The Crucial Role of Analytics and Bioassays to Fast-Track mRNA-LNP Drug Development

December 01, 2023

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The scientific community has always known the potential of the customized RNA or mRNA and the development and approval of the mRNA vaccines by Moderna and BioNTech/Pfizer during the COVID-19 pandemic have ignited a wave of inspiration and determination within the pharmaceutical industry. It has spurred the exploration and expansion of therapeutic innovations across a spectrum of diseases, from cancer treatments to personalized vaccines. The RNA-LNP (Lipid Nanoparticle) drug development is rapidly evolving with endless possibilities. However, translating these scientific promises into tangible medical breakthroughs is far from straightforward. The lipid nanoparticles can encapsulate RNA within, protect RNA from degradation, facilitate cellular uptake, and enable precise delivery to target issues.  However, it is a complex process and despite the extraordinary progress achieved, it is essential to acknowledge that mRNA technology remains nascent, with no standardized manufacturing protocols. Amongst the number of challenges is the formulation of mRNA delivery systems, the lipid nanoparticles. It requires meticulous measurements, analysis, and experimentation. The analytical tools and bioassays can drive advancements in the precise development of RNA-based therapies. Pharmaceutical companies and biotech start-ups are heavily investing in RNA-LNP research and development. Partnerships, collaborations, and funding initiatives have surged.

Adam Crowe, Manager of Analytical Development at Precision NanoSystems (PNI), stated that as the technologies facilitating LNP-RNA therapeutics are still in the early phase, there is no definitive line between proof-of-concept data to the final product, which may be surprising to small-to-medium-size-biotech-companies-looking-to-expand and scale their ongoing ventures.

"The small-scale manufacturing, which is between 1 ml and 10 ml, is not problematic, but it's not easy to replicate processes and technologies to large-scale production of tens of liters of formulations," says Dr. Crowe, who, along with his teams develops novel analytical assays for LNPs and nanomaterials for drug delivery. "Existing compositions have proven effective, but the intellectual property (IP) and licensing agreements is a complex landscape which makes accessing these compositions complex for new groups looking towards moving to the clinical phase."

"Companies that aim to develop drugs using these new modalities should also understand the multiple variables that could cause the project to fail. Finding new material sources to bring to a CDMO isn't enough."

"There are at least 30 to 40 parameters to consider, and even a small one-parameter change could be catastrophic. Some of the variables are the RNA itself, LNP composition, and the manufacturing process."

Evading impurities in lipids

According to Dr. Crowe, the N-oxide-based impurities originating from the ionizable lipid responsible for deactivating the mRNA are one of the emerging challenges to the potency of an LNP.

Each LNP consists of four components that serve a purpose. One of the four lipid components is the ionizable lipid. Depending on the pH, this species modifies its charge. As a result, they have significantly improved the performance of the new generation of LNPs; however, the issue is that ionizable lipids are inherently more reactive. When a tertiary amine, a reactive species, is present, a lot of strange chemistry can happen, one of which is the main impurities shown to be present in the lipid nanoparticles.

"Their reaction with RNA could inactivate the RNA itself."

The chemical production of the PEG lipid component and Ionizable lipid present another impurity difficulty. Although they cannot be derived from animal products, they are frequently synthesized in poor purity (between 60% and 70%). That is not a problem in an academic lab, but knowing which impurities are down to 1% or 0.1% weight abundance becomes a significant challenge when you want to create a medicine.

"Unfortunately, that can put down a lot of projects," says Dr. Crowe. "That's a critical area of analytics where we use different High-Performance Liquid Chromatography (HPLC) approaches. Charged Aerosol Detection (CAD) is the industry standard, but liquid chromatography–mass spectrometry (LC-MS) is also used to identify what's in the materials. We invest a lot of effort in generating high-quality material, as it is typically not done in the initial stages of customer engagement. We also spend much time understanding its solubility, degradation behavior, and reactivity. The material must be observed under various circumstances. Is it reacting with the RNA or other elements—this is a difficulty that keeps getting harder to solve. A project may fail due to other factors, such as polydispersity and particle size. Polydispersity refers to the range of particle sizes produced during LNP production. The PDI determines how significant the variation of sizes at which LNPs are created is, as they are never formed at a single size. If the PDI increases, something is wrong with your manufacturing process because your particles aren't acting as you want them to—they're fusing, aggregating, and acting differently.

"Another problematic area is encapsulation efficiency," says Andrew Kondratowicz, bioassay team lead of Precision NanoSystems.

"How far away is your payload? Where is it positioned? Is it positioned where the particle is supposed to be, attached to the outside, or perhaps free in solution? Here, it's crucial to remember that the LNP's primary function is to deliver and protect the RNA. There are numerous immunogenic and other issues if the RNA is outside if it isn't within, and it truly serves no use if it isn't inside. This is an excellent example of what encapsulates at a small scale and can only work at a large scale."

The significance of the 'biological readout'

Having a "biological readout" is one of the critical areas when establishing these processes and optimizing the composition, looking at the different ways you can produce and manufacture it. You must be able to determine if the formulation has the desired effect, according to Crowe and Kondratowicz.

The substance's potency must be meticulously evaluated every time you make a slight alteration, something Kondratowicz believes needs to be noticed. "The bioassays are the canary in the mine of how successfully your process development is progressing," explains Kondratowicz. The LNP scaling process may be monitored for hundreds of variables using bioassays, and if something is amiss, we can alert our clients immediately. Also, regulatory organizations require this to submit a new drug product. It would help if you had a bioassay in place to ensure a program's success because every formulation scales differently.

Crowe comments: "Scaling LNP-RNA sequence production is so fraught with factors that can go wrong, and so Andrew's analytical ability to screen at high capacity ensures that even if a batch fails – which is a common possibility given the complexity of the materials and how new the market is - PNI can help get the clients' projects back on track.

Kondratowicz adds, "To ensure the RNA quality itself, PNI can do formulation-independent mechanisms of getting the RNA into the cell so that, at the very least, we go into the LNP formulation process with good RNA and avoiding a scenario where later in the project something goes wrong and then realizing it was the RNA. So, we at least remove the variable to benefit the project. Although we have the capacity to create more than 100 milligrams on site, for larger batches, we can screen for the desired results and then tech transfer the project to a different supplier who can replicate it on a much greater scale.

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Final Outlook

The era of mRNA-based genomic medicines is the next big thing. There is a huge development in the next-generation mRNA vaccine designs using self-amplifying RNA (saRNA) and circular RNA (circRNA) molecular formats. It might need the RNA, which is 100 times less with increased efficacy and performance. In addition, the circRNA will not have the Poly-A tails and 5' caps that cause many quality problems in manufacturing RNA. But the analytical processes required will get much more complex, and PNI is unique in its ability to analyze both of these already.

Partnering with end-to-end solution providers like PNI can provide tailored strategies to support fast-emerging industry needs, which will be crucial given the high level of uncertainty still surrounding the best methods for process development and manufacturing of mRNA-LNP.

The future of vaccine development, oncology, and personalized medicine will undoubtedly be impacted by mRNA technology. PNI aims to assist academic research labs and biotechnology leaders by providing access to a unique proprietary lipid library and the knowledge necessary to create a custom lipid nanoparticle formulation for transforming genomic medicine.

PNI's GenVoy Delivery Platform comprises off-the-shelf RUO reagents such as GenVoy-ILM™ and a lipid nanoparticle portfolio of proprietary lipids available as custom formulations. In addition, LNP manufacturing is scalable to advanced preclinical and clinical scales with NxGen™ NanoAssemblr® technology.

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