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Lipid-based nanoparticles (LNPs) are effective non-viral vectors for delivering messenger RNA (mRNA) products, most notably used for production of vaccines against the recent SARS-CoV-2 pandemic.
In a recent live webinar, now available on demand,
Jérémie Parot, Research Scientist at SINTEF Industry, demonstrated a robust and high-throughput kit-based analysis of nucleic acids extracted from LNP-mRNA samples that were subjected to a series of stressing conditions.
During the Q&A portion of the webinar, we were unable to get to all the submitted questions. In this blog, I share answers from Jérémie and myself to the remaining questions to help you further improve your RNA analysis.
- You looked at a specific lipid composition for the LNP. Can you comment on how your methodology would perform with different lipid compositions?
In the study presented in the webinar, we looked at 2 different ionizable lipids. However, in addition to the data from the MC3 lipids discussed in the webinar, we also looked at the list of LNPs with different lipid components. We plan to present the complete study for the other LNPs later in the year. For other compositions of lipids and mRNA, we need to ensure that the extraction method works, and that the quantification is unaffected by RNA-lipid interactions. The preliminary results indicate the method has great platform potential. No changes have been needed for the analysis method so far.
- Did you have a control where you just used Triton but no heat? I am curious if Triton itself is enough for releasing the mRNA.
That is a very good point. Our focus during the study was to compare the integrity of the mRNA throughout different stressing conditions. The heat is likely unnecessary and was used to speed the extraction process. It could very well be that other extraction methods that do not use heat would also work. For other projects, we will continue investigating extraction methods to further improve sample preparation.
- I have a couple questions: 1) What are major factors that influence the baseline of the detection? 2) Is 0.3% the final concentration of Triton X-100 in the lysis buffer?
First, the baseline will mainly be influenced by degradation products and impurities. If users apply the RNA 9000 Purity & Integrity Kit from SCIEX, minimal interferences due to reagent or gel preparation should be observed, ensuring a low baseline. As with any sample preparation, users must apply stringent protocols and approved standard operating procedures (SOPs) to minimize sample degradation or the introduction of foreign particles into the sample, which could increase the baseline. Users are strongly encouraged to include positive and negative controls. In this case, the RNA 9000 ladder can be considered a positive control and the capillary electrophoresis (CE) grade water, provided in the kit, a negative control.Second, the concentration of the stock Triton X-100 lysis solution was 0.3%. The final concentration of Triton X-100 for extracting RNA from LNP-mRNA in our studies was 0.2%.
- I noticed in your electropherograms the overall signal for the LNP-mRNA is much lower than that of the free mRNA. Were the different samples prepared at the same concentration? Can you comment on this change in signal intensity?
That is a good observation. As described in our study design slide (#12), our main goal for this first study was to understand the integrity of the RNA in the LNP-mRNA over a series of stressing conditions. For the stressed LNP samples, the RNA concentration in the beginning of the experiment (prior to stressing) was the same, which allowed us to compare these samples and understand the RNA integrity under different conditions. As demonstrated by our results, we observed that RNA extracted from LNP-mRNA samples stored at 4ᵒC showed severe RNA damage. Samples that were stored at -80ᵒC showed a profile that was similar to the free mRNA or starting material.The differences in free mRNA compared to extracted mRNA after synthesis could be linked to a variety of factors. A main difference was that the free mRNA and the formulated mRNA had different concentrations. For the aim of comparing the integrity of the formulated mRNA and the purity profile, these differences were out of scope. For future experiments, we are thinking of using free mRNA that is as close as possible to the formulated mRNA in terms of concentrations.
- Have you considered trying different intercalating dyes to see if the amount of HMW product changes?
That’s a great point. We evaluated Orange G and SYBR Green II dye, which is a component of the ssDNA 100-R Kit from SCIEX. Between those 2 dyes, we did not observe large changes in the HMW products. Since SYBR Green II provided improved signal intensity and therefore assay sensitivity, we decided to use SYBR Green II in the assay and in the commercially available kit.
- How many different sample preparation procedures have you tested? How much better was Triton X-100 compared to other procedures?
We have tested more than 5 different sample preparations. So far, Triton X-100 offers the best results in terms of reproducibility, feasibility and extraction of the mRNA from the LNPs without affecting the RNA measurement.
- We noticed that if lipids are not removed from the LNPs prior to CE analysis, then the capillary life is decreased. Is that a common observation?
Based on our experience, the lipids do not negatively affect the CE measurement. We do not remove the lipids prior to injection onto the CE system as this step has a high risk of affecting the mRNA and related quantification. You might want to check other components you have introduced in your method, such as the detergent, which could potentially damage the capillary. Users should exercise caution with the type of solvents or solutions applied for the RNA extraction from LNP-mRNA, and with subsequent injections of these solvents into CE instruments in general.
- Can the RNA 9000 Purity & Integrity Kit be used for DNA-based therapeutics?
Yes, the kit can resolve single-stranded nucleic acid products in the range of 50–9,000 bases within a 30-minute separation run, and it works with both the PA 800 Plus system and the multi-capillary BioPhase 8800 system. Scientists and QC personnel can take advantage of this platform in R&D by using the high-throughput BioPhase 8800 system or the PA 800 Plus system for QC applications.
- After Triton incubation, do you remove the LNP components before injection in the equipment?
No, we do not remove the lipids before injection onto the CE system. For us, this step seems too complicated and comes with a high risk of damaging the mRNA. Moreover, based on our experience, the lipids do not seem to affect the CE measurement.
- While the LNP components will not affect the separation, will they affect the sample injection?
We have not observed that happening, and we are confident they do not affect the injection based on their size and properties.
- Did you have a control to confirm that all your LNPs had been destroyed by the detergent?
That is a great point. It is fairly complicated to have such a control, but it is well known that using 0.5x of Triton X-100 will destroy all LNPs. Currently, the most important question for us is whether the Triton concentration could affect the mRNA, mRNA measurement or the techniques used to quantify the mRNA. With the Triton concentrations we used, we haven’t seen such effects. For future studies, it makes sense to better understand mRNA extraction efficiency.
- What concentration range was covered in your linearity study?
We determined a linear dynamic range (LDR) of 97.7 ng/mL to 50E3 ng/mL. We also tested higher values, but found slight saturation with those, which led us to exclude higher concentrations in our curve.
- Has the RNA 9000 Purity & Integrity Kit been qualified on both the PA 800 Plus system and Biophase 8800 system?
Yes, this kit has been qualified on both instruments. To make it easier for scientists to implement it into their workflows, the kit is sold as a single part number that works on both platforms.
- Did you check the recovery of RNA after extraction from the LNP?
Assessing the recovery of RNA from formulated LNPs is not straightforward. To assess the RNA extraction efficiency, different extraction procedures can be used. Ideally, different analytical techniques to measure mRNA concentration, such as CE and LC-MS techniques, should also be compared. We have tried both aspects and found a good correlation between different quantitative methods. There is room for further experiments to verify whether the RNA extraction is at high efficiency across different lipid compositions and LNP concentrations, so that it can be used as a platform method. We aim to provide an update later this year.
- Can this analysis be performed using the PA 800 Plus system with the PDA detector?
Yes, it is possible to perform this analysis using the PA 800 Plus system with the PDA detector. We evaluated this approach using absorbance as a detecting method. The separation resolution is comparable between the 2 detector systems, while the sensitivity is 100–1,000 times lower compared to using the LIF detector.
- How long does it take to run about 10 samples, including sample preparation and analysis?
The length of the run time depends on the platform, which in this case would be the multi-capillary BioPhase 8800 system or the single-capillary PA 800 Plus system. When using the BioPhase 8800 system, setting up the reagent plates will vary from 5 minutes to 10 minutes depending on the number of samples. The sample preparation time takes around 10 minutes, which includes the time it takes to mix reagents, heat for 5 minutes and cool the sample. For LNP-mRNA samples in particular, a lysis buffer solution containing Triton X-100 is added for 20 minutes prior to utilizing the heat denaturation step. The run time is about 25 minutes per 8 samples on the BioPhase 8800 system or per 1 sample on the PA 800 Plus system.
- Slide #16 contained an overlay of free mRNA (red trace) compared to the LNP-mRNA complex digested using Triton X-100. Can you elaborate on the difference in response and the efficiency of Triton X-100 digestion? It appears the efficiency is somewhat low.
You are correct. The differences in the free mRNA compared to the extracted mRNA after synthesis could be linked to a variety of factors. A main difference is that the free mRNA and the formulated mRNA had different concentrations to begin with. For our aim of comparing the integrity of the formulated mRNA and the purity profile, this was not a major concern. For future experiments, we are thinking of using free mRNA that is as close as possible to the formulated mRNA in terms of concentration.
- Has the RNA 9000 Purity & Integrity Kit been used to qualify mRNA therapeutic modalities besides LNP-mRNA?
Yes, the RNA 9000 Purity & Integrity Kit has been used to characterize the CRISPR/Cas9 gene editing system. The kit was able to show the main product of the Cas9 mRNA and single-guide RNA (sgRNA) components of this gene editing system along with the nucleic acid impurities on both the PA 800 Plus system and the multi-capillary BioPhase 8800 system.
- Is SCIEX planning a higher-throughput instrument that accommodates the simultaneous analysis of 96 samples??
At this point in time, we are not yet planning a 96-channel CE instrument. Please feel free to reach out to your SCIEX representative to elaborate on your high-throughput needs.
- Can you explain why the mRNA sample at day 92 stored at 4°C also looks good?
It is known that LNP-mRNA samples can still be “good” after 3 months at 4°C, as was the case with our study. However, to be fully confident, additional tests are necessary to make sure that the transfection, for example, would still work as desired.
- Can you suggest an economical way to determine the mRNA encapsulation ratio?
Understanding how much of the mRNA was encapsulated is not an easy task. While different options exist to quantify RNA from LNPs with different characteristics—such as limit of detection (LOD), limit of quantification (LOQ), sensitivity, reproducibility and accuracy—determining how much was encapsulated compared to how much was present in the sample requires additional control experiments. We are investigating this question but cannot yet provide a definitive answer.
- From your unique perspective, what is the current status and near-future prediction of 1) nanomedicine, 2) nanotechnology in vivo imaging and 3) molecular nanotechnology?
That is a great question. It is unfortunate that we did not have time to address this during the Q&A after the live webinar, as this question is best answered as part of a panel discussion, which could easily take an hour. However, a series of great discussions is coming up that will dive deeper in that direction. Also, please feel free to reach out to SINTEF or your local SCIEX contacts if you want to hear more about the scientific work or how commercially available solutions might help with your specific research.
- Will the slides be available for review?
The webinar is now available on demand. In addition, the information is currently being summarized in a SCIEX technical note that will soon be available through SCIEX.com. Please feel free to reach out to your local SCIEX representative for further information.