November 1, 2023 PAO-10-23-CL-03
ONPATTRO® (patisiran, Alnylam Pharmaceuticals), an RNA interference (RNAi) therapeutic for the treatment of the polyneuropathy of hereditary transthyretin-mediated (hATTR) amyloidosis in adults, was the first nucleic acid–based drug formulated as an LNP to receive U.S. FDA approval (in 2018). FDA authorization of the two mRNA-based COVID-19 vaccines from Pfizer/BioNTech and Moderna clearly demonstrated the safety, efficacy, and potential of mRNA products and galvanized further development in the sector.
Countless clinical trials are underway for many different types of RNA-based therapeutics and vaccines. DNA-based products and CRISPR-based gene-editing solutions that rely on guide RNAs are also advancing through development. Many of these candidates rely on LNPs as a carrier vehicle owing to their ability to stabilize the nucleic acids and facilitate their entry into cells.
The production of nucleic acid–LNP products is influenced by the nature of the nucleic acid and the specific lipids used to generate the LNPs, with the ionizable lipid the most important of the constituent lipids. The stability and processability of the particles are directly influenced by the properties of these components. An efficient and consistent mixing process, an appropriate method for solvent removal and concentration of the LNPs, effective solutions for particle surface modification, and appropriate purification technologies are also essential.
These different aspects of a nucleic acid–LNP manufacturing process can generally be highly controlled at small scale, but achieving consistency can be challenging as the process is scaled. A thorough understanding of the entire process is necessary for successful scale-up, which can only be realized via real-time monitoring of critical process parameters.
Continuous manufacturing provides an ideal solution for achieving robust, scalable nucleic acid–LNP production. Typically, continuous processes involve much smaller quantities that interact for much shorter periods of time, which is hugely beneficial for lipid-based products. In addition, these attributes — combined with real-time monitoring — result in a higher degree of control, affording consistent product quality throughout longer runs. Minimal human intervention reduces risks of error and contamination, as would containment in a closed system. Continuous manufacturing also minimizes holding time (in the range of seconds) between steps, reducing the risk of destabilization and degradation. As a result, LNPs can be generated with a tight and consistent particle distribution and high encapsulation efficiency. For continuous processes involving ultra-small volumes, such as those implemented using microfluidic devices with restricted volumetric throughput, scale-up can be achieved by running multiple processes in parallel.
While batch processes for nucleic acid–LNP production typically comprise anywhere from six to nine unit operations, with continuous nanoparticle generation, it is possible to fold all of those unit operations into a single process within a closed system. Hold steps and testing required between individual steps are eliminated. As importantly, with real-time analytics, it is possible to divert or halt processes midstream if needed to make adjustments, significantly reducing waste. Rather than losing an entire 200-liter batch that is only tested at the end of the process, only milliliters would be lost with a continuous manufacturing process.
LNP formation is more complicated than some typical continuous processes. Not only the properties of the nucleic acid but the surface charges and steric stability of the lipids employed influence particle formation and ultimately particle-size distribution. Similarly, the ratios of the different lipids and the ratios of the lipids to the nucleic acid can impact the stability of the nanoparticles. It is therefore critical to develop a product formulation and continuous process that together enable precise control of these parameters.
DIANT Pharma began its journey toward continuous nucleic acid–LNP manufacturing with the receipt of multiple awards for research being performed at the University of Connecticut on liposome nanoparticles. Roughly $5.5 million was awarded for this work over the course of several years, much of it driven by the increasing interest of the FDA in advanced manufacturing solutions –– particularly continuous processing. The focus was simplifying LNP production through automation and the development of a truly continuous process. Patents were filed through the university, with the core patent granted in 2019. DIANT Pharma formed soon after.
Much of the success achieved to date can be attributed to the establishment of a team with expertise in engineering, nanoparticle technology, and the design-of-experiment approach to process development. This combination of capabilities enabled rapid prototyping, in-house programming of sensors used for real-time monitoring (process analytical technologies (PAT)) and the collection of information on the relationships between process parameters and material attributes.
The goal at DIANT Pharma from the outset has been to develop an end-to-end nucleic acid–LNP manufacturing platform leveraging a closed system and an abundance of PAT. The focus has been on a single, continuous process stream that incorporates not only particle generation but downstream operations, such as solvent removal, buffer exchange, particle surface modification, particle purification, and fill/finish.
The successfully established process builds on the bottom-up, ethanol injection strategy combined with DIANT’s proprietary 2TFF module that takes advantage of single-pass tangential-flow filtration (SPTFF) technology. This technology does not include any recirculation of the process solution and reduces biofouling, enabling a truly continuous operation for extended periods of time.
The DIANT Pharma system for the production of mRNA–LNPs and other pharmaceutical-grade nanoparticles offers the benefits of a smaller production footprint complemented by reduced facility and storage requirements, ready scalability, reduced human intervention, elimination of holding tanks/times, and, specifically for this process, greater particle size control.
In fact, DIANT’s continuous nanoparticle generation system not only eliminates numerous batch-based unit operations — it also generates very high-quality nanoparticles with improved properties compared with those typically obtained from batch processes. The inline/atline sensors incorporated into the system provide real-time process data (e.g., temperature, pressure) throughout the entire process. As a result, users have extensive insight regarding process performance and the ability to understand what is happening during the continuous flow of material, which provides a high level of process control.
In addition, only one set of operators is required to run the entire process, saving time and money on personnel and personnel training. Furthermore, because the system is fully closed, cleanroom requirements are also reduced. As a result, we consider DIANT’s approach to be revolutionary rather than evolutionary. It represents a unique solution for nanoparticle generation and has the potential to change the way in which this process is implemented in the pharma industry, including for the production of mRNA–LNPs with therapeutic and vaccine applications.
At the heart of the DIANT continuous LNP production solution is a single injection site for formation of the nanoparticles that is designed to enable easy process scaling. The proprietary DIANT Jet technology includes a turbulent jet mixer that operates in co-flow for the rapid mixing of an ethanol phase containing the lipids with an aqueous phase containing the nucleic acid. It affords a high degree of mixing under controlled conditions, allowing fine tuning of the particle size for different applications and tissue targeting and a very low polydispersity (a polydispersity index of typically 0.1 or lower) for any given formulation.
Furthermore, nanoparticles generated over a range of volumetric flow rates have similar characteristics, making this solution highly desirable from a risk-mitigation perspective. In addition, DIANT’s lab and research unit (LARU) uses the same technology and geometry as the large-scale LiFT system, eliminating the need for extensive process optimization when scaling up from R&D to clinical and commercial production. The mixer has specifically been tuned across the system to provide consistent results. As a result, the LARU can be used for the majority of product/process development with transition to the LiFT system only when larger quantities of product are required.
As importantly, the technology can be used to produce much more than just nucleic acid–LNPs. In addition to LNPs and other lipid-based nanoparticles, DIANT’s system can be used to generate polymeric nanoparticles, polymeric micelles and other polymer conjugates, polymer/lipid particles, nanosuspensions, and nanoemulsions.
Currently, the DIANT system for the production of nucleic acid–LNP products supports particle synthesis and downstream processing up to the point of fill/finish activities. It is, therefore, end-to-end adaptable, which will be fully achieved once it is possible to integrate a fill/finish assembly with the existing solution.
The difficulty in doing so at present does not reside with the production technology but the need to conduct release testing, such as sterility testing, before going to a fill/finish assembly. The assays currently approved for use by regulatory authorities are lengthy, with turnaround times of hours to days. DIANT Pharma is looking for potential control solutions that could be built in that would provide the necessary level of sterility assurance and meet regulatory compliance expectations. The FDA might, for instance, accept new qualified sensors that determine bacterial counts in real time. If such a PAT solution were introduced and deemed compliant, it could pave the way to integration of fill/finish activities with the existing DIANT system.
With respect to RNA synthesis, there are other companies developing continuous processes leveraging RNA synthesizers. If such a solution is developed and accepted by the regulatory authorities, DIANT would likely be able to integrate this step with the existing system to achieve an even more completely end-to-end RNA–LNP continuous production platform. A true end-to-end solution would also incorporate lipid synthesis. DIANT is currently exploring various collaborations and partnerships with companies that may allow expansion of the current offering as a more complete end-to-end approach with respect to nucleic acid and lipid synthesis.
DIANT Pharma is committed to working closely with customers — and potential customers — to explain and demonstrate how the DIANT platform enables continuous production of nanoparticles and the numerous benefits it provides. Many customers provide material with which DIANT generates LNP products for their evaluation. This first step provides the opportunity to build relationships and help clients better understand and appreciate the technology.
Companies that choose to purchase a DIANT system are then provided as much support as they require, including assistance with the performance of feasibility studies, product development, and process optimization and scale-up. If needed, collaboration with customer scientists can begin with gaining better understanding of the particle properties to identify the optimum particle size, encapsulation efficiency, and other attributes. Many clients also appreciate assistance with optimization of downstream processes, including SPTFF, sterile filtration, and so on.
Evaluation of manufacturing and/or commercialization strategies is another aspect of our support services. Results could potentially include integration of new sensors or algorithms to meet specific customer needs. The goal at DIANT Pharma is for all customers, regardless of size, to get to know and trust us and truly understand who we are and the value we provide through not only our unique manufacturing system but our unique and focused expertise.
The DIANT Pharma manufacturing platform for the continuous production of nanoparticle-based products reflects the company’s commitment to advancing continuous manufacturing through the development of new techniques and technologies. We are eager to work with customers to demonstrate the effectiveness of our solution and prove its value from the lab through clinical to commercial production, which we believe will only become even clearer as more data is generated and the field matures.
Antonio Costa, Ph.D. is chief executive officer at DIANT Pharma Inc., an early-stage company that provides continuous manufacturing technology, and an assistant research professor at UConn in the Pharmaceutical Sciences department. His work focuses on continuous manufacturing approaches, downstream processing of nanoparticle formulations, physicochemical characterization, process analytical technology integration and systems engineering. He is an author on 12 peer-reviewed publications and inventor on two granted patents and six patent applications.
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