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In a recent webinar, available on demand, Jingtao Zhang (PhD), Scientific Director, and Daniel Turner, Scientist at Catalent^® Pharma Solutions, presented their approaches to addressing stability challenges of mRNA-based products.

In the presentation, they showed how lipid nanoparticles (LNPs), widely used vehicles for mRNA drug substances, were affected by pH, excipients and freeze/thaw (F/T) cycles. Furthermore, they provide insights on method optimization of their capillary gel electrophoresis (CGE) assay using the BioPhase 8800 system to assess mRNA integrity. Their capillary electrophoresis (CE) method is used for evaluating the stability of mRNA under different conditions to make informed decisions on formulation development.

In this blog, I summarize their answers to the most pressing questions to help you improve your mRNA-LNP analysis.

mRNA integrity analysis with CE

Can you provide details for the conditions of your pre-injection rinses?

Prior to starting a sample sequence, we perform an acid wash, a water wash and then fill the capillaries with gel. As part of our optimized method, we use 70 psi for 2 minutes for the acid wash, followed by a water wash at 70 psi for 2 minutes. To fill the capillaries with gel, we use 50 psi for 5 minutes. Once the capillaries are filled with gel, we run the separation method briefly—for 2 minutes—to pull out any small impurities before moving ahead to any samples. In addition to pre-injection rinses, we found it extremely important to use sample loading solution to dilute samples prior to injection.

Where can the sample loading solution be obtained from?

The sample loading solution is a catalogue item, which we obtained directly from SCIEX (P/N 608082). It helped us improve the peak shape and resolution of our mRNA samples and the RNA ladder. We use it to dilute samples instead of using water. It acts as a denaturant and prevents secondary structure formation.

Can you elaborate on your settings for the pressure injection of mRNA?

We used 1 psi for 5 seconds. However, you can adjust the duration depending on your needs. We have also used electrokinetic injection in the past, which applied -1.0 kV for 6 seconds to load samples into the capillaries. This method also worked well and required less sample, but we decided to stick with pressure injection since it provides higher reproducibility in our experience.

Is there a size limit for mRNA analysis on the BioPhase 8800 system?

I’m not aware of a specific size limit on the BioPhase 8800 system. The ladder from the RNA 9000 Purity & Integrity kit covers a range from 500 nt to 9,000 nt. While most mRNA we worked with is within this range, larger mRNA can be analyzed when extending the run time, with the caveat of working outside of the calibration curve range which would impact sizing accuracy. This would be something interesting to investigate.

Which capillaries do you use for mRNA analysis?

The capillaries we used for the BioPhase 8800 system are bare-fused silica (BFS) capillaries as part of the BioPhase 8800 BFS capillary cartridge (P/N 5080121). The cartridge contains 8 pre-installed capillaries, each 30 cm long, and a detection window 20 cm from the inlet. Liquid-based temperature control of the separation temperature is incorporated with these cartridges.

mRNA extraction from LNPs

Can you comment on the transferability of your extraction method with the Triton X-100 and temperature settings for CGE analysis to other mRNA products?

In general, the method can be transferred to different mRNA products. However, I suggest optimizing the surfactants and the temperature settings since different mRNA products can be more susceptible to secondary structure formation, aggregation or formation of multimers, and may have different sensitivity towards temperature. You really want to make sure that your surfactant concentration is suitable for your mRNA product. In case you are getting poor recovery, you would want to investigate surfactant types or concentration. Since different mRNA products exhibit different sensitivities to temperature, I recommend optimizing sample incubation temperatures. For instance, a test range of 40 to 70 degrees Celsius, including different incubation times for a given temperature, is a good starting point. I also recommend optimizing your cartridge temperature as well to reduce the formation of secondary structures.

Did you find that the recovery of mRNA from your LNPs is dependent on the type of ionizable lipid used?

We tried several types of ionized lipids and different compositions of LNPs. For the ones we tried, we have not experienced significant issues related to recovery using an optimized extraction method. We did observe that non-optimized extraction methods, including sample preparation such as surfactants level and denaturants, could affect recovery.

LNP stability

Based on your experience, does the size of the RNA, for instance >9,000 nt, affect the stability of the LNPs?

The size of the mRNA is expected to affect its stability. A major mode of mRNA degradation is by breakage along the phosphodiester backbone. The degradation is proportional to the number of nucleotides: The longer the mRNA, the more liabilities for potential breakage. Another important aspect to consider is potency. Any adduct on mRNA will lead to a potential loss in the ability to accommodate the translation machinery. Similarly, breakage of phosphodiester bonds will automatically lead to a loss of ability to translate into full protein products.

In your opinion, how many F/T cycles would be sufficient for testing LNP stability?

I think one should consider its usage for determining suitable stability strategies. After manufacturing, LNPs are usually stored and shipped under freezing conditions. That means they undergo at least one F/T cycle. From my experience, 3 F/T cycles is the norm for testing stability for LNP product usage. Ideally, you want to test 3 to 5 F/T cycles.

Are you able to comment on other approaches regarding stability, such as lyophilization?

The approach we currently take is focused on preserving the formulation through freezing. This adds inconvenience and increases costs. During lyophilization, water is removed from the LNP system, and a product could be stored in refrigerated conditions, potentially even at room temperature. The requirements for upholding a suitable cold chain would be dramatically reduced. Alternative solutions for storage of mRNA products, like lyophilization, are therefore very interesting. A lot of work still needs to be done, particularly regarding lyophilization of LNPs.

Can you comment on how excipients affect mRNA integrity?

Various excipients—lipid and non-lipid excipients—play an especially important role in stabilizing the product overall and can also affect the active ingredient, the mRNA. For instance, it is known that secondary structures of mRNA are pH dependent. Excipients, such as buffer salts, modulating the pH, can therefore affect the mRNA structure. Lipid excipients are crucial to the drug’s efficacy and tolerability. Because of that, a lot of attention needs to be paid to their quality in addition to stability reasons. Impurities in excipients can lead to degradation of mRNA. A now well-known example is the reaction of mRNA with aldehydes, which can exist as impurities in lipids. When evaluating excipients, my suggestion is to decouple the effects of the excipients from those related to the excipients’ quality.

Future outlook

Is there any carrier system being used for mRNA other than LNPs?

Up until today, LNPs leveraging ionizable lipids are the most clinically validated system for mRNA delivery. LNPs come in different flavors, usually using ionizable or cationic lipids as a key component, complexing the negatively charged mRNA cargo. Some LNP research focuses on the usage of biodegradable lipids to improve the biocompatibilities while others have focused on targeted systems for enhanced efficiencies. Other systems can be used for non-viral delivery, such as polymers or cell-penetrating peptides (CPP). In some cases, polymeric systems can be coupled with LNPs to gain the best of both worlds. It’s an exciting field with a lot of ongoing research.