The only reason I showed like this is because the purple gloves show a better background.
YANG LU: So with our new approach, all these potential issues are solved. First of all, there is no secondary tube. So we don't need to worry about to completely fill the secondary capillary. As long as we snap the primary capillary tube and make sure the snaps or the small pieces are completely filled, then we're having a good set volume. And also, there's no contamination issue during the sample transfer.
YANG LU: And there's no concern about sample lost. By using this approach, using the same amount of 32-microliter of whole blood, we can have at least one backup sample. And that can be used to overcome the potential over-dilution or contamination issue during sample analysis. So this solves a lot of problem that's been caused by the regular capillary method. So here, I want to show some example.
YANG LU: It's more like a feasibility example we developed at Frontage. And here's the result. We tested the drug midazolam using all three kinds of techniques-- the conventional samples; the 10-microliter sample, which includes the sample transfer; and also the Frontage method using the snap one capillary method. So this is just has occurred. So because of time, I will just not going into much detail.
YANG LU: But from here, you can see the curve over a range of 1 to 1,000 nanograms per mL of midazolam. The curve looks really good. And our square is almost close to 1. And we have three [? interruns, ?] and they have no problem. So the big comparison comes from the QC sample. Because we prepared three sets of QC, one used in the regular pipette QC, which represents the conventional sample collection procedure.
YANG LU: And also we compared the Frontage method, which is named in this table 3.3 microliter capillary, and also the usual way of using capillary is the 10 microliter. And you can see they are very consistent about the accuracy and reproducibility. Within three [? interruns ?] of 18 QC sample for each QC level, the result of bias is within 5%. And the percent CV is also one digit. So they are not only accurate, but also reliable and reproducible for each of them.
YANG LU: So the good example for this one actually comes from-- the very basic question is the extraction procedure. Using the 10-microliter capillary and the 3.3 microliter capillary, they provide pretty much the same-- 100%-- recovery ratio. And this will also show that the dilution-- even we use dilution procedure to dilute the sample, we also have no problem using the capillary tubes. And you can see on the right side, we show represented chromatogram for double blend, black, LQ, and a mid-range QC.
YANG LU: And that is when we say we have to-- but just keep in mind, so this one only used literally two microliter of real plasma sample. So we do need to fine tune the instruments and fine tune the method to have a good sensitivity and signal-to-area ratio. So there might be a concern that how can we make sure that the prescored primary capillary tube can provide consistent volume for each small snap.
YANG LU: So actually, this is just example how we measure the consistency. We use 36 replicates of these tubes. And we've prescored them, filled them with water, and tried to measure the volume of each snap. And we can see this is very consistent. And this is something we did in-house. But actually, we don't have to do that for each tube we need to use in real practice, because these prescored capillary tubes are commercially available.
YANG LU: And according to our request, we can purchase the prescored capillary with different volumes-- either 3.3 or 2 or 5, just according to the necessary. OK? So I will save this a little bit, but I just want to mention one thing is, the capillary microsampling technique is not only for animal studies.
YANG LU: It can also be applied to human studies. And that can also save a lot of trouble during sample collection. The only one concern for this capillary tube for human study is, unlike animal study, the human study usually have a lower LQ. So the sensitivity requirement is even higher and requires more validation and method development work. So to summarize the capillary microsampling parts, the capillary microsampling has been attracting more and more attention in bioanalysis research.
YANG LU: It provides an additional method to reduce the sample volume. And of course, additional sample method development and validation needs to be covered regarding the new method incorporated. As Xiaonan just mentioned, there's no clear rule about what needs to be measured based on the regulatory requirements at this moment. But the new release-- the guidance-- for bioanalysis work has mentioned some of the microsampling techniques.
YANG LU: And if you are interested, you're more than welcome to review that. And also, capillary-microsampling-based analysis can provide data as reliable as the conventional sample collection procedures. And also, the microsampling techniques-- no matter if DBS or CMS-- is aimed to provide accuracy, but also very friendly approach in bioanalysis.
YANG LU: So before I end my talk, I just want to show one more slide. This is just a dummy example. Let's just say if we want to have four different dosing groups, and we need to have 12 time points, we only need to use 40 animals to generate 480 data points. So that is very, very efficient. And that can save a lot of trouble, no matter from the scientific or ethical reasons. And that will be my talk about capillaries.
YANG LU: And thank you.
MARTHA POWELL: Thank you for that very insightful presentation, both of you. I hope our audience found it of great interest and value. If you do have any questions, please continue to submit them using the Q&A tool on the right-hand side of your screen. So we've received a number of questions so far. And we'll just address some of these live on air. So first up, is there any gap between the previously validated methods and the new FDA guidance issued in 2018?
XIAONAN TANG: I can speak for the DBS part. I have carefully reviewed the new guidance that is issued May this year. And I find that because we have done a thorough job for previously validated method, we can be confident to say that there is no gap between our previous validated methods and the new guidance. So we're in good shape.
MARTHA POWELL: That's great.
YANG LU: So--
MARTHA POWELL: Sorry. No, no. Go ahead.
YANG LU: Yeah. I just want to mention a little bit from the CMS point of view is the current practice and the regulatory requirements, I just mentioned in my talk, there's additional validation parameter we need to check, including the recovery and stability and absorption issue. So because, not like the DBS method Xiaonan just mentioned, a capillary tube is still a plasma method. So it's very similar to whatever we need to measure during validation for a plasma method.
YANG LU: The only difference is we need to consider even more that it's new or that it's unique to the microsampling technique.
MARTHA POWELL: That's great. Thank you. So our next question, how reproducible are the capillary volumes?
YANG LU: Right. So that's actually a very good point. And that's actually the key to producing reliable and accurate data. So as I just mentioned, I would just talk about the conventional-- or the regular-- capillary method and the Frontage method. The capillary method, usually the set volume capillary tube or the second recovery tube is commercially available. And there is a quality controlled process.
YANG LU: And usually what we receive these capillaries, we need to measure the consistency. A way to do that is very similar to what I showed in my slide is we will try to fill some of the capillary tubes, 10 microliter, for example-- and weigh them before and after we fill that with water. And we can have an idea how reproducible the volume is. And usually, it's very consistent. And for the Frontage method, the same thing.
YANG LU: We can do that in-house. And as I said, it's also commercially available. But again, we need to check the reproducibility of sample volumes by measuring the sample weight before and after we fill the tube with water.
MARTHA POWELL: Perfect. Thank you. So next, how do you avoid contamination when the same puncture is used on all the spots at different concentrations?
XIAONAN TANG: So we take the carryover issues very seriously. So during the method development, we try to make sure there's no carryover, and there is no contamination from the same punch. So what we do is every time we used a punch on a high-concentration spot, we would punch a disk at the blank areas of the same filter paper. And then we would use the punch again on the following samples.
XIAONAN TANG: And in this way, we can avoid cross-contamination between the puncher. And the other factor is we want to make sure the blood is completely dried before they were punched or extracted. And that way, the cross-contamination can be greatly reduced as well.
MARTHA POWELL: That's great. Thank you so much. So the next question is a bit of a long one. What was the process used to score the capillary tubes for each sample used for analysis? And during the scoring of the capillary tubes, what measures were taken to prevent cross-contamination from sample to sample?
YANG LU: Right. So that's a very good question. So when we prescored the capillary tube-- that's the Frontage method-- is we measure the exact length on the capillary tube. And we use just a blade to score the capillary tube. But that's just an in-house method. As I mentioned, this kind of prescored capillary tube is commercially available. And we can just simply order whatever volume we need to use in practice and try to do that.
YANG LU: And talking about the contamination-- as I said, actually, one of the advantages of the Frontage method-- the one-capillary method-- is to prevent contamination. Because during the regular capillary tube method, there is a transfer staff. And our method avoids that. And we can simply just use a pair of tweezers to snap the tube. And the only thing we need to keep an eye on is not to snap the capillary tube too hard and to avoid potential splashing.
YANG LU: And if we can make sure of that, because the outside of the tube should be very clean, and everything should be contained in the capillary tube. So if we can avoid splashing during the snapping, the contamination should not be a big issue. And our result actually showed that.
MARTHA POWELL: That's great. Thank you so much. So I'm afraid that's all we have time for today. But we'll address any of the other questions offline. So if you do have any questions, please feel free to send them in to webinars at bioanalysis-zone.com. I'd like to once again thank both of our wonderful presenters, Yang and Xiaonan, as well as you, our listeners, for your time and your questions. So if you'd like to let us know your thoughts on today's webinar, then please feel free to complete our feedback survey in the resource widget, or tweet us @Bioanalysis.Zone.
MARTHA POWELL: And don't forget to visit us at www.bioanalysis-zone.com for many more webinars. So thank you again for attending. And goodbye.
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