Navigating a pandemic has highlighted just how important accelerated vaccine development can be. In a milestone moment, the approval of the first COVID-19 vaccines ultimately altered the course of this global health crisis while also showcasing the rapid response potential of messenger RNA (mRNA) therapeutics.
While it may seem like the mRNA technology underlying the COVID-19 vaccines was an overnight success, these formulations came from decades of innovative scientific research into mRNA’s safety and potential. However, getting highly sensitive RNA molecules into cells without degradation while maintaining safety, potency, and efficacy has been a longstanding challenge.
Lipid nanoparticles (LNP) have solved many of the problems researchers faced over the years and are a key element in making mRNA vaccines a reality. The mRNA vaccines for COVID-19 have driven explosive growth in the development of RNA-based vaccines and simultaneously made LNPs a mainstream drug carrier for complex polynucleotide- and peptide-based therapeutics.
The LNP 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 (LDL), LNP 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 LNP are complex delivery systems, their favorable toxicity profile, efficient encapsulation of a variety of genomic payloads (or multiple payloads), and ability to be engineered to target a specific cell type present new opportunities for emerging nanomedicines.
Key considerations in LNP process development and manufacturing
With growing global interest, demand for LNP is at an all-time high. The move from a niche to a mainstream application has increased investment into LNP bioprocessing development to establish reliable, robust, scalable, and compliant manufacturing processes. As the quality by design (QbD) and design of experiment (DoE) approaches to process development gain momentum, vertically (up/down) scalable platform production technologies, predictive process models, and automation are providing deep process knowledge.
Manufacturers need to evaluate both upstream and downstream steps at scale to gain end-to-end process insight across the entire manufacturing workflow. This assessment is critical to identifying any gaps or unanticipated effects on product critical quality attributes (CQA) resulting from process or analytical changes. 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 be the difference between success and failure on the path toward commercialization.
The impact of downstream process development on bioactivity
While the goal of process development is to define and optimize critical process parameters while ensuring process scalability for long-term success, the size and complexity of LNP-based nanomedicines can make production a challenge.
Nanoparticle morphology can be impacted by downstream filtration processes which influence 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 (CPP) 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 ― including particle size, polydispersity index (PDI), and drug encapsulation efficiency (EE%) ― guide formulation and process development to ensure product identity, potency, and safety are maintained across all developmental stages.
Traditional methods for nanoparticle manufacture have involved turbulent mixing processes where organic solvents containing LNP 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 turbulent production techniques and improve the consistency and reproducibility of nanomedicine production. NxGen™ technology has made microfluidics both accessible and practical for drug development and manufacturing by enabling flow rates thousands of times higher than conventional microfluidic designs while maintaining controlled mixing conditions. Non-turbulent flow brings the fluid streams containing the lipids dissolved in an organic solvent and the nucleic acids dissolved in an aqueous buffer together in a controlled manner, creating a solvent polarity change and triggering the formation of LNP loaded with RNA. Precise control of the chemical and physical environment enables highly predictable, time-invariant mixing for reliable and repeatable nanoparticle self-assembly.
NxGen™ technology is available across a range of NanoAssemblr™ systems with increasing throughput to support LNP formulation through all drug development stages ― from preclinical and clinical to commercial production ― while also meeting 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 scalability 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 versus static technologies.
The next stage in the downstream workflow after LNP-RNA assembly is tangential flow filtration (TFF), which encompasses both diafiltration and ultrafiltration. 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 then concentrates the therapeutic to its final formulation.
Development of a self-amplifying RNA-LNP COVID-19 vaccine
saRNA vaccines are considered the next generation of mRNA vaccines because of their ability to promote the self-replication of mRNA within target cells. This approach offers the advantage of a lower dose, significantly reducing raw material requirements and manufacturing burden.
In 2020, our two candidates of saRNA-LNP vaccine formulations, LNP-1 and LNP-2, were developed using the NanoAssemblr™ manufacturing platform for the made-in-Canada COVID-19 saRNA vaccine. Both formulations maintained similar CQA including size, PDI, and encapsulation efficiency across all scales of production. However, LNP-1 outperformed LNP-2 in vitro, with comparable results in vivo.
When optimizing downstream TFF parameters and considering their effects on particle characteristics, it was found that LNP-1 particle size and PDI increased while LNP-2 size and PDI remained stable with scale-up. In addition, LNP-1 showed suboptimal stability during storage compared to LNP-2. This finding highlights the importance of optimizing process parameters during scale-up and led to the selection of LNP-2 as the lead vaccine candidate for further development, even though LNP-1 showed slightly higher bioactivity.
Without stability and scalability data, LNP-1 would have likely advanced in the pipeline. This cautionary example emphasizes the need for due diligence in downstream process development to mitigate the risk of failure during scale-up and prevent costly delays on the path to commercialization.
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, serving as a roadmap for the development of other LNP-based nanomedicines targeting a range of diseases. The LNP-2 lead vaccine candidate is undergoing further evaluation.
Process development is a challenge for any drug developer, and working with mRNA vaccines is no different. Process optimization is needed to achieve a sustainable, cost-effective, and robust manufacturing workflow that ensures the safety and efficacy of the end product. Platform technologies such as the NanoAssemblr™ system enable small-scale modeling of unit operations that are predictive of performance at scale to accelerate process optimization.
The ability to accelerate the development and commercialization timelines for new mRNA vaccines and other LNP-based therapeutics has the potential to ensure global readiness against future pandemics and bring life-saving treatments to patients faster.