(M1330-07-39) Optimized Prime: A Transformative LNP Composition for Scalable RNA Vaccines

Purpose: Lipid nanoparticle-encapsulated RNA (RNA-LNP) vaccines have played a pivotal role in the global response to the COVID-19 pandemic (1). With proven safety and efficacy, RNA-LNPs are now at various stages of development against a variety of other infectious diseases (2). However, limited access to potent ionizable lipids, a key component in LNPs, and the expertise required to formulate and scale LNPs present real barriers to entry in this field. To overcome these challenges, we developed and optimized a novel ionizable lipid and a corresponding LNP composition for the delivery of RNA in vaccine applications. Using a scalable mixing platform, we formulated vaccine-candidate LNPs with both mRNA and self-amplifying RNA and investigated their physicochemical attributes and in vivo immunogenicity.

Methods: RNA-LNPs were prepared on the NanoAssemblr™ Ignite™ system, using a custom lipid mix including an ionizable lipid from our lipid portfolio, at a total flow rate of 20 mL/min. LNPs were diluted with a physiological buffer (pH 7.4), concentrated using 30 kDa Amicon® filters, and stored at -80°C in a cryopreservation buffer. RNA-LNP size and polydispersity were determined by dynamic light scattering and encapsulation efficiency by RiboGreen™ RNA assay. We determined in vitro protein expression in HEK293 cells by flow cytometry and we investigated in vivo immunogenicity by intramuscular injection of BALB/c mice with RNA-LNPs, with a booster dose on day 28. Tail bleeds were performed on day 21 and mice were culled by cardiac puncture on day 42 followed by spleen harvesting. To demonstrate the scalable nature of both the novel LNP composition and the mixing process, we took our lead SARS-CoV-2 self- amplifying RNA-LNP vaccine candidate and prepared batch sizes of 100 mL at 48 L/h using the NanoAssemblr™ commercial formulation system.

Results: The novel LNP composition, combined with a scalable mixing process, resulted in high-quality RNA-LNPs with reproducible particle sizes of 60 to 90 nm, polydispersity indices < 0.2, and RNA encapsulation of > 80%. When stored in cryopreservation buffer at -80°C for 6 weeks (wk), we observed no detrimental effects on physicochemical attributes and in vitro potency. We completed in vivo immunogenicity studies using SARS-CoV-2 self-amplifying RNA and haemagglutinin mRNA-LNPs. A strong immune response was mounted against both antigens, where we observed antigen-specific IgG titers of 10⁶ ng/mL, and IFNγ responses > 100-fold higher than naïve mice. Finally, we found that RNA-LNPs prepared on a commercially relevant scale were comparable to those prepared at the benchtop scale with respect to physicochemical attributes and immunogenicity.

Conclusion: In this study, we developed and optimized a potent and scalable LNP composition for vaccine applications, demonstrated using different RNA coding for clinically relevant antigens. All the RNA-LNPs had high-quality physicochemical attributes, and strong biological activity in vitro and in vivo. Consequently, we envisage this new LNP composition will be used in a wide range of preclinical drug development activities including antigen screening and in vivo proof-of-concept studies. In addition, we have derisked the scale-up of this composition and the mixing process, showcasing how these technologies can be used to accelerate drug development timelines.