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Rapid Viral Titer using Microfluidic Resistive Pulse Sensing

A presentation at ISEV Infectious Diseases 2021

…and here’s a transcript for your reading pleasure!

Hello, my name is Jean-Luc Fraikin, I am the CEO of Spectradyne. Thanks very much for tuning into my talk today. You have probably heard of our technology for EV quantification, but today I’m going to describe how Microfluidic Resistive Pulse Sensing (MRPS) delivers accurate and rapid measurements of virus concentration instead, far faster than using live biological titer.

Let’s start by reviewing why virus concentration is important in the first place. The reason is basically because virus concentration is a critical experimental variable and when you control it, you have tighter experimental outcomes. The value of this really shows up in two key areas.

First, when we are using virus as a therapeutic, so obviously gene therapy, for example. In these situations, concentration of the virus equals the dose of the drug. So it is a critical experimental variable, and it’s important to control it for safer, more effective products.

Second, when virus is being used as an assay readout, so, for example, diagnostic testing. In this case, the concentration is the test result itself, so the better you can measure it, the higher sensitivity and specificity you will have in your in your application.

So the bottom line is basically virus concentration is a critical variable that has to be controlled, and when it is, we end up with tighter experimental outcomes, and faster science. Which is good for everybody.

00:01:23 Overview of methods

So how is viral titer usually obtained? The gold standard is live biological titer. This is a tissue culture technique, and if you’ve ever done it, you know how painful it is. At best it takes hours to get your answer, but it can take as long as days and is very error prone. It requires many replicates as a result, and just the whole tissue culture infrastructure presents an enormous time and dollar cost also. So, we want to try to do better than this.

Optical particle analysis methods are challenged by this application because virus are weakly scattering biological antiparticles just like extracellular vesicles (EVs). Not to mention they live in polydisperse complex samples that easily overwhelm optical methods. So we need better than that too.

And here’s where Microfluidic Resisted Pulse Sensing, or MRPS, from Spectradyne comes in. It delivers fast and accurate concentration, and it is affordable and easy to use.

Our technology comes in the form of the nCS1TM. It is a benchtop instrument that delivers accurate concentration, starting around 50 nanometers diameter and up. It uses electrical detection, not optical techniques to measure the particles, and it leverages everything there is about microfluidics that makes it so exciting. It is easy to adopt, and because the microfluidic cartridges are single-use disposables they are contamination-free: You don’t have to clean anything in between measurements to avoid cross contamination. Compare that to working under a Bunsen burner for example, to do phage titer. One other bonus is that there are only three microliters of a sample required for analysis.

Let me give a quick plug for an upcoming webinar that will be hosted by SelectBIO that we will sponsor. It is called Fast and Accurate Virus Quantification Including SARS-CoV-2. This webinar will take place on Tuesday, February 9th 2021, at 11:00 AM Eastern Time. And we will have Dr. Zoltan Varga from the Research Center for Natural Sciences in Hungary speak and describe his measurements of SARS-CoV-2 using MRPS. You can register either by emailing us to get the link or to follow this registration link to go to the SelectBIO registration page.

00:03:40 How the nCS1 Works

Let me briefly describe how the nCS1 works in case you are not familiar with it. The technology is called Microfluidic Resistive Pulse Sensing (MRPS). Particles are counted one by one in this technique, and concentration and size are measured as directly as possible.

What we do is flow all the particles through a bottleneck in the fluid flow, through a constriction, and every time a particle passes through that bottleneck, we get an electrical signal that looks like this on the right side here.

So we measure two things about it. The size of the signal tells us how big the particle is, and then how quickly the particle went through the constriction tells us the volume of sample that we’ve measured. So with counts and volume measurements we can determine concentration correctly.

Here is a longer measurement, showing many particles being detected. You can see that there are a few different sizes of particles in here. You can read that directly off the histogram.

When we convert counts to concentration, the output looks like this. This is a plot with concentration on the Y axis and particle diameter on the X axis. You can see this sample contains 4 different populations of particle sizes. This was just a mixture of polystyrene beads, 50, 90, 120 and 150 nanometers in diameter, and this combination of resolution, accuracy and ease of use is just not available with any other method. At this point we have a lot of literature to help convince you of that.

00:05:04 Measurement Examples

Let me show you some measurement examples, but first I will give a plug for Dr. Zoltan Varga’s talk, in this session a little bit later at ISEV-Infectious Diseases. He willl be describing briefly the measurements of SARS-CoV-2 using MRPS. That will be at 2:50 PM Eastern and please tune into that, and if you want to learn more, join our webinar.

The first example I will show is a measurement of human adenovirus 5 and I wanted to start by just showing you what the raw data looks like. What we have is a baseline here and then every time a single particle goes through, we get an upward spike that is highlighted by a red dot, just like I showed you before. So here we are counting virions one by one-You can see your virus, in treal time, being detected.

Now when we convert that to a particle concentration versus size plot, this is what we get. In this case I am showing you three replicates of the same sample of the adenovirus 5 virus and we can see there is a clear, identifiable population from the virus in the sample, and there is high repeatability between the replicates.

And this measurement is quantitative over any size range. Of course, you are probably interested in measuring the concentration of the virus, but this technology also allows you to quantify off-peak particulates, which can be important depending on the application, which I will describe.

When we measure just the concentration over the size range of the virus, we see very accurate, reproducible measurements. And just as a reminder, this is absolute concentration measured in about 5 minutes per sample with no tissue culture required. So this was a super easy, you know, 15 minute measurement set.

00:06:48 Application 1

Next, let me describe what you can do with this technology. The first example is to assess purity of a virus preparation. In this case this is a commercial customer that is preparing viruses and we measured three different stages of purification. First was the crude preparation in dark blue at the bottom, and you can see there is no peak in the size distribution. There is a slight peak emerging after first step in purification and then after purification number two, you can see that the virus has been enriched and now stands out over the background in this sample. This is an incredibly important measurement for this customer because it allows them to tweak parameters of their purification processes to optimize the production of their materials.

00:07:28 Application 2

Finally, I want to highlight another key application of this technology: Because the measurements are so quick it allows you to gain essentially real time measurement of virus concentration for optimizing production. This measurement here is showing you a crude myxoma virus preparation. You can see the sample contains a broad background of random particles, probably cell culture debris and proteins, and there is a clear signature from the myxoma virus right where we expect it, around 260 nanometers diameter. When we zoom in on this, you can see there is a clear peak, and we can quantify just the virus in this sample as being 2 × 108 particles/ml. So what this allows you is in just five minutes to take a sample of your production sample, as you’re producing the virus and measure the tighter in a few minutes and then wait until the concentration is optimal and maximized before harvesting the virus. This is incredibly powerful value to this application.

00:08:32 nCS1 Performance

Finally, I’d like to show you a couple of slides describing the performance of the technology. This first example shows a retroviral drug formulation, a serial dilution. You can see the viruses are showing a clear peak in the distribution here, and as we plot the concentration as a function of dilution factor, we get a very linear response in the measurement. Last, I will show you the dynamic range of the technology. This shows measurement of a single sample ranging from about 65 nanometers up to five or six microns in diameter. You can see the concentration in this sample spans over 9 orders of magnitude within a single sample, and there’s just no other technology that can currently deliver this kind of measurement.

00:09:18 Diverse samples measured

Our customers measure all different kinds of samples of different viruses. If you don’t see yours on this list, then email us to learn more or send us a sample so we can show you what it looks like in high resolution.

00:09:29 Conclusion

In summary, Spectradyne’s technology delivers accurate viral titer in just a few minutes per measurement without any of the hassle of cell culture, and this allows you to do better science faster. Which is great!

Please contact us to learn more-We love demonstrating our technology so send us a sample to see for yourself. Thanks for your time, and I hope you enjoy the rest of the conference.