Accelerating the Development and Scale-Up of mRNA Vaccines

The need to accelerate vaccine development has never been more important as we continue to navigate life amidst a global pandemic. The approval of the first COVID-19 vaccines was an enormous milestone for messenger RNA (mRNA) therapeutics that altered the course of the pandemic and highlights the rapid response potential of the technology. While it may seem as though the mRNA technology underlying the vaccines was an overnight success, it was based on decades of scientific research and innovation, making synthetic RNA safe for injection. However, getting these highly sensitive RNA molecules into cells without degradation while maintaining safety, potency and efficacy was a major challenge. Lipid nanoparticles (LNPs) have solved many of these problems and are now the key to making mRNA vaccines a reality. The demonstrated clinical efficacy of the COVID-19 mRNA vaccines has driven explosive growth in the development of RNA-based vaccines and concomitantly propelled LNPs into the mainstream as an effective drug carrier for complex polynucleotide- and peptide-based therapeutics.

The LNPs used in the COVID-19 vaccines are composed of positively charged ionizable lipids which undergo an electrostatic interaction with negatively charged mRNA molecules. The LNP shell effectively encapsulates the mRNA, forming a protective barrier against metabolic enzymes. Mimicking endogenous low-density lipoproteins (LDLs), LNPs are taken into the target cells by endocytosis. Within the endosome, the pH-sensitive ionizable lipids facilitate endosomal escape and release of the mRNA payload into the cytoplasm. While LNPs are complex delivery systems, their low toxicity, ability to efficiently encapsulate a variety of genomic payloads (or multiple payloads) and be engineered to specifically target a type of cell present new opportunities for emerging nanomedicines.

With growing global interest, demand for LNPs is at an all-time high. The move from a niche application to mainstream has increased investment into bioprocessing development efforts to establish reliable and robust manufacturing with clear scalability and compliance goals in mind. As the Quality by Design (QbD) concepts and Design of Experiment (DoE) approaches gain momentum in process development, opportunities to leverage vertically (up/down) scalable platform production technologies, predictive process models and automation are providing deep process knowledge. Importantly, evaluation of both upstream and downstream steps at scale is needed to gain end-to-end process insight across the entire manufacturing workflow, which is critical to identify any gaps or unanticipated effects resulting from process or analytical changes on product critical quality attributes (CQAs). With nanoparticles, scale-up of downstream formulation and fill-finish operations can have huge impacts on functionality and stability. Therefore, paying attention to downstream considerations can make the difference between success and failure on the path towards commercialization.

The Impact of Downstream Process Development on Bioactivity

The goal of process development is to define and optimize critical process parameters while ensuring process scalability for long-term success. The production of LNP-based nanomedicines can be challenging because of their size and complexity, since nanoparticle morphology can be impacted by downstream filtration processes that can impact the bioactivity of the resulting drug product. Therefore, a thorough understanding of how to mix the lipids and RNA to form the nanoparticles in a robust and reproducible manner is key to successful LNP formulation and delivery. Critical process parameters (CPPs) such as flow rates, temperatures and mixing ratios can affect the physicochemical characteristics of the resulting nanoparticles. Appropriate analytical and biological assays to assess how changes in processing variables affect nanoparticle properties, which include particle size, polydispersity (PDI) and drug encapsulation efficiency (EE%), are needed to confirm that product identity, potency and safety are maintained across all developmental stages to guide formulation and process development.

Traditional methods for nanoparticle manufacture have previously involved turbulent mixing processes where organic solvents containing LNPs meet the aqueous solutions of RNA in an uncontrolled manner. However, heterogeneous particle size, inconsistent encapsulation and poor batch-to-batch reproducibility pose barriers to scale-up. Non-turbulent microfluidic mixing devices were developed to overcome the shortcomings of these production techniques to improve the consistency and reproducibility of nanomedicine production. Precision NanoSystems (PNI) has made microfluidics both accessible and practical for drug development and manufacturing with with their NxGen™ technology, which enables flow rates thousands of times higher than conventional microfluidic designs while maintaining controlled mixing conditions. Non-turbulent flow brings together the fluid streams containing the lipids dissolved in an organic solvent and the nucleic acids dissolved in an aqueous buffer in a controlled manner, creating a solvent polarity change and triggering the formation of LNPs loaded with RNA. Precise control of the chemical and physical environment enables highly predictable, time-invariant mixing for reliable and repeatable nanoparticle self-assembly.

PNI has implemented NxGen™ technology across a range of NanoAssemblr® systems to support LNP formulation through all drug development stages with increasing throughput, from preclinical, clinical to commercial production that also meet phase-appropriate regulatory requirements. The conserved mixing element across production volumes offers developers a risk-based approach to chemistry, manufacturing, and controls (CMC) studies since operations modeled on small-scale preclinical instruments can be more easily translated to large-scale platforms. This helps to minimize variability during tech transfer and reduce the number of engineering runs prior to cGMP manufacturing. Of course, process development is often not linear and may require movement between scales to revisit and reoptimize process parameters. Scalable technology that supports this flexibility enables rapid and streamlined process development and thus offers significant advantages over static technologies.

The next stage in the downstream workflow after LNP-RNA assembly is tangential flow filtration (TFF), which encompasses both ultrafiltration and diafiltration. For nanoparticles, diafiltration is used to exchange the organic solvent used during formulation for a buffer that is suitable for storage stability and administration. Ultrafiltration is used to concentrate the therapeutic to its final formulation concentration. The recent development work at PNI to produce an LNP-encapsulated self-amplifying RNA (saRNA) COVID-19 vaccine highlights the importance of evaluating the impact of downstream processes during scale up.

Case in Point: Development of a Self-Amplifying RNA-LNP COVID-19 Vaccine

In 2020, Precision NanoSystems received a grant from the Canadian Strategic Innovation Fund (SIF) with the goal of developing a made-in-Canada COVID-19 saRNA vaccine. saRNA vaccines are considered the next generation of mRNA vaccines because of their capacity to self-replicate their mRNA within the target cells. This has advantages over mRNA vaccines because the required saRNA for each dose is considerably less (roughly 20-fold), which significantly lowers the raw material and manufacturing burden.

Two candidate saRNA-LNP vaccine formulations, LNP-1 and LNP-2, were developed using PNI’s NanoAssemblr® manufacturing platform. Both formulations maintained similar CQAs including size, PDI and encapsulation efficiency across all scales of production. However, LNP-1 outperformed LNP-2 in vitro, with comparable results in vivo. Upon shifting to the optimization of downstream TFF parameters and their effects on particle characteristics, it was discovered that LNP-1 particle size and PDI increased while LNP-2 size and PDI remained stable as production was scaled. In addition, LNP-1 showed suboptimal stability upon storage compared to LNP-2. This led PNI to select LNP-2 as the lead vaccine candidate for further development, even though LNP-1 showed slightly higher bioactivity, and highlights the importance of optimizing process parameters during scale-up.

Without the stability and scalability data, it is likely that LNP-1 would have advanced in the pipeline, leading to some very different outcomes. This cautionary example emphasizes the need for due diligence in downstream process development to mitigate the risk of failure in scale-up that can impose costly delays on the path towards a commercial product. The vertically scalable NanoAssemblr® platform greatly accelerated the development of a novel saRNA COVID-19 vaccine and provided an agile mechanism to transition from preclinical to cGMP manufacturing, which serves as a roadmap for the development of other LNP-based nanomedicines targeting a range of diseases. The PNI LNP-2 lead vaccine candidate is undergoing further evaluation in support of a Phase I clinical trial commencing this year, less than two years since the project began.

Process development is a challenge for any drug developer, and mRNA vaccines are no different. Process optimization is needed to achieve a sustainable, cost-effective and robust manufacturing process that ensures the safety and efficacy of the end product. Platform technologies such as the NanoAssemblr® enable small-scale modeling of unit operations that is predictive of performance at scale and accelerates process optimization. The ability to accelerate the development and commercialization timelines for new mRNA vaccines and other LNP-based therapeutics holds tremendous potential to ensure global readiness against future pandemics and bring life-saving treatments to patients faster.

Learn more about Precision NanoSystems’ COVID-19 saRNA-LNP vaccine in the virtual symposium Vaccines from Concept to Clinic: New insights into accelerating the development of RNA vaccines.