Stability Studies of Lipid Nanoparticles Using Analytical Chemistry

Messenger RNA (mRNA) is the intermediary between protein encoding DNA and the proteins produced by ribosomes. mRNA sequences can be engineered to encode certain proteins that can be translated and expressed within cells. There are many applications of mRNA therapeutics such as the development of mRNA-based vaccines (Hogan, 2018). Although mRNA has become a promising alternative to traditional vaccine approaches, it is fragile and can degrade easily within cells. A delivery agent is needed to package the mRNA payload and efficiently deliver the mRNA in vivo. Lipid nanoparticles (LNPs) are commonly used as delivery agents to protect the payload and release it within the target cells. The four lipid components of LNPs are an ionizable cationic lipid such as Dlin-MC3-DMA (MC3), a neutral helper lipid, cholesterol, and a PEGylated lipid. This experiment tests the stability of the lipids in LNPs under different temperature and storage duration conditions. Lipid and mRNA samples were extracted from LNPs and stored for 4 weeks at 4°C, 25°C, and 37°C. The lipid samples stored at each temperature were evaluated for degradation using liquid chromatography mass spectrometry (LCMS) every week for three weeks after the initial storage period. Both high and low lipid concentrations were tested. LCMS was used to look for the presence of lipid components and their fragments in the organic lipid extract samples from LNPs. It was found that as the storage time increased, the signal intensity of both the cholesterol and MC3 compounds decreased. The inverse relationship between signal intensity and storage time shows that the longer the extractions are stored, the more the lipids will degrade. It was also noted that the samples stored at cooler temperatures generally had a higher intensity. Experimental results provide insight on the required storage conditions for LNPs and mRNA vaccines.

Hello everybody, my name is Lina Alkarmi, I’m a bioengineering undergraduate student, and today I will be giving an overview of my URSP project where I researched the stability of lipid nanoparticles using analytical chemistry techniques. With the recent Pfizer/BioNTech and Moderna Covid-19 vaccines, mRNA vaccines have been in the spotlight. mRNA is the intermediary between protein encoding DNA and the proteins produced by ribosomes. Lipid nanoparticles, or LNPs, are commonly used as delivery agents to protect the mRNA payload and release it within the target cells. Typically there are four components of LNPs, which are an ionizable cationic lipid, a neutral helper lipid, cholesterol, and a PEGylated lipid, which you can see the structures of on the right. MC3 is a commercial ionizable lipid commonly used in LNPs. LNPs are produced through microfluidic mixing, where the lipid components in ethanol are injected in one channel and the aqueous mRNA is injected in the other channel, so the cationic lipid will ionize and the particles will be formed. The stability of these nanoparticles is an important aspect of mRNA therapeutics because after the LNPs are formulated, they need to be kept stable for use so they can be effective. My research focuses on studying the stability of the lipids in LNPs under different conditions. My first research question focused on the effect of storage temperature on lipid stability, while my second research question focused on the effect of storage duration on lipid stability. For my experiment, after LNPs were formulated, the lipids and mRNA were extracted from the LNPs. I then stored these extractions for a period of a month at 4, 25, and 37°C, and tested their stability using LCMS over the course of 3 weeks to look for changes. I tested lipid samples with high and low concentration. Liquid chromatography mass spectrometry is an analytical chemistry technique that combines high performance liquid chromatography with mass spectrometry. HPLC is used for the separation of components in an organic mixture. It works by applying pressure to carry a sample through a column and detector. Depending on the polarity of a sample and the solvent system used, different compounds will elute off the column at different times. After coming off of the LC, the eluent will be hit with a large voltage which will turn it into an ion spray. The ions will be detected and measured according to their mass charge ratios. In the first quadrupole, Q1, the initial mass will be detected in the precursor scan. The second quadrupole is the collision cell, where the ion beam is hit with nitrogen gas that will interact with the molecule and break it apart into different products or daughter ions that will be filtered in Q3. For example, MC3 can be broken into fragmented products that can be observed. LCMS can also be used to look for degraded products. The process can be used to look for the presence of a compound in a solution. For my experiment, LCMS was used to look for the presence of lipid components and their fragments in the organic lipid extract samples from LNPs. For my samples, I looked for the presence of cholesterol and MC3 in the lipid extractions. I tested the lipid samples at the three temperatures 4 weeks, 5 weeks, and 6 weeks after extraction. In the chromatograms, taller peaks, or higher signal intensity, indicate a higher presence of compound with less degradation, and vice versa. Here on the left we have a chromatogram depicting the signal intensity versus retention time for the MC3 compound in the low concentration LNP samples. In the chromatogram, the tallest three peaks correspond to the most recently extracted lipids that were stored for 4 weeks. As the storage time increased, the intensity decreased, shown by the lower week 5 and week 6 peaks. At week 6, the MC3 compound was not detected at all, so it was completely degraded 6 weeks after extraction. The same trend was observed in the high concentration samples, where the intensity decreased as the storage time increased. The inverse relationship between signal intensity and storage time shows that the longer the extractions are stored, the more the lipids will degrade. The same trend was observed when looking for the presence of cholesterol in the high and low concentration samples. The lipid extracts stored for longer had a lower presence of cholesterol in them, shown by the lower signal intensity. It was also noted that the samples stored at cooler temperatures generally had a higher intensity. The LCMS data gathered for the samples indicates that after LNP extraction, the lipids began to degrade. It was found that a longer storage period resulted in less intensity and more degradation for the lipids, and the samples stored at higher temperatures generally had a lower signal intensity. Future steps would include testing fresh LNP extracts every week to monitor their degradation. Thank you to Dr. Buschmann, Dr. Alishetty, Zach Beaulac, and Manuel Carrasco for their guidance during this project. These are the sources referenced throughout this presentation, and thank you for watching!