Read time: 8 minutes

Lipid-based nanoparticles (LNPs) are effective non-viral vectors for delivering messenger RNA (mRNA) products or other drug substances.

In a recent webinar, Jérémie Parot (PhD), Research Scientist at SINTEF Industry, demonstrated the robust and high-resolution kit-based analysis of nucleic acids extracted from LNPs using capillary electrophoresis (CE).

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 technical experts from SCIEX to the remaining questions to help you further improve your RNA analysis.

Sample preparation

What was the final concentration of Triton X-100 in your study?

The concentration of the Triton X-100 stock solution was 0.3% and the final concentration for extracting RNA from LNP-mRNA for the data presented in the webinar was 0.2%. You can find a titration experiment, in which Triton X-100 concentrations from 0% to 1% were tested, in this technical note. Generally, we recommend testing different concentrations for your specific formulation to ensure the best assay performance.

Have you evaluated detergents other than Triton X-100 and can you share your findings?

We have tested more than five different sample preparation procedures. So far, Triton X-100 offers the best results in terms of reproducibility, feasibility and extractability of the mRNA from the LNPs without negatively affecting the RNA measurement with CE. To our understanding, it is also the most frequently used detergent for mRNA extraction prior to CE measurements mentioned in peer-reviewed publications.

Can you comment on how your sample preparation performs with different lipid compositions?

In the webinar, we share data from two different ionizable lipids—MC3 and SM-102—used for encapsulating FLuc mRNA. However, in addition to these data, we also looked at LNPs with different lipid components (not shown). The preliminary results indicate that the method has great platform potential, meaning the mRNA extraction works reliably and the quantitation is unaffected by RNA-lipid interactions. So far, no adjustments were needed for the sample preparation and analysis. However, we strongly recommend testing different detergent concentrations, heating temperatures and incubation times for your specific formulation and mRNA to ensure the best assay performance. Some mRNAs might be less stable and need to be handled more carefully with regard to heat or might need more heat or longer heat exposure to avoid the formation of secondary structures that can result in additional peaks with slower migration time.

CE measurement and kit

Which major factors influence the baseline of the detection?

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. We strongly encourage users to include positive and negative controls and use 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. In this case, the ladder of the RNA 9000 Purity & Integrity kit can be considered a positive control and the CE grade water, provided in the kit, a negative control. In a  , scientists from Catalent were discussing how they improved the baseline for their measurements of mRNA. They improved their method and baseline stability by implementing pre-injection rinsing cycles, for instance.

Have you considered trying different intercalating dyes to understand if the detected amount of HMW product is dye-dependent?

We evaluated different dyes and did not observe notable changes in the detected amounts of HMW products. Since SYBR Green II provided improved signal intensity and, therefore, assay sensitivity over other tested dyes, SYBR Green II was used in the assay and in the commercially available RNA 9000 Purity & Integrity kit.

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 platforms, the PA 800 Plus system and the BioPhase 8800 system. To make it easier for scientists to implement the kit into their workflows, it is being sold as a single part number that is compatible with both platforms.

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 two detector systems, while the sensitivity is 100–1,000 higher using a LIF detector compared with a PDA detector.

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. We also applied the kit for the separation of linear and circular RNA products here.

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. With the kit, we were able to separate the main product of the Cas9 mRNA (~4500 nt) and the single-guide RNA (sgRNA, ~100 nt) from other nucleic acid impurities on both the PA 800 Plus system and the multi-capillary BioPhase 8800 system within one injection as described here. We also looked at separating circular RNAs from their linear precursors with success by using the kit, as described here.

How long does it take to run eight samples, including sample preparation and analysis time?

Analysis time: eight samples can be run in 25 minutes on the BioPhase 8800 system (3.125 min/sample), while one sample can be run per 25 minutes on the single-capillary PA 800 Plus system (25 min/sample).

Sample preparation time: for both platforms, the sample preparation will take approximately 25 minutes for eight samples. This includes time to mix reagents (5-10 min), heating and cooling the samples for 10 minutes, respectively and setting up reagents for the CE measurement (~5 min).

In our lab, we noticed issues with capillary lifetime if lipids were not removed prior to CE analysis. Can you comment on that?

Based on our experience, the lipids used for LNPs 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 quantitation. You might want to check other components you have introduced during your sample preparation, such as the detergent, that could potentially damage your capillary. We recommend exercising caution with regard to the type of solvents or solutions applied for the mRNA extraction from LNPs and subsequent injections of these solvents into CE instrumentation.

While the lipid components will not affect the mRNA separation, could they affect the sample injection?

We are confident the lipids will not affect the injection because of their size and physical-chemical properties. So far, we have not observed any effect indicating otherwise.

Study design

What concentration range was covered in your linearity study?

For the data presented in the webinar, a linear dynamic range (LDR) of 2.1 orders of magnitude, ranging from 0.0122 µg/mL to 1.56 µg/mL was established. The lower limit of detection was determined at 6.1E-3 µg/mL.

In the meantime, we performed additional experiments evaluating different buffers for the dilution of standards with the goal of assessing the total mRNA content and free mRNA and calculating a full/empty ratio. The standard curve to measure the total mRNA concentration in the mRNA-LNP samples ranged from 7.8–1,000 µg/mL, covering an LDR of 2.1 orders of magnitude. This standard curve covers the expected total mRNA concentration of ~400–600 µg/mL in a typical mRNA vaccine drug product. The other standard curve ranged from 0.78–100 µg/mL, also covering an LDR of 2.1 orders of magnitude. Details of the study design can be found in the technical note here.

The concentration range for the standard curve can be adjusted depending on the expected total mRNA concentration and encapsulation efficiency.

Did you perform a recovery experiment to understand the efficiency of your mRNA extraction procedure?

It is complicated to have such a control, especially when LNP formulation and CE measurements are not done in the same lab or in close proximity. However, it is well known that using Triton X-100 will disrupt and solubilize LNPs at the concentration used for our samples. Recently, the technical team at SCIEX looked at better understanding the sample preparation and the accuracy of the CE method in quantifying mRNA. You can find the study looking at the extraction here and assessing quantitative accuracy here. Further work to verify the recovery is being planned, and we hope to be able to provide updates later this year.

Did you evaluate the mRNA encapsulation ratio with CE?

Understanding how much of the mRNA was encapsulated is not an easy task. After the webinar went live, the SCIEX technical team worked on answering this question with a CE-based workflow. The findings were published in this technical note.