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Stability and in vivo performance aren't just affected by lipids.
For messenger RNA (mRNA) therapeutics and vaccines formulated as lipid nanoparticles (LNPs), the excipients receiving the most attention are the encapsulating lipids. While these compounds are crucial to the stability and delivery of mRNA-LNPs, the non-lipid excipients also play crucial roles in the function of final drug products. They impact the morphology and stability of these delivery systems, which in turn affects in vivo performance and the overall usefulness of mRNA drugs developed using nanoparticle technology.
Non-lipid excipients used to formulate mRNA-LNP products include solvents, salts, and sugars/cryoprotectants, according to David Jung, senior manager RD&I, nucleic acids at Evonik. Overall, these excipients serve the function of controlling the pH during the initial mixing and formation of the particle and ensuring stability upon storage of the material, he notes.
More specifically, rapid mixing is the predominant technique for lipid nanoparticle–mRNA formulations: an ethanol phase (lipid components) and an aqueous phase (mRNA molecules, usually in acetate or citrate buffers) are mixed under specific conditions (pH and flow rate), explains Luca Bruno, segment marketing manager, MilliporeSigma, the Life Science business of Merck KGaA, Darmstadt, Germany. Rapid mixing, he adds, allows scalability and reproducibility of mRNA-LNP formulation.
Organic solvents and residual components are then removed by ultrafiltration. The mRNA-LNP drug substance is then formulated using a more physiological buffer, typically tris(hydroxymethyl)aminomethane- or phosphate-based together with salts such as sodium chloride or potassium chloride to adjust the ionic strength, Bruno observes.
Storage conditions (as aqueous solutions, frozen, or lyophilized) must also be carefully considered in the context of mRNA-LNP clinical translation, according to Bruno, because they can affect the long-term stability of mRNA-LNP formulations. “Sugar stabilizers (sucrose in particular, trehalose or mannitol as alternative options) are commonly used for this application,” he says.
In addition to assuring long-term stability and in vivo performance, non-lipid excipients for mRNA-LNPs also play an important role in controlling manufacturing processes, Jung comments. “The selection of these excipients must therefore be done carefully. Typically, they are initially chosen based on their chemical and physical properties (i.e., pKa and lyophilization characteristics, freezing point/state) because these attributes will determine compatibility with the drug substance. Because these choices may also impact the morphology of mRNA-LNPs, which can affect overall efficacy and stability, modifications may be required as determined [by] experimental testing,” he says.
The susceptibility of mRNA to degradation requires that formulation buffers be free of any ribonuclease contamination. Testing of excipients for the absence of nuclease activity is therefore the preferred approach, Bruno stresses. Other product-quality attributes should also be determined, including endotoxin content and bioburden, as they play an important role in controlling the risk of contamination by these materials.
Beyond the product-quality attributes, Bruno emphasizes the need to consider supplier related aspects for any excipients used in mRNA-LNPs, including non-lipid excipients. “Supply robustness and availability of supporting documentation are both essential if mRNA-LNP developers are to navigate regulatory challenges and ensure smooth manufacturing scale up and progression through clinical development to commercialization,” he adds.
Since the approval of the COVID-19 mRNA vaccines and demonstration of their effectiveness, interest in mRNA-LNP therapeutics and vaccines has risen dramatically from all perspectives. In particular, the rise of the mRNA technology due to the COVID-19 pandemic and the manufacturing scale needed to produce vaccines shifted the attention to mRNA manufacturing needs, according to Bruno.
“The development of non-lipid excipients has primarily focused on the quality attributes based on the specificity of the technology,” Bruno notes. For instance, the need for endonuclease-free materials required establishment of additional product-release testing methods and services to support drug manufacturers.
There has also been a significant amount of effort placed on assessing non-lipid excipients that may improve the long-term stability of mRNA-LNP formulations, observes Jung. He points specifically to the use of different cryoprotectants to improve refrigerated storage stability. Lyophilization is also being explored as a means for eliminating cold-chain requirements, with several clinical-stage studies underway to assess the impact of lyophilization on the stability and in vivo efficacy of traditional mRNA-LNPs.
Separate efforts are also underway to create novel delivery mechanisms for RNAs that are not dependent upon LNPs, Jung adds.
A key focus in the mRNA field is on lipid excipients and that will continue to be true for some time, contends Aditi Mehta, head of mRNA process and delivery, MilliporeSigma, the Life Science business of Merck KGaA, Darmstadt, Germany. “Despite the success of the COVID-19 vaccines, we still have not reached the full potential of mRNA therapeutics, and there are several hurdles. Insufficient targeting of organs beyond the liver or vaccinations, an acute immune response against LNP administration, stability issues, and the need of extreme low temperature for storage are key bottlenecks in the development of LNP medicines,” she says.
These issues, Mehta observes, can be addressed through improvements in lipid and LNP design, (e.g., optimization of linker chemistry, addition of degradable bonds, more optimal synthetic routes, better LNP components, etc.). “Strategies such as design of novel ionizable lipids based on large libraries produced with combinatorial synthesis have been aiding the understanding of structure-function properties and enabling the design of unique lipids with improved properties (targeting, stability, tolerability),” she states.
Data continues to be published on new lipid libraries that demonstrate delivery to different tissues/cells (1), Jung agrees. Targeting of mRNA-LNPs to specific tissues or cells can be achieved through either passive or active means. A majority of studies investigating ways to target LNPs have focused on passive targeting through modification of the LNP size, surface, and composition. Substitution or addition of an extra moiety (polymeric or lipidic) has also been shown to be beneficial for achieving passive targeting toward specific organs, according to Mehta.
“Active targeting approaches leverage conjugation of lipid components with small molecules, antibodies, proteins, or aptamers,” Mehta comments. She points specifically to flexible platforms and highlights the ASSET platform developed in the lab of Prof. Dan Peer at Tel Aviv University (2). “This type of approach enables customized targeting towards any cell receptor and, therefore, any tissue or organ,” she says.
Mehta does caution, however, that while there has been a tremendous push (and success) in this area over the past few years, there is still a long way to go and many challenges to overcome.
While lipids have predominated the mRNA delivery space, they are not the only materials that can provide effective delivery of these sensitive and highly charged drug substances. Hybrid lipid–polymer delivery systems, for instance, have been developed to harness and combine the advantages of both lipid and polymer nanoparticles, according to Mehta.
“Polymers possess an inherent high degree of chemical variety that is not feasible with lipids, increasing tremendously the possibilities of chemical functions, such as promoting endosomal escape, controlled released of RNA, targeting, and finally stability,” she explains. Lipids, meanwhile, show great potential for enhancing bioavailability, improving pharmacokinetic profiles, and increasing biocompatibility.
Strategies investigated to formulate hybrid systems include lipopolyplexes, hybrid LNPs, and others. As an example, Mehta notes that incorporation of biodegradable polymers such as poly(beta amino esters) in LNP formulations leads to improved and specific lung delivery, highlighting the potential of hybrid lipid-polymer nanoparticles (3).
Evonik has a research agreement with Stanford University to develop and commercialize the charge-altering releasable transporter (CARTs) technology initially developed by the Waymouth lab. These polymeric materials can effectively encapsulate mRNA and then rapidly degrade through controlled self-immolative reaction to release the mRNA, according to Jung.
“The first generation of CARTs showed mRNA delivery efficacy in a range of cell types in both in vitro and in vivo models,” Jung notes. He adds that subsequent generations of CARTs incorporating lipid side chains on the CART backbone structure (4) showed an improved delivery efficiency.Further generations of CARTs that are still under development, says Jung, have shown specific targeting capabilities in preliminary testing (5,6).
Cynthia A. Challener, PhD has been a freelance technical writer for over 20 years and contributes regularly to BioPharm International and Pharmaceutical Technology.