The success of mRNA vaccines has sparked significant interest and investment in using mRNA technology to treat a broad range of diseases, including cancer, infectious diseases, and rare conditions. Yet when “naked” RNA is injected, it degrades quickly. To address this issue, scientists have developed, and now widely use, the strategy of encapsulating mRNA into lipid nanoparticles (LNPs). Because LNPs protect mRNA, facilitate delivery to target cells, and support cell uptake by endocytosis, they promise to revolutionize global healthcare.
As researchers from both academia and industry work to discover the next advances, they must address growing challenges related to process development, scaling technology, regulatory approvals, and GMP guidelines. These challenges need to be addressed alongside scientific research for RNA-based therapeutics to achieve their full potential.
Key considerations for process development
The LNP encapsulation process is intricate and demands precise control over the mixing of two liquid streams—one containing lipids in an organic solvent such as ethanol, and the other containing RNA in an acidic buffer—in order to induce spontaneous self-assembly. When combining these two liquid streams, developers must consider how to best achieve scalability and reproducibility. Microfluidic mixing offers controlled and rapid nonturbulent mixing of the LNPs and RNA, making it an effective method for formulating LNPs.
For this innovative class of therapeutics, three key process considerations are critical to evaluate and optimize during process development.
1. Flow rate: The size of RNA-LNPs after the encapsulation step strongly depends on the flow rate of RNA and lipids through the mixer. As mixers scale up, flow rates must increase. If the flow rate of reagents is too low for a given mixer size, the resulting RNA-LNPs will typically be larger than desired. When scaling the mixer, a key activity is to screen a range of flow rates to ensure the new mixer prepares RNA-LNPs with sizes consistent with bench-scale results.
2. In-line dilution: The ethanol used to solubilize the lipids prior to encapsulation destabilizes the RNA-LNPs and must be removed. Typically, the ethanol concentration is reduced by dilution, and then ethanol is removed by filtration. At the bench scale, simply pouring the concentrated RNA-LNP sample in phosphate-buffered saline or another buffer is sufficient. However, the larger batch sizes, the increased RNA-LNP hold times that result, and the logistics of fluid transfer make pouring RNA-LNPs into buffer infeasible. The solution is to introduce in-line dilution into the process.
3. Downstream processing: The RNA encapsulation step is one of about 15 steps needed to prepare the final drug product. Downstream purification also requires scale-up and optimization. Methods for removing ethanol at the bench scale such as dialysis or spin filters do not extend to the commercial scale. The introduction of tangential flow filtration (TFF) to solve this issue introduces process parameters that require optimization (filter chemistry, shear rates, etc.). Nanoparticles are especially sensitive to the high shear forces present in TFF filters, yet if the shear forces are too low, the filter membrane may foul.
Process and analytical insights for GMP manufacturing
Comprehensive analytics form the backbone of consistent, scalable, and efficient GMP manufacturing. Our analytics and stability studies assess a product’s identity, size, purity, potency, and stability. Using advanced analytics, we can predict how RNA-LNP formulations will perform in specific patient populations, allowing for adjustments to optimize efficacy while minimizing potential adverse effects. These tests are essential for evaluating the shelf life and performance of RNA-LNP formulations in personalized medicine.
Determining batch size requires ensuring sufficient sample quantities for comprehensive testing throughout the manufacturing process and final product release. GMP standards necessitate strict quality control measures, with batch data playing a key role. Batch documentation serves as a detailed record of processes, materials, and quality measures, ensuring that each batch meets the highest standards of safety and efficacy. This documentation acts as a “manufacturing GPS,” guiding companies through regulatory compliance and helping them refine processes to deliver exceptional therapies to patients.
Advantages of automated platform technologies
Automated technologies for mRNA-LNP drug manufacturing streamline development and production, reducing human error and providing precise control over critical operations. Real-time monitoring, data analytics, and algorithms drive process optimization and quality control. These systems generate comprehensive data records and traceability logs, capturing each step of the manufacturing process in real time. When issues arise, the availability of detailed data aids in troubleshooting, ensuring quality assurance throughout. Digitized batch records and electronic systems support data integrity, simplify regulatory compliance, and enhance workflow transparency.
Automated platforms are designed to handle large-scale manufacturing needs. When critical processes are automated, production volumes and batch numbers can be increased while product quality and integrity are maintained.
Establishing a manufacturing partnership
Large-scale LNP drug production is a complex, resource-intensive process. Collaborating with established technology providers allows developers to leverage their expertise in technology transfer while optimizing manufacturing processes and assisting with regulatory submissions for GMP operations. This support is crucial whether the goal is to produce billions of doses of mRNA-based vaccines, or to develop tailored RNA-LNP therapies for personalized medicine.
Manufacturing collaborators have infrastructures that can scale processes efficiently, reducing development costs and accelerating time to market. Building a manufacturing collaboration also enhances supply chain resilience. Interruptions or failures can lead to noncompliance with regulatory authorities such as the FDA and EMA. Secure supply chains ensure consistent and dependable production flows and act as a safety net against unforeseen circumstances.
Experienced manufacturers engage with regulatory agencies early, gaining insights into technologies and innovations that accelerate development. This proactive approach supports changing industry needs and speeds drug development.
LNP technology will help shape the future of oncology treatment, infectious disease response, and cell and gene therapy development. The industry will require a blend of advanced, cost-effective manufacturing technologies for this future. Navigating this evolving landscape will involve integrating scaling strategies, automated platforms, process controls, and analytical insights to drive innovation in new therapeutic areas.
Scaling strategies for GMP and commercial manufacturing
A consistent and reproducible manufacturing process is crucial when scaling up to clinical and commercial production. Variations in larger-scale production can impact a formulation’s stability and effectiveness, demanding careful engineering and raw-materialss control. As production scales up, advanced equipment and specialized facilities become vital for maintaining product quality attributes.
Implementing advanced technologies such as automated platform solutions allows for small-scale modeling of unit operations. This approach can help predict performance at scale and expedite process optimization. Additionally, early investigation of process parameters helps establish process ranges and critical controls, ensuring GMP compliance and meeting the requirements of a defined target product profile, ultimately preventing program delays.
Investing in flexible equipment and instrumentation enables biopharmaceutical manufacturers to adapt their facilities to meet changing needs, which is important in a rapidly evolving market. Platform technologies, including single-use components, facilitate standardized manufacturing workflows and quick configuration for different products across various scales.
During development, scale-down strategies offer several benefits. By designing and characterizing scale-down models, manufacturers gain insights into process behavior, identify potential challenges, and establish critical controls before scaling up. This approach reduces the risk of costly errors, accelerates process development, and helps ensure final products meet quality and safety standards.
Clinical trials and commercialization also benefit from scale-down capabilities, which assist in evaluating different dosage levels and patient populations. Manufacturers can simulate and adjust processes to accommodate variations in drug dosage, tailoring products to meet the specific needs of different patient populations. Addressing the challenges posed by scaling down production requires advanced manufacturing technologies, robust quality control measures, diligent regulatory compliance, and specific facility considerations.
Beyond infectious diseases—Emerging cell therapy applications
While the therapeutic applications of RNA-LNPs are perhaps best typified by the approved prophylactic vaccines for SARS-CoV-2 (Comirnaty by Pfizer and Spikevax by Moderna), a rapidly emerging application is the use of LNPs for cell therapy applications. As an example, genome editing of CD34-positive hematopoietic stem cells (HSCs) is increasingly being acknowledged for its therapeutic potential in addressing inherited hematological disorders.
Cytiva, which now includes Precision NanoSystems, recently introduced the RNA delivery LNP kit, which is designed for use with the NanoAssemblr Ignite and Ignite+ nanoparticle formulation systems. The new kit, which offers a preoptimized ionizable lipid mix for rapid payload screening and RNA drug delivery validation, complements the GenVoy-ILM product line, which includes reagents and off-the-shelf LNP kits. According to Cytiva, the new kit lowers the barrier for vaccine developers by providing protocols and proof-of-concept data for mRNA and saRNA delivery in infectious disease models.As HSCs are difficult to source and maintain in culture, this exacerbates the shortcomings of conventional transfection methods such as electroporation, especially in the context of maintaining sufficient cell viability and yield for effective therapeutics. LNP technology enables efficient delivery of genetic material to HSCs and is scalable to support acceleration to the clinic.
Currently, the CRISPR-Cas9 system is commonly used for genome engineering, whereby a Cas9-enzyme-encoding mRNA and a single-guide RNA are delivered to the cells either by electroporation or a viral vector. However, these conventional methods possess potential drawbacks in safety, such as the immunogenicity and cytotoxicity associated with viral vectors or the cell death resulting from electrical pulses required for electroporation. In contrast, LNP technology promises to enable efficient cellular transfection of RNA. Furthermore, recent studies have focused on the advantages of LNP-based methods over traditional methods such as electroporation for ex vivo HSC and CAR T-cell therapies.
For these emerging cell therapy applications, maintaining a high standard of quality for the LNPs remains essential. Many, if not all, of the quality and scaling considerations that are present for infectious disease applications of RNA-LNPs will likely be present for producing LNPs that can be used in genetic engineering of HSCs or other cell types of interest. As these cell therapies progress through the development pipeline, investment in automated, scalable, and robust LNP manufacturing strategies remains prudent.
Ian Johnston is a field application scientist for RNA-LNPs at Cytiva.
