The Fastest Kinetic Screening Available for Your Antibody Discovery – April 9, 2020

0:00:00.0 John McKinley: I'd like to welcome everybody for joining us on our webinar today. I'm your host, John McKinley, and I'm really glad that you could all make it here during these trying times. Here at Carterra, we are working with many organizations and labs that are working on therapies and vaccines for COVID-19, including the Gates Foundation. Please reach out to us if you're working on any COVID-19-related projects, so we can collaborate with you. A couple of housekeeping issues before we get started, in the upper right-hand corner of your WebEx app, there's an icon that will allow you to view the screen full-screen mode. Please click on that. Also, if you have any questions, use the chat function to send them directly to me, your host, John McKinley, and we'll address those questions at the end of the webinar.

•  Dan Bedinger - Application Science Team Lead

0:00:49.4 JM: I'd like to now introduce our presenter, Dan Bedinger, he's our Application Science Team Lead. Dan has a PhD from UC Davis in cellular and molecular physiology. He has 20 years experience in the generation and characterization of therapeutic monoclonal antibodies. Dan has spent time at Abgenix, Xoma, and other companies. Dan has a broad experience with many different label-free biosensors. Now, I'd like to hand it over to you, Dan.

0:01:23.8 Dan Bedinger: Thank you all for joining us today for this talk. So I just wanna give a brief overview of the topics I'm gonna cover today. We're gonna start with a platform overview of the LSA, and then a brief discussion of what kinetic binding constants even are and why we measure them. And then I'll talk about two examples of high throughput kinetics analysis, the first one being an example of anti-PD-1 antibodies, and the second example, it will be a multi-antigen characterization with long off-rate. And then we'll end with a little summary. So this is the LSA instrument. We call it the Lodestar Array. Carterra likes to have a navigation theme for our names for things. It is a benchtop instrument. It's quite large, but it can sit on a benchtop, and we have a cart that we sell it with, that it sits on, that holds the PC and the waste and some storage for consumables that sit below it.

• LSA is really a game changer in high throughput SPR

0:02:29.9 DB: The LSA is really a game changer in high throughput SPR because of its very unique fluidic configuration. So the system uses two relatively independent fluidic modules that both address the same sensor chip, so they move robotically on and off the sensor surface. So the first system, and this is really one of the big differentiators and proprietary technologies to Carterra, is the 96-channel continuous flow micro-spotting head. Which actually creates 96 independent flow cells on the chip at a time. And then there's also a single-channel manifold that can inject one sample over the entire array surface.

0:03:15.0 DB: So the way the system works is that the 96-channel manifold can print or capture or immobilize up to four different sets of 96 onto the array at the same time creating a 384-spot array. And then the single channel module can dock onto that and do the single injection, we call it one-on-many, that exposes all of the spots on the array to the same fluid at the same time for either kinetics or epitope binning. And the two modules can switch on and off the chip surface during the run automatically.

0:03:54.3 DB: So the LSA is really optimal for doing some of the most common applications in antibody screening and characterization, those being kinetics and affinity analysis, competition-based epitope binning, peptide or mutant mapping and quantitation. So in addition to having a hardware architecture that really streamlines and makes these processes easy and efficient. We've put a lot of effort into generating really industry-leading software that makes both for quick processing of these really large data sets, but also really excellent visualization and utility of the data with the... We have a software module called Kinetics, and then we have a sophisticated epitope analysis tool that has some great visualization techniques like these network plots that you can see here.

• 96 needle manifold

0:04:50.1 DB: So going a little bit more into detail of the system and how it operates, this... What you're looking at here, this is a video I'm about to start, is the 96-needle manifold that loads the samples to the 96-channel manifold. So you can see it goes into a microplate position, and there are three plate positions on the deck that it can access and then float the sample over the chip surface. So this is really important in that it's very different than other microarray approaches, which are traditionally deposition-based. This is actual flow, so the sample goes from running buffer to sample then back to running buffer. And when it's doing that, it actually cycles the sample back and forth over the surface for a long time. So there is no flow rate to volume trade-off. You can have a very long contact time and maintain a high flow rate the entire time.

0:05:42.8 DB: Also, I'm gonna restart the video, after the sample is used for the immobilization or capture step, it is returned to the microplate, so you get the vast majority of your sample back to reuse again or use in a different assay. So you can see here, we've created a 196-array. Now, the needles are going into a second position on the plate, and the printhead has indexed to a different location on the array. And if it does that, up to four times, you can create the higher density 384-array seen there. So once you've created your array with the 96-channel manifold, that un-docks and moves out of the way, and the single-channel manifold can come and dock onto the chip, and so that's shown here.

0:06:32.6 DB: So here we're flowing one fluid over the entire surface of the chip. In this case, it could be an activation solution for prior to immobilization or a preparation of what we call a lawn. Which is like the same capture molecule on the entire surface of the chip. Once an array is created, the single flow cell can re-dock onto it, and then you can flow that single sample over the entire surface of the array. So this is what makes it so incredibly efficient both in terms of time and sample consumption. Because for that one 250-microliter volume injection that cycled back and forth over the surface during the association phase, you're getting to collect data for all of the immobilized ligands at the same time. So that would be a concentration of a kinetic series or an analyte injection on an epitope binning assay. We call that one-on-many format.

• Manifold To Create The 384-Array

0:07:33.1 DB: To highlight a little bit more about how this is ranged, the top image, the pink vertical rectangles represent the individual flow cells of a single docking of the 96-channel manifold. And then down below is a completed 384-array where we've done three additional inset prints or capture steps with the manifold to create the 384-array. In either image, you can see that there's a horizontal blue rectangle, and that is actually showing the inter-spot references. So these are unprinted spots that are used to subtract things like refractive index changes from the data stream, and so those are all collected in real time. And in the bottom, you can see there's four ROIs or spots above and below each reference, and so there's eight references or eight active spots that are subtracted from each reference, or well, the other way, the reference is subtracted from each of those eight automatically. So there's 48 total reference spots. And then once the single flow cell docks, you get this flow of one sample over the entire array and collect all 384 active spots of data at the same time.

• High Throughput Kinetic Analysis

0:09:00.0 DB: So this layout really has a lot of advantages in terms of running high throughput kinetic analysis. We've touched on a few of them. It requires very little ligand. One, the ligand that you utilized is returned to the plate, so you actually get it back, but also that single 200-microliter volume in the plate can be used to capture for a long time, up to about half an hour. So if you have a low concentration crude sample and you want to use a high affinity capture reagent on the chip surface to enrich it into the array so that you can get your kinetic analysis, it's very good for that. It also has very low antigen requirements because you're only injecting one analyte volume per analysis of your entire set of 384 antibodies, it becomes very efficient, at least on a per antibody basis for usage. Also, because all of the data is being collected in parallel, time is not usually a limiting factor. Most kinetic assays are run overnight and are completed by early morning the next day at the latest, so you don't find yourself making very many decisions where... Well, in terms of limiting the number of concentrations or designing the assay to reduce the time. You really design them based on the way you want your data to look.

0:10:23.1 DB: Also, the array allows you to power your analysis or increase your n. If you have less than 384 antibodies, it's very easy to spot the same antibody multiple times into the array, and this allows you to generate real replication of data within an experiment. So if you can spot an antibody eight times in the array, you have a very good sense and you can get a mean and standard deviation of the actual kinetic rate constants you observe in that analysis. Also, it allows you to do things like printing the dilution series of your antibodies, so that you can observe the binding kinetics at different densities, which is very useful. The system also has an integrated analysis software package that allows you to really rapidly analyze the kinetic data and have streamline modes of presenting really powerful figures and images of the data.

0:11:23.0 DB: So to take a step back, what are we measuring when we're doing kinetic screening or kinetic characterization? So the typical one-to-one binding interaction is A plus B goes to AB in a direction that goes in both orientations, so there's an association and a dissociation phase. And during an injection, they can reach an equilibrium, so the on-rate or association rate constant, the ka, and that is the driving constant towards formation of the complex, and the dissociation rate constant or little k little d or off-rate is the driving rate constant that drives the dissociation. And then the big K big D or the equilibrium dissociation constant or affinity is the dissociation constant divided by the on-rate or the association constant. So it's the reciprocal of those two parameters. Typically when we're actually doing a global kinetic set of data, we're not directly calculating the KD or extrapolating it from the off-rate and the on-rate parameter which are actually in the model.

• Therapeutic Antibody Screening

0:12:38.0 DB: And so why are we interested in this property in terms of therapeutic antibody screening? And that is that if you have an antibody that has a fast on-rate, it can have an increased speed of onset of action. So that can be the driving factor of efficacy and affect various other pharmacodynamic properties of how the antibody will function. Also, antibodies with very slow dissociation kinetics can lead to a long duration of effect. There's cases where antibodies continue to show physiological effects well after the serum levels of the drugs have dropped below a time where they should be able to act because they're still residing on whatever their target is in the body. Also, overall, high affinity antibodies can reduce dosing requirements. So if you have a lower demand for serum concentrations of drugs based on affinity, then you can achieve that physiological effect at lower concentration.

0:13:45.1 DB: Now, we're gonna go and do an example of a capture kinetics assay. And this is a very standard assay format for us to run on the LSA. Some of the details are here. So we made a anti-human IgG Fc lawn surface, meaning that we used the single flow cell to create a capture surface over the entire array area using this polyclonal anti-Fc antibody and used 13 micrograms of that antibody to prepare that surface. And then we capture the antibodies to the array. And these can either be purified, diluted antibodies like they were in this case, or they could be supernatants. Either way, we used well less than a microgram of each antibody to perform this capture step. And then after the array is created, we do a titration series of antigen. In this case, it was a PD-1, which is a 17-kilodalton protein. And to do a eight-point dilution series starting at 1 micromolar and going down to 450 picomolar, we used a total of 7 micrograms of antigen, so it's incredibly antigen efficient for the amount of data we collect. And the LSA has three 384-well plate positions in it, so if you're doing capture kinetics, you can run that on up to 1152 samples.

0:15:18.5 DB: So this is what we think high throughput kinetics should look like. This is a 384-antibody array shown. In this case, we only had about 40 antibodies, so each one is spotted in 8 to 12 replicates. And this is the data from the experiment I just described. So this was set up in an afternoon, ran overnight, eight concentrations of antigen in a 3-fold series that used 7 micrograms of antigen. If we zoom in on that a bit, we could highlight one of the features of the software. So it's very common that people will report rate constants traditionally in SPR that are potentially inaccurate or have some unflagged liabilities. We've tried to reduce that a lot with the LSA Kinetics software, where it flags various things for you automatically. So in gray are shown clones where the total binding signal observed was below a certain threshold, so they're considered non-binders and their rate constants will not be reported in the data tables. You don't have to manually exclude those. Also, things that have poor fit where the standard deviation of the residuals is beyond a certain cut-off you could specify, they're flagged as yellow here, and it's something you need to consider whether you wanna use those rate constant values.

• If The Antibodies are Hitting Kinetic Limitations of the Assay

0:16:48.5 DB: Also, if the antibodies are hitting kinetic limitations of the assay, they are flagged. So in this case, there's two different cases, the top one being an antibody that has a very slow dissociation where we essentially didn't have a fit table dissociation profile over the 15-minute time period this data was collected, and therefore, it's flagging it as being below a limit. The default limit in the software is one times 10 to the minus five for the KD, which is pretty reasonable for most screening assays, and so it will flag those and here they're shown as purple and report the actual affinity as a limit function, say, less than 50 picomolar, for example. You really don't have enough data to support an analysis that's finer than that. And then the other case here is shown with an antibody where we didn't inject a high enough concentration of antigen to accurately determine the KD. So based on the model, it doesn't think we've injected a concentration of antigen over the KD, therefore, we may not have enough association rate data to accurately predict it, so it will flag that for us as well. It will report a rate constant, but it flags it as or something for us to be aware of.

0:18:06.9 DB: So this slide shows one clone that was spotted at 12 locations on the array from three slightly different dilutions, and it shows that one of the powers of the array is that you can generate highly replicated data and determine very accurate rate constants based on higher confidence from these multiple replicates. Also, this shows that the data is equivalent from various locations in the array. These 12 replicates were not right next to each other in the array. They were actually at several different distinct positions within the array, so it shows that the data is very consistent across the array. And then one feature in the analysis software is that it automatically has a statistics page that will automatically calculate the mean and the standard deviation for all of the rate constants and the affinity and the Rmaxs as well.

•  Another Advantage of This Assay Format

0:19:05.6 DB: So another advantage of this assay format is that when we do a broad concentration series, like for us, the standard is to start at about 1 micromolar then do this 8.3-fold serum dilution, so we're covering a very broad concentration range. And that gives us a really broad range of sensitivity and measure kinetics for different antibodies. So there's about, in this case, there's about a 20,000-fold range we were able to describe where you can see, for the high affinity antibody on the top...

0:19:38.0 DB: Really, we probably could have omitted the top three concentrations of antigen and gotten essentially the same kinetic result, but for the lower affinity antibodies, that 145-nanomolar antibody affinity down below, we needed every one of those high concentrations. And so oftentimes when you're doing this type of biosensor analysis, you need to kind of do optimization experiments where you pick your smaller subset of concentrations and even hope they apply to the whole set or maybe sometimes go back and run the optimized concentration series for individual clones because doing everything at a really high concentration and over a broad range would use too much antigen and take too long, but on the LSA, that's really not a trade-off, because everything's analyzed in parallel. That one single injection volume of that high concentration gives you data to everything. You can apply other generalized approach for these series and a very robust one.

0:20:40.2 DB: So one of the great visualization tools in the software is the iso-affinity plot. This is a great way to view kinetic diversity within your panel of antibodies. If you're not familiar with the plot, the way this works is that we have the off-rate on the X-axis and the on-rate on the Y-axis, and the diagonal lines that are shown as blue represent single affinity, single KDs. And so as you go towards the upper left, the antibodies are of higher affinity and they're lower affinity down to the lower right, and you can see the kinetic distribution within those groups. You can see here at the one times 10 to the minus five off-rate line, we have a number of clones that pegged against that axis. They hit that limit that we were applying.

• Collaboration Between Carterra

0:21:29.7 DB: So the data set I just went over is actually included in this recent publication of PLOS One, which was a collaboration between Carterra, Adimab, and Amgen, and we compared the affinity of these antibodies using the LSA, the Biacore 8K and two solution phase, the measurements using the KinExA and MSD assays. And so it's a great review. It's a great demonstration of how we measure kinetics on the LSA and how that compares to the Biacore. Essentially, you get the same answer on the equivalent chip type, but it also goes into some interesting details about transport dynamics using different chip chemistry. So I highly recommend this read and we thank our collaborators for this good work.

0:22:26.6 DB: So the next example in kinetics I wanna go over is an experiment we did where we measured very slow off-rate clones using very long dissociation times to three different antigens. So when you're measuring slow off-rates and you wanna collect in long dissociation times, you have to have a very rigorous approach to experimental design. Replication is very important, so you wanna have multiple blanks and multiple injections of the concentrations you are measuring to make sure that there is no drift or aberrations in the data that are skewing the off-rate that's being observed. This is really important for every biosensor, not just the LSA, as even a very small change in the slope of the curve when you're measuring something slow have a dramatic impact on both the estimated off-rate and the overall affinity. So in this case, we did a dissociation for an hour. You can see we did duplicates of two concentrations with two blanks in between each injection, and we were able to accurately determine the off-rate was between 1.5 and 1.7 times 10 to the minus five for this clone.

0:23:50.8 DB: So I'm gonna actually walk through the process now of how we set up this experiment. So in the Navigator software, so this is the instrument control software, we've tried to make everything wizard-based and color-coded to make it very easy and approachable and straightforward to set up an assay. And if you work with the LSA, you'll realize that actually the architecture of the system where you're either injecting 96 samples at a time or one sample at a time, allows for a really straightforward setup where you don't have to split samples across multiple levels and coordinate complex arrangements, against kind of this you place it somewhere obvious and that's where it uses it. It also color-codes things, so you can see the analyte, or sorry, the ligands in this case are orange, and that coordinates to this mock sensorgram down below where we have the injection times of the various savers. So to do this setup, we need three vials, one with the activation solution, we can do this EDC and Sulfo-NHS mixture, and then that would be the first step where we activate the chip. And then it will flow the antibodies. In this case, it's antibodies that were diluted in 10 millimolar sodium acetate pH 4.5 of various dilutions. Run for 10 minutes and then the single channel comes back onto the surface and we would do the blocking injection, which is ethanolamine or the quenching step. And then a regeneration cycle to a conditioned surface prior to the analysis.

•  Setup The Actual Kinetics Run

0:25:26.4 DB: Then to set up the actual kinetics run, it uses a similar wizard-based system where everything's color-coded. Again, we have our mock sensorgram here, where we have the baseline time, association, dissociation, regeneration condition, and stabilization phase. And then these are all of the samples you need. So we need one, two for regeneration in the sample block, so this is on the... These two plate positions up here are for the single needle side, and they hold usually a sample block in what we call Bay 1, and this has positions for 50-ml conical vials, 15-ml conical vials and 1.5-millimeter vial. And then there's a microplate position. Here we have a microplate that has wells that contain each of our two antigen concentrations for three antigens and a bunch of blank wells for doing blank injections. So once you've run that analysis, this is what you would see on the first page of the software, of the analysis software.

0:26:32.2 DB: So there is a table here called the main Legon table, and this is where you define the name of the antibody that's present on each spot or ROI. You can define them in different groups, so if you're spotting antibody, say, from two different sources or against two different targets, you can define them as a set, and then this table is single channel injections from the actual kinetic assay. So here we have concentration information and sample names, and again, these are groups, so in this case, we have three antigens with a few injections that have scrolled off, so we have antigen one, antigen two and antigen three and then they're all injected in duplicate sets, not in order. So we were doing one injection at 400 ANI molar and then tenfold dilutions to do another injection at 40 ANI molar. So usually, in the previous example, we recommend doing large dilution series, a really broad kinetic range because the main focus in this analysis was on off-rate, we chose to do replicates of two concentrations and we had three antigens. So this was about a 24-hour run, just with these injections, and that was about the most time we had for this experiment.

0:27:56.5 DB: So, once you have everything set up in this table the way you want it, and you're looking for groups to find, you can click on this batch-now tab here. And that brings you to this pane, and this is where we can use these bars to set the parameters that will perform essentially the entire analysis. So these shaded region on the right and left the crop are the regions that's in the shade that will be cropped out. The next pink line, you can see right here, is the y-alignment line, that is where it's going to zero the data to. Green bar is the injection start point. So when you're using this offer in this view you can zoom in to set that very accurately. The red bar is the injection end line, so that's where your association phase ends and it will assume the rest of it is dissociation. Once you've set all those up, you can click this start batch analysis button up at the top, when you click that, it runs and at that point it does the referencing, the blank subtraction and the kinetic fit all as one automated process. Depending on your computer and how complicated the assay is, that will run from about three seconds to 20 seconds to do that, and it will move you to the results page, in this case, its on the array view tab.

0:29:29.7 DB: And so this is on the kinetics page, so we need to find on the data page the anti-like and like in groups, that enabled us to have these tabs on the analysis. So what we're seeing here, each graph is one of the spots on the array and the different colored injections are the injections of the different antigens. And you can see we're on the all-tab of the analyte set, looking at antibody set too, so it works with tabs, so you can click on a tab and then just see the relevant data to that. Another feature of this array view, this was one of the most powerful views in the software, is you can set how many rows and columns you want for this display. If you set it to be too large, say you wanted to look at all 384 spots at once, it will display that, but you end up with relatively small figures and it's hard to really get a great sense of what you're seeing. So it's nice to be able tune and to scale things to a visually pleasing size, so say we're gonna click through the antigen tabs now to see how these different antigens compare. So if we click on antigen one, we can see just the antigen one as you are seeing but the raw data, which in this case is the red curve, and then the model fits, which is the black data. Then if you click on antigen two, you see the blue antigen two data then antigen three.

0:31:00.7 DB: So just looking at this, since we're looking at the same clones in the same lay out, you can see that this clone and duplicate here has very different kinetics to antigen two and antigen three. It's much more rapid to antigen one. So these kind of things can be easily visualized in the software. The other tab on the kinetics page is the kinetics-analysis-tab, and this is a view where you can really dig into the numbers and fit and adjust parameters. So if you highlight a row in the table, it will display the sensorgrams, the fits. This is a point where you could adjust the injection start and stop point if you want to, it also display the residuals, so this is the difference between a kinetic model and the observed data is shown here. Then you can also toggle a variety of fit parameters, you can change the minimum allowable KD limit. You can float parameters like you can add a book shift component, and you can even select a different fitting option, so you can fit either just the dissociation, you can fit what's shown here the KA and KD simultaneously, or you can have a mass transport coefficient to the fit. Again, like on the array view, it has the same analyte tabs, so there's antibody set one and two, and the analyte one, two and three are all.

• Way of Easily Generating Multiplex Data

0:32:30.2 DB: So it's a way of easily generating multiplex data, so let's say you immobilized antibody sets from two different projects and you really are only interested in kinetic analysis of the cognate antigen for that set. By just clicking set one, set two and then looking at the analyte here, you'll have a simplified table where you don't have to worry about comparisons. But then again, if you wanted to look at poly-specificity, you just can click across the other antigens or at all, and it will display all of them in the same table.

0:33:14.0 DB: So, to summarize here, we think the LSA has a major advance in high-throughput kinetic screening in characterization, it really enables people to do this analysis, and a much larger number of clones layer in there screening process and do it, very easily and with very low energy requirements. The ability to take, large numbers of crude samples say hybridoma supernatant for example and generate, a very detailed and complete kinetic characterization on the entire panel, it is game-changing and powerful. Not to mentioned it that reduces the amount of material required versus other methods, to do a 384 analysis example in published PD 1 example uses less than 1 percent of the sample that's required on the Biacore K and is about 50 times faster.

0:34:17.1 DB: Again the kinetic analysis we perform isn't just a crude screening, it's really a detailed, kinetic analysis to all of your clones and we do, full concentration series typically. We did a very robust picture of that and, the software that we use to analyze it is very powerful that these really excellent visualization approaches with array view and the, ISO affinity plot in different techniques. The statistics page that generates your means and standard deviations for all of your rate parameters of your replicates, there's the ability to generate scatter plots, for, any parameter that you've defined in the software you can plot them on the x and y axes for your, analysis. And also, once you've completed the entire analysis, you can export the whole thing to Microsoft Excel it's just the button that says export to Excel. And you have all the kinetic constant tables, all the figures we've created like the ISO affinity plot and the array view images. So that all come out in one simple summary which is great for electronic notebooks and sharing with your coworkers. I want to thank you all for attending this webinar, I hope it was useful, looking forward to answering your question. So yeah thank you and stay safe, here's my contact information if you want to reach out to me at dbedinger@carterrabio.com.

• LSA and SPR analysis 

0:35:48.2 DB: If any additional questions I'm always happy to talk about the LSA and SPR analysis in general, and for general inquiries can reach us at info@carterrabio.com. Thanks again and stay safe.

0:36:06.2 JM: Thank you Dan that was a great presentation.

0:36:08.9 DB: Thank you.

0:36:12.9 JM: We have some questions coming in, okay here's our first question. How low can RU, Rmax be? What are the rule on Rmax and are they the same as on the Biacore? 

0:36:29.1 DB: Yeah so, I feel very comfortable doing kinetic analysis in most cases on sense grams that are down to about 20 RU in height, and in general the rules are the same as the Biacore. I believe they kind of give a specific specification, I tend to think of it more as... It really depends on your system you know the, larger your antigen is and the faster the on rates are of your clones, the easier it is to elucidate the kinetic limitations that you can come into with mass transport effects. Which is the main benefit of mobilizing at low densities. So, one thing that's great about the LSA is it's very easy to immobilize things in titrations where you cover, a broad range of surface density. So that's kind of the ultimate test to see if you're having mass transport is if you immobilize something that, multiple densities over a broad range to look at the kinetic set to see if your apparent on-rate changes dramatically across then you know that you're having a mass transport limited system.

0:37:40.3 DB: If it doesn't really change then you know that you're in a kinetic zone or the diffusivity of your antigen is such that it's not causing that problem. So I found even with clones that seemed to have, a fairly significant mass transport limitations that are in the, mid ten to the six, on rates, with fairly large antigens. Once you get down to about, that 130/150 RU range, the kinetic behaviors much more uniform and that if you titrate up from there in terms of surface density, you'll see more of those effects. For other clones, you have a much bigger window as they have slower on-rates. Is the another question? 

•  Types of Samples can be Used for Antibody Kinetics

0:38:28.4 JM: Okay, we have another question, what types of samples can be used for antibody kinetics, for example can I use crude samples like bacteria lysate? 

0:38:40.2 DB: Yeah absolutely so we have a number of customers that use the LSA for screening crude primary samples. Bacterial periplasmic extracts, can be used we have customers that screen single chain, fragments that are v 5 tagged on them they make a high affinity anti v 5 capture surface. And then, can capture the periplasmic extracts with the SFC FU to the surface. The LSA is actually really ideal for that because of the way, it cycled the sample back and forth, with that very limited amount of sample you get from a PPE you can take about 100 micro liters and include a one-to-one and running buffer and then capture for 20 to 30 minutes onto the array. And end up with a very enrich surface even from the extremely low concentration sample. At which point, you can then go do fully for kinetic characterization of those. And the same is true for, hybridoma or other types of supernatant. We do find the bacterial periplasmic extract, tend to work better than and bacterial lysate.

•  Deal With Nonspecific Binding of Antigens

0:39:56.5 JM: Here's another question, how do you deal with nonspecific binding of antigens, does it frequently limit your ability to test certain systems? 

0:40:08.2 DB: No, not typically. In general, the chip hydrogel surfaces have relatively low levels of non-specific binding, there will be occasional proteins that have some interaction with the matrix, and do have non-specific binding. But it's almost never blocks us from being able to do the analysis. There's a number of approaches you can take to reduce non-specific binding, such as changing the ionic character of your running buffer by adding like, ammonium sulfate. You can change the chemistry that you use to quench the chip surface by using diethanolamine instead of ethanolamine, which reduces the charge of the surface. Simplest thing to do is just increase the concentration of DSA in the running buffer, for probably, the majority of cases that's sufficient to block non-specific binding. You can also add things like soluble dextran to the running buffer to try to sop up some of that non-specific interaction, so there's a variety of approaches to deal with those issues.

0:41:17.0 JM: We have time for one last question. Can you analyze kinetics to multimeric proteins like the SARS-CoV-2 spike protein? 

0:41:29.1 DB: Yeah. So kinetic analysis to multimeric proteins is always going to be more complex, obviously, than monomeric proteins. If you have a multivalent analyte, it may be impossible in some cases to fully eliminate avidity and to get things to behave completely in a one-to-one interaction format. With that being said, people do in our screening, the SARS-Covid-2 spike protein, again, kinetically with the LSA to antibodies, and I think maybe it's a bit more of a qualitative thing where you can clearly differentiate strong binders from weak binders. And things that bind very stably from things that have more dissociation, so there may be a bit of a qualitative aspect to it. Also, there's some approaches you can do like immobilizing things on a planar chip-type RCMD T-chip.

0:42:28.5 DB: If you do a low density immobilization of your antibody on a planar surface, the actual spacing of the molecules and their lack of flexibility in terms of orientation, and make it to where, it's relatively unlikely that the single molecule advantage in the pint to one antibody can be positioned, so that it will be able to bind another epitope to a different antibody on the surface. So you're sort of constraining the molecule and spacing them out. So often times, if you immobilized antibodies in the dilution series are a low density on these planar chip-types and you're injecting dimers or trimers. You can find conditions where the kinetics largely reverts to a one-to-one interaction. I think that's the approach people would take in that case.

0:43:23.5 JM: Thank you, Dan. We'll make sure to reach out to you, to answer all the questions that we weren't able to address today, and I encourage all of you to please register for upcoming webinar on SPR fundamentals. I will be sending you an invite for that shortly, and thank you again, Dan.

0:43:43.8 DB: Thank you, John. Thanks everybody.