SCIEX
Duration: 26:03 Min
Comprehensive mRNA-LNP Characterization with Capillary Electrophoresis & Mass Spectrometry
Transcript
0:01
Hey. Hello, everyone. My name is Fang. I'm going to show multiple different workflows regarding
0:34
the mRNA lipid nanoparticle characterizations by both capillary electrophoresis and mass spectrometry.
0:42
Thanks, right, we have heard. We have got a very detailed CQA characterization for the
0:48
mRNA part, but as a comprehensive – I don't know why it's running automatically – but
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compared – Sayegh, as a comprehensive analytical solution provider, we essentially provide,
1:01
at least here, right, demonstration of the different concepts to the entire workflow
1:06
for the mRNA lipid nanoparticles from plasmid DNA characterization, right, that's the raw materials,
1:12
and to the IVT-expressed mRNA integrity and encapsulation efficiency. And different
1:18
mRNA CQAs, I will touch a little bit upon, again, the poly(A) and 5' capping efficiency using an
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orthogonal method. And then the other part of the lipid nanoparticles, the ionizable lipids,
1:32
which is another core component to make a successful and – sorry, why is it running – and
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to make a successful and stable mRNA lipid nanoparticles. And last, right, what happens
1:47
after those mRNA lipid nanoparticles are injected into the patient, right, the MAD-ID,
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right, post-administration tracking. And we also have a concept to – a proof concept workflow to
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track what happens after the ingestion. So, without further ado, let's dive onto the first part,
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right, the raw material, plasmid DNA. And plasmid DNA is a critical starting point
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for protein productions, mRNA, and also some viral vector products. And here we essentially
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are going to showcase a brand-new kit, our chemistry workflow, DNA 20 kb plasmid and linear
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kit that's actually only launched last month. It is using intercalation dye for the detection
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and is compatible with our single-platform – single-capillary platform legacy P100 plus
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system and also the multi-capillary BioPhase 8800 system. So, we have a miniature version of the
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BioPhase 8800 on the booth, so feel free to check out that. And because we're using LIF detection,
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the sensitivity can be very good. It goes down to on the nanogram per microliter – or picogram
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per microliter scale. And when it comes to plasmid analysis, one of the key points is not
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actually the sizing, but rather the topology distribution. How much supercoil, open-circular,
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or linear, or the aggregates of those supercoils exist in our sample. So, with this new kit,
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we're able to demonstrate with a single-platform method that we can analyze plasmids ranging from
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2 to 20 kilobases with the same method for a high-throughput environment. With this method,
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we can separate the supercoiled, open-circular, and linear species, or sometimes the multiples
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of the supercoiled if they exist in the sample in this one-platform method. So, this is more for
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high-throughput. I have one method to run all kinds of plasmids. If we do need, as if needed,
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a calculation is needed, a higher resolution is needed, we can always optimize the injection
4:03
volume, the separation voltage, and sample concentration to achieve a more suitable method for the
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sample type. So, there's a lot of area for the optimization, too. So, it depends
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on the environment you have. It depends on the sample requirement you have. The method can be
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very flexible. But it's one ready-to-use kit. Another thing, right, we have the supercoiled
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plasmids. And before it actually can be used either for mRNA IVT or protein production,
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it needs to be linearized. And then, the next step for the plasmid DNA acquisition will be,
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after linearizing, is my reaction complete? Do I have other fragments that I missed during
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the initial assessment? So, the same kit can also be used for linear DNA quantification.
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And if we have that running the same method, it also helps us to identify some of the peaks
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in the original plasmid samples. So, by aligning the migration time, and, of course, if we use
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a ladder together with it, we can determine or estimate the size of our linearized mRNA.
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This is not a full mass spectrometry or sequencing, but it gives us a rough estimate, oh, am I having
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the right product, roughly? And the linear DNA resolution is also very tunable. It depends on
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the capillary length, right? In this particular case, it's showing a 30-centimeter capillary.
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If we use the same material, the same ladder, but running a slightly longer capillary,
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the 50-centimeter-long capillary, we can achieve basically baseline resolution even between 7,000
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and 8,000 base pairs. And in this particular case, the plasmid we used and linearized is 7.8
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kilobases. It sits right in between the 7,000 and 8,000 base markers. So, that's linear.
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And another thing, right, we have known from both regulatory and previous studies that
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host genome DNA can be critical in the sample. If we have too high or too big or too small,
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then it causes problems later on. So, because we have a high-resolution size separation
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and the LIF detection provides a very high-sensitivity quantitation method,
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with these two coupled, we're able to actually do a host genome size analysis of a sample,
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like how much is left and what size are those host genome DNAs. And because the cutoff is 200,
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right, by spiking a 200-base pair marker in the sample, we can clearly see how much below
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200-base pair and how much above 200-base pair. Another thing is this separation is
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sequence-independent. We don't need to know the sequence. We don't need to design a product or
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cell-specific primer or targeting to understand and determine the quantitation of a host genome's
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DNA. So, that's about plasmid DNA. And moving on to mRNA, do we have the right product being produced
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during the IVT process, right? Then the BioPhase ADN-100 system or the single capillary P100
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system, when it's coupled with RNA 9000 purity and integrity kit, it allows us to use a CGT-LIF,
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so against the LIF-based detection, to estimate the mRNA size when using it with a marker,
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to quantify the intact mRNA amount or percentage purity. Or another thing is if we run a standard
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curve, we can actually also determine or estimate the absolute quantification of the mRNA, and with
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that data, by running a degraded sample and intact sample, we can even estimate the percentage
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encapsulation efficiency. So, for encapsulation efficiency, right, because that's the ultimate
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final drug product in one of the CQAs, and the common method will be the ribonuclease assay.
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Following the same sort of philosophy and workflow as the ribonuclease assay, we do a
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serious dilution of our intended mRNA to generate a concentration versus signal standard curve,
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and then we run two samples, right? The total mRNA from a free, essentially degraded,
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mRNA sample and the free mRNA from formulated mRNA lipid nanoparticles. And because now we have the
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area counts or peak intensity from those two samples and calculate against our series diluted mRNA,
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we can determine the encapsulation efficiency as well as the integrity of the formulated mRNA
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versus the formulated lipid nanoparticles to get some insights on that as well.
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Thank you.
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and standard curve, we can have a microgram per mL or milligram per mL concentration for the mRNA
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in both situations, and with a mathematical conversion, right, the total mRNA minus the free
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mRNA divided by the total, the exact same calculation that we follow in the RiboGrain assay,
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we can also calculate the percentage encapsulation efficiency. So, here's an example,
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right, for this particular case, and it demonstrates how we can use the ladder and
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this kit to estimate the size. The sample we are using here is a Firefly Luciferase mRNA,
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so Fluc mRNA, and the theoretical size for this mRNA is 1.9 kilobase. And with running the ladder
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and the sample in the same sequence, we can use the built-in algorithm in the BioPhase software
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to estimate the size of those peaks. And the main peak is our Fluc, intact Fluc mRNA,
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that's 1.9 kb as theoretical size, and we can average about 1.94 kb. And with a minor species
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that's about 100 base pairs less, and with the hypothesis identity for peak two is actually
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the tailless version of that mRNA, and because for this particular one we know the tail length
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for that mRNA. And number three is actually what we just observed is accurate for the mRNA,
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so that gives about 2.4 kb. And so, that demonstrates how we can use it for size
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discrimination. And then when we deform, right, the mRNA lipid nanoparticle with Triton X solution,
10:27
incubation, and then our SLS, so sample loading solution, those two will break up the lipid
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nanoparticle and also denature the mRNA to make them more straight. And then now we observe the
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single intact mRNA in the sample as the aggregate peak, right, is minimized. And with that,
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we are actually – so, this is, again, the purity assay for size discrimination. So,
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because it's a purity assay, another key component we typically look for in purity analysis assays is
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this method stability indicating. If I do have a bad sample or a stressed sample, can the assay
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tell me that this is a bad sample? So, what we did was actually incubate the mRNA lipid nanoparticle
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at 37 degrees for two days and five days, respectively, and then doing the same denatured
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breakup, right, deformed mRNA lipid nanoparticle on the same analysis, and we can clearly see two
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distinctive peaks, the 0.9 kb and the 1.1 kb show up in the fragment site. And as we get longer and
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longer in the heat stress compared to two days and five days, we also saw these two peaks increase.
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And with the total quantification, right, for intact lipid nanoparticles, the control sample,
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which is always stored at minus 80 degrees, will have 92 percent intact purity. As we heat
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stress the sample, we can see the intact percentage decrease significantly, and those two are the
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fragments we were able to identify from the control to the stressed sample. So, it qualified
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it as a good purity indicating method because it tells different stability. Now, compare those
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two samples again, right, coming back, we have that standard curve, and we're able to add in
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all these peaks together because we do believe those are still intact mRNA, free mRNA in there.
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So, the free mRNA in the sample is 21 micrograms per mL. The total mRNA after the denaturing
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comes back 436 micrograms per mL. Using that calculation, it gave us an encapsulation efficiency
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of 95 percent. To demonstrate, right, if this is another orthogonal method that we can rely on,
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while we're doing mRNA integrity assay, just adding a standard curve, we can get additional
13:02
quality attributes. We actually ran the RiboGrain assay, which is considered to be the gold standard,
13:08
the go-to method, right, for encapsulation efficiency. Based on that assay, this sample
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gives about 92 percent encapsulation efficiency, and with the CE method, it's 95 percent. So,
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it actually correlates pretty well, and we actually ran some other stressed samples with
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30 and 70 different encapsulation efficiency with RiboGrain, and it came back around 40 and
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70 percent, too. So, it correlates well, even though it's not an exact number match between the
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two assays. So, moving on, for the poly(A)-tail and 5'-perm capping, I think we just heard fantastic
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workflows on that, so I'm not going to dive too much. And what CE provides is an orthogonal method,
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and what we're able to show is, for the poly(A)-tail, after the same digestion we just heard,
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is we can do a single nucleotide resolution from 9 nucleotides all the way up to actually
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160 nucleotides. That's what we were able to demonstrate. If we have a sample to go even further,
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maybe we can also do that, right? But for this particular sample, it has a 120-nucleotide tail,
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theoretically, and this is the poly(A)-tail distribution we were able to observe.
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The center is located at 120-121 nucleotides, as expected, and then we saw almost nominal
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distribution from 97 all the way out to 156. All of them are single base resolution, so we can group
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them for quantification, or we can report the individual nucleotide distribution in that
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poly(A)-tail. And then the capping and uncapping, right? Because this CE workflow provides single
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nucleotide resolution, and between the capped and uncapped nucleotide or digestive product,
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there's also one nucleotide difference. So, we are also able to separate the uncapping
15:09
versus the capping structures. But if we do want to know exactly what is the structure
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for the uncapped species, right? Is it 2-phosphate, 3-phosphate, or all that modification,
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we still need to rely on mass spectrometry. So, this is just another way to monitor the sample.
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It's not intended to replace any deep characterization method. If we need to know that information,
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we still need to go back to LC-MS using the phenyl column and also the non-TOF 7600
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mass spectrometry. So, this is just another orthogonal method. So, enough of the mRNA core, and then
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moving on to the lipids. There's generally four types of lipids involved in making a lipid
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nanoparticle, right? The structure, the cholesterol in the ionized lipid, and the packed lipids.
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In here, we focused on, for the deep characterization, we focused on the ionizable
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lipids and using the ALC-0315 as an example because that's one of the popular ones that's
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being used, I think, in the COVID vaccine and a couple of other products and pipelines we worked
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with our collaborator. So, focusing on the ALC-0315, what I'm going to show is how can we
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use LC-MS workflow to do a very clear, right, unambiguous identification of the impurity in
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that sample, and also how can we monitor the quantification of that lipid. So, here is just
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one example to begin with. In here, this shows the ALC-0315 structure. So, this particular part,
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is the head group, and that's the tertiary amine that generally can be oxidized and cause big
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problems, right? Another thing to keep in mind is the ionizable lipids are 100 percent synthesized.
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So, with that in mind, we generally, with a synthetic molecule, right, a lot of times it
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comes with a wealth of impurities that with small differences cannot be removed in a typical
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purification process. So, the lipid raw material control can be very critical, and
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quoted by our collaborators, right, that sometimes the quality of that ionizable lipid can actually
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determine the destiny of the mRNA lipid nanoparticle project. And with that in mind,
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when we look at the LC-MS, right, so what happens in this particular study is we formulated the
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ALC-0315 ionizable lipid and incubated it at 60 degrees Celsius as a heat stress,
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and for three days and five days. Then we run the control
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sample, the heat stress sample, in one chromatography sequence. And by comparing
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the difference and relying on the mass spectrometry for identification, we identified the 12.1 minutes,
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that's the normal, right, the pure ALC-0315. And as we can see, compare the control versus
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the stressed study, three or four, right, there's more down here that's much lower abundant,
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but these four peaks change relatively higher abundance. And based on MS1 identification,
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we're able to identify, okay, one is loss of SO group, and second is loss of the head group,
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third one is oxidation, and the fourth one is loss of alkyl chain. But then the question is,
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which one of them are the problematic one, right, PQA versus CQA? Which one is critical that we
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need to monitor and control tightly? Based on literature, we know oxidation can be problematic,
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but where is this oxidation happening? Is it on the N-terminal, which is the big one,
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where the corporates can be the huge problem for the mRNA lipid nanoparticle,
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because it's intact with the mRNA, and for the modified mRNA, causing loss of efficacy,
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or is it some other position on the chain that's less disruptive? And then for that, MS1 is not enough anymore,
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because no matter where the oxidation happens, it generates the same aspects. With the EAD,
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right, electron association dissociation… electron association dissociation fragmentation technology
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that's unique to the 7600 Xenoscope system, we're able to find unique fingerprints on the lipid product,
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the ALC-0315, and by piecing those fragments together, the software we use is molecular profiling,
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and that will spit out the ion formula based on M over Z ions, and then we're able to clearly
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identify which pieces, right, each one of the fragments correspond to, and confidently identify
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that the oxidation is happening actually on the N, the head group, which is probably a problematic
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one, and then we can continue to monitor and characterize that particular modification during
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the process of production or when we procure the material. And then another one to demonstrate
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the power of the workflow is we obtained, right, our collaborator actually got the ALC-0315
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and 9th bold lipids from three different vendors. We ran the same workflow and did a deep
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characterization of those raw materials, and we're clearly able to provide valuable information for them
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to make a decision, right, because as we can see, vendor two has the lowest purity percent,
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while vendor one provides 98.2 percent of the intact or intended molecules, and also when we
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look at the different modifications and impurities, they are actually ranging from 0.01 percent to
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0.3 percent, adding all of them together, reaching about 1.7 percent of the impurity.
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And I'm not showing the data here, but even with the 0.01 percent impurity, we're clearly seeing
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all the fingerprint ionization fragments to give us unambiguous identification of that particular
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modification. So, that's the lipid raw material, right? That gave us confidence to pick the right
22:07
material and to make the right lipid nanoparticles. But what happens afterward?
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Following the administration, lipid nanoparticles can travel to different parts of the body
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and undergo different metabolite changes, and frequently, right, ionizable lipids,
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because it's a non-endogenous cationic lipid, can actually be used as a surrogate for
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quantitative analysis of lipid nanoparticles in invaluable samples. Here, we are demonstrating
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by spiking the lipid nanoparticles into plasma samples and incubate them. It's not actually a
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clinical sample, but it's just a spiking matrix. And, again, we're using LC-MS, and the software
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is a molecule profiler for those metabolite identifications. And, right, again, as I mentioned
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previously before, there are four types of lipids to make a typical lipid nanoparticle. Here,
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it demonstrates the fragment, right, metabolite product that can be identified in this workflow,
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right, the cationic lipids. In this case, it's shown as ALC-0315. The sterol lipids in here is
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using cholesterol, standard cholesterol, and the helper lipids, and we're using phosphatidylcholine
23:30
in here with a 36-carbon chain. And then, the PAK lipids, that's the PAK2000. So,
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with the same workflow, we can identify all four kinds of lipids that we put into the lipid
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nanoparticles. And then, using the ALC-0315 as the surrogate, because cholesterol exists in the
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sample, too, and it's not easy to tease out which one is from the lipid nanoparticle,
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while the cationic lipids, because it's a non-endogenous one, we can follow the percentage.
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And in here, we essentially mix the lipid nanoparticles at different levels into the
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plasma. And then, after incubation, right, monitoring, mimicking that PKPD process,
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we're then using solid phase extraction to extract the lipids back out, essentially clean
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up the sample from the plasma, and using the LC-MS workflow to analyze it. And all the data
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points at each level of concentration were done in triplicate. And using the extracted ion that
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can be essentially identified or input in the processing method in the molecular profiler,
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we were able to quantify the different samples, and then we can see a very good linearity across
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four logs of concentration. When we have four logs of linearity, it basically indicates that
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we can quantify down to 0.1 percent with high confidence of these particular ionizable lipids.
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So, with that, essentially, we're able to showcase workflows, maybe not exactly the
25:16
sample you are working on, but generally, right, plasmid DNA calculations, mRNA integrity,
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encapsulation efficiency, structuralization of the mRNA, including poly(A)-tail and the capping
25:31
structures, and lipid impurity identification, and post-administration lipid nanoparticle
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monitoring. So, all this gathered, right, that's essentially provided on the three instruments,
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the single-capillary legacy P800 plus system, the multi-capillary BioPhase AD 800 system,
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and our Xenoscope 7600 mass spectrometry. So, those two, we actually have a miniature on our booth,
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and if you find any of the information interesting and helpful, feel free to stop by and find out
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more details. With that, I'll take any questions.