The Lunatic from Unchained Labs

Unchained Labs

I originally came across the Lunatic when investigating Quanterix’s flow cells. Quanterix flow cells are manufactured by ex-Sony DVD processing unit Stratec. Stratec also manufacture flow cells for the Lunatic, a UV-Vis spectrophotometer made by Unchained Labs. And the name alone was enough to make me want to investigate further. Unchained was recently acquired for $435 million by Carlyle. The company expects to generate $75M in 2021 and has 170 employees.

Unchained have a large portfolio of instruments. None of which I’ve ever heard of, it’s fascinating to me that companies like this can find relatively healthy niches while I assume having a relatively small market share.

Unchained has a storied history, having acquired Trinean, Freeslate, and AVIA Biosystems. They have a portfolio of products covering UV-Vis, scattering, Raman based particle identification, and lab automation. I get the general impression that their focus is on drug development, which might explain why I’ve not heard of them…

In this post, I wanted to delve into “The Lunatic” a little. This is a UV-Vis spectrophotometer which they position against the Nanodrop.

A Brief History of UV-Vis for Nucleic Acid Quantification

The Lunatic is a UV-Vis spectrophotometer and appears to be marketed against the Nanodrop as a tool for detecting impurities in nucleic acids. The basic UV-Vis method looks at nucleic acid absorbance at 260nm and compares this to compares this to the absorbance from proteins and other contaminates peaking at 280nm.

260nm is obviously way down in the UV range which means you need a UV capable spectrophotometer. This implies a UV light source, and UV compatible optics (fused silica) making the instrument more complex, and expensive.

Typically these A260/A280 measurements would be taken using a UV-Vis spectrophotometer such as the Beckman DU530 (which I’ve been tearing apart on my blog). These instruments use light sources covering the visible and UV range. A grating splits this out and a slit is used to select a small range of wavelengths. The grating therefore has to physically move so as to direct a single part of the spectrum toward the slit.

The instrument will scan across wavelength passing the monochromatic light through the sample and measuring absorbance. This is detected with a single sensing element (a photodiode):

Scans take some time, because a stepper has to physically move the grating to select a wavelength. The photodiode then registers the amount of light absorbed one wavelength at a time. These instruments have a few issues, scans can take more than a minute, and you generally need at least 100ul of material.

The Nanodrop provides a solution to these issues, requiring only 1ul of material, and giving a readout in a few seconds. It does this by using a different architecture:

Essentially spread spectrum light is sent through the sample, some is absorbed and the remaining light comes out the other side. Only then is a grating used to split the light and the absorbance spectrum measured. The spectra is registered on a linear image sensor (CCD, or potentially a CMOS sensor in newer instruments). No moving parts are required, and measurement is near instantaneous.

Rather than using a cuvette (sample container) on the Nanodrop the sample is directly sandwiched between two fiber optic cables, which allows for very low sample volume:

For general purpose UV-Vis applications, this approach likely has a number of disadvantages, but for nucleic acid quantification it appears to work just fine.

The Nanodrop doesn’t require any consumables, and from what I understand is a pretty robust instrument, rarely requiring servicing. 

The Lunatic

The Lunatic sets itself up as an alternative to the Nanodrop. This is a microfluidic chip based system which performs a similar function to the Nanodrop, and similarly is marketed toward nucleic acid quantification:

There are different chips available for the Lunatic, but they all essentially draw the solution into a detection region. As far as I can tell there’s no active fluidics in this system, it’s all driven by capillary action:

Unchained labs suggest that the Lunatic has similar sensitivity and reproducibility to the NanoDrop. But that the Nanodrop shows significant variation between instruments:

In some ways this feels like a calibration issue, in that the Nanodrops are showing the necessary reproducibility, but that they are not typically calibrated to the same extent as the Lunatic. It’s also not clear to me that the Nanodrop is the right instrument to compare the Lunatic with, perhaps Denovix who position themselves as a higher sensitivity alternative might make a more interesting comparison. The Lunatic does appear to have a higher dynamic range than the Nanodrop, and this maybe useful in some scenarios.

The major disadvantage of the Lunatic of course is that you need to buy chips to use it. From what I can tell, this works out at about $4 per sample. The chip likely as some advantage in terms of ease of use and clean up, but overall I find it hard to see a strong advantage over the Nanodrop and other platforms.

But with this instrument being marketed toward drug discovery, perhaps even a small edge over the Nanodrop coupled with a healthy portfolio of instruments, is enough.

A quick look at some Theranos patents

This post previously appeared on the substack.

I’d guess by now everyone in Biotech has heard of Theranos and John Carreyrou’s excellent book on the company. John now has a podcast on the trial currently underway which I’ve been following with interest. I suspect that anyone who has worked in Biotech will recognize the personality types and dynamics at play. In any case, it’s fascinating to see things play out from this unique perspective.

Recent episodes have discussed Theranos’ use of modified Siemens ADVIA instruments. The claim is that Theranos were saying they were using their own instruments, but actually just processing finger stick samples on modified Siemens ADVIA systems. In defense of this position, some seem to argue that the ADVIA modifications were a significant trade secret, which they only wished to divulged when the patents covering their modifications were published.

So, while it’s not really my area of expertise, I decided to go and have a look, and generally have a poke around in Theranos’ patent portfolio.

The first patent I came across that mentioned the ADVIA system, really just seems to say “you could use a smaller sample container”. It has a single independent claim:

So you can put inserts into the sample container to make it smaller and it can have a angled floor. The other claims are mostly around the cavity geometry and coupling the container to a sensor… the patent doesn’t explicitly discuss the ADVIA (there are no examples). But it does say that assays may be performed on commercial equipment including instruments from Siemens and the ADVIA specifically.

Overall, it’s difficult for me to see how the above patent is novel.

Other patents mention that the ADVIA was used for reference measurements. But beyond this I couldn’t see other references to the ADVIA…

But I decided to poke around a little further and found one or two other interesting things including a patent that focuses on the use of their instrumentation in space, leading with:

“Open top test tube and sample containment will encounter challenges to work effectively in the weightless or microgravity environment found on a space station or on a spacecraft”

Other patents are full of hundreds of exciting pictures of the mechanical system, or discuss testing for Zika virus. But nothing jumped out as particularly novel.

One patent that did seem a bit more interesting was this one. Here they describe an approach called TOSCA (two-step chemiluminescent enzyme immunoassay). They present this as an alternative to regular competitive binding assays. This method “eliminates the need to mix a conjugate and a sample before exposing the mixture to an antibody, which may be desirable when very small volumes of sample and conjugate are used”

Essentially this is a minor modification to the competitive binding approach. In the competitive approach, antibodies are attached to a support (surface). You flow in a labeled Antigen along with your sample. If your antigen of interest in present in the sample, it will compete with the labeled antigen. This results in reduced signal at higher antigen concentration.

The Theranos TOSCA approach is exactly the same, but you flow in your sample first and the labels second. This should give antigens in the sample a better chance of binding to antibodies. I guess the idea is that at low concentration you have more antibodies available than antigens in the sample. So you try and bind all sample antigens you can. Only then do you flow in a labeled antigen to “fill in” any antibody sites which are not occupied.

They show data where the TOSCA approach appears to show stronger signal:

Later in the patent they discuss calibration approaches, for example using a known spike-in. These plots show sensitivity down to about 10 pg/ml. Beyond this response looks pretty linear to me. Elsewhere they describe “low concentration” as being in the 10 pg/ml range. So this doesn’t appear to be an approach that in designed to be competitive with some of the fg/ml detection approaches.

There are hundred of patents to look at, but in my brief search I didn’t come across anything I found hugely exciting. Still, I’ll be be keeping all this in mind as I continue to listen to reports on this fascinating case.

Mesoscale Diagnostics

This post previously appeared on the substack.

I initially came across Mesoscale when looking at Quanterix. Like Quanterix, Mesoscale make very high sensitivity claims for their immunoassay, in the fg/ml range. Mesoscale Diagnostics has also been around for much longer that Quanterix having been founded in 1995. They are reported to have an annual revenue of ~$100M

In particular I came across Mesoscale, because they show very 1 fg/ml sensitivity on HIV p24:

Mesoscale HIV p24 detection from here.

These results are similar to the 1 fg/ml results shown by Quanterix, which suggest that Quanterix is not unique in their sensitivity claims.

The Mesoscale approach uses electrochemiluminescence (ECL) to detect antibodies. Here we’re electrically initiating a chemical reaction which will emit light. We can imagine this has certain advantages over fluorescence. In particular we don’t have any background fluorescence to deal with, or any excitation illumination to filter. ECL has in fact been using for single molecule super-resolution imaging. Though I imagine that with each reaction only emitting a single photon, the advantage of this over other super resolution approaches is limited.

If you’re familiar with Roche’s Cobas e411 and this all sounds somewhat familiar that’s probably because they use the same basic approach. Mesoscale successfully sued Roche for infringing their patents in a legal situation that seems both fascinating and horribly complex

Interestingly, there’s a teardown of a Mesoscale instrument on YouTube. The instrument uses a fairly typical 2000s vintage Princeton/Roper cooled CCD camera with 20 micron pixels. Comments on this video were also fascinating, indicating the the plates cost ~$1000. As these plates require embedded electrodes, we would expect them to be a high cost consumable. But this still seems rather expensive…

Comments also suggest that “multiple competitors…can outperform and do assays cheaper” and “Bio-rad’s multiplex Elisa is pretty much as good”. The Bio-rad kits I could find however only report sensitivity down to ~5pg/mL on HIV p24 which is an order of magnitude worse than Mesoscale. But of course for many other assay’s like Quanterix the sensitivity is likely limited by available antibodies in any case.

For the most part I was interested in looking at Mesoscale to see how unique Quanterix/Simoa is. In terms of raw sensitivity, Quanterix does not appear to be unique. Mesoscale platform is showing similar single digit fg/ml sensitivity. The Roche Cobas e411 p24 assay also gets close, with sensitivity in the 60 fg/ml range. 

It seems likely that Quanterix has a cost advantage over Mesoscale. But Cobas e411 tests in the range of $20 appear to be available. It’s therefore possible that Quanterix may still have an advantage here if they can show very high sensitivity at low cost.

SomaLogic

This post previously appeared on the substack.

Somalogic are developing a aptamer based protein detection platform with a microarray-like readout. Aptamers have been around since the 1990s, the basic methods being far older still. The basic idea is that you evolve a DNA or RNA sequence that will specifically bind to a protein of interest. Aptamer’s have a troubled history and I’m not really aware of many successful commercial applications based on aptamer approaches.

Somalogic claim to have solved the aptamer problem by adding 4 additional modified nucleotides to the existing natural set. The basic method was published in 2010. This gives them 8 basic monomers to work with (still a lot less than 20+ amino acids, but in the ballpark of the ~10 that have been proposed as a minimally viable set).

The heart of this paper shows that they can find aptamers for various proteins using modified nucleotides where they previously couldn’t:

Kd values here range from 10⁻⁸ to ~10⁻¹⁰ (smaller is better). This in the range of high affinity antibodies (but not very high). These values also compare well to those for regular aptamers, for example these in with 10⁻⁸ to 10⁻⁹ range.

However even antibodies have a relatively high false positive rate (for example, 0.4% for COVID19 tests). A false positive rate of 1 in 250 may not seem like much. But when you’re looking at ~10000 proteins, you’ll get 40 false positives. Of course, this doesn’t exactly map to the Somalogic approach where you’re looking to quantify expression levels rather than obtaining a yes/no answer.

So, particularly coming from a background in genomics, the Somalogic readout is likely to be noisy. This is likely the case for any large-scale aptamer/antibody platform. Olink, by linking two affinity reagents may be able to reduce this. Nautilus, by using multiple affinity reagents sequentially, may also have a route to a less noisy readout.

But what Somalogic may have, is more resolution on their readout. Unlike Olink and Nautilus, the Somalogic platform doesn’t appear to be using a single molecule approach.

Essentially they fish out the aptamers that bound, and then pull these down onto a microarray like platform (hybridizing the aptamers to probes on the surface). Microarray’s (and hybridization) always seems like a process fraught with issues, with environmental issues (like temperature and ozone) effecting results. All issues, you rarely see with sequencing.

So, there’s potential for binding issues between aptamer-protein and with the aptamer-array. The upside is that the array gives you a large dynamic range (I believe they suggest 12 orders of magnitude). So rather than having to readout billions of molecules (as Nautilus or Olink might) you can get this information from the Somalogic platform by imaging each protein (aptamer type) once. I.e. an array with on the order of 10,000 spots. A fairly small (and likely cheap) array by DNA microarray standards.

Final Thoughts

Somalogics secret sauce seems to be in their modified nucleotide aptamers. I would guess at best these are one order of magnitude better than regular aptamers. So you can make the argument (like Nautilus) that you could just use a couple of lower affinity aptamers rather than one slightly higher affinity one.

Aptamers do give Somalogic a natural way to generate a high dynamic range readout (using microarrays). That’s neat, but brings its own issues. And overall, I suspect compensating for artifacts may be harder than in single molecule platforms (like Nautilus and Olink).

All these platforms are likely to have issues with binding affinity, background signal, and other environmental factors.

These are issues that a true protein sequencing platform could potentially overcome.