In part thanks to the clinical success of messenger RNA (mRNA) vaccines for the SARS-CoV-2 virus, RNA-based therapeutics are a rapidly expanding class of genomic medicines. The mRNA and lipid nanoparticle (LNP) delivery technology underlying some of the groundbreaking COVID-19 vaccines offer flexibility and speed in development, production and application ― key advantages which could change our current approach to prophylactic vaccines and enable new treatments for cancers and beyond.

With investment and interest in RNA-LNP therapeutics at an all-time high, scalable manufacturing processes are needed to support the growing number of investigational programs ranging from novel personalized therapies to formulations for a rapidly deployable, large-scale pandemic response.

However, since it’s a relatively new technology, manufacturers of LNP-encapsulated RNAs have barriers to overcome. Many of the growing pains are reminiscent of the early days of monoclonal antibodies (mAbs), when the industry struggled with low titers and poor purification yields that resulted in costly and inefficient commercial manufacturing. Process development was key to overcoming these challenges for mAbs and it will be a critical factor in driving RNA-LNP therapeutics forward as well.

For RNA-LNP therapeutics, the LNP encapsulation process is complex. Two liquid streams ― one containing lipid dissolved in an organic solvent (i.e., ethanol) and another with RNA in an acidic buffer ― must be mixed with precise control to induce spontaneous self-assembly. Microfluidic mixing is the predominant method to formulate LNPs, as it offers controlled and rapid non-turbulent mixing of the LNP and RNA species along with scalability and reproducibility (1-4).

Enabling technology platforms, such as NanoAssemblr™ instruments equipped with NxGen™ microfluidic technology, can produce batches of RNA-LNPs from microliter to liter volumes across a wide range of flow rates to ensure robust and reproducible particle production at both small and large scales. In particular, the NanoAssemblr™ Ignite™/Ignite+™ and Blaze™/Blaze+™ systems are easy-to-use platforms that facilitate a risk-based, cost-efficient approach to rapidly exploring the design space during process development as part of Quality by Design (QbD) best practices.

Downstream of the RNA-LNP formulation step, there are key process considerations that are unique to this new class of therapeutics, which are important to evaluate thoroughly and optimize at scale during process development. Here, we highlight three important process considerations across the RNA-LNP manufacturing workflow which include limit-size behavior, in-line dilution, and downstream tangential flow filtration (TFF) (Fig 1).

Fig 1. Key unit operations for RNA-LNP industrialized manufacturing.

Characterization of limit-size in RNA-LNP process development

One key goal of process development is to maintain critical quality attributes (CQAs) ― such as size, polydispersity (PDI), and morphology ― while critical process parameters (CPPs) like total flow rate (TFR) increase with scale to achieve higher throughput.

The limit size, defined as the minimum nanoparticle size achievable for a specific LNP composition and RNA payload, is a parameter of RNA-LNP formulations that is influenced by microfluidic process parameters (5-7) and typically ranges from 40 to 120 nm. An essential activity during process development for a new RNA-LNP formulation is to characterize nanoparticle size distributions relative to the mixing flow rates to construct a limit-size curve. Once sufficiently high flow rates have been met, any further increase in flow rate does not change the particle size. In theory, that means operating above the minimum limit-size flow rate should produce the same size RNA-LNP particles (7). This limit-size information can help in selecting flow rates, defining operational limits and optimizing throughput to produce nanoparticles with consistent CQAs that can withstand minor CPP fluctuations.

The new benchtop NanoAssemblr™ Ignite+™ system equipped with both the NxGen™ and NxGen 500 microfluidic mixers can be readily implemented to conduct limit-size characterization experiments for novel RNA-LNP drug candidates at flow rates representative of large-scale manufacturing. By using simple benchtop workflows and small volumes (<10 mL), performing limit-size characterization is fast and economical. Extending the capabilities beyond the NanoAssemblr™ Ignite™ system with higher flow rates (up to 200 mL/min) and larger volumes (up to 60 mL) enables formulations to be made on demand to support rapid and cost-effective process development.

After limit-size behavior has been defined, these findings are next translated to larger scale instruments, such as the NanoAssemblr™ Blaze™/Blaze+™ (up to 115 mL/min and 10 L) systems. Using the NanoAssemblr™ Blaze+™ system reduces the cost of process development and accelerates timelines by allowing users to perform large-scale formulations on a lower-cost system in less time than more traditional clinical systems. The material produced at this scale can support further analytical characterization of lead RNA-LNP drug candidates, initiation of formulation stability studies, and evaluation of upstream and downstream procedures including TFF, while enabling larger cohort in vivo animal studies during preclinical evaluation (7).

In-line dilution offers a consistent, scalable option for LNP stability

After initial RNA-LNP formulation steps, the ethanol percentage is typically 20% to 33% of the total volume. This concentration prevents the nanoparticles from organizing into a stable state, with LNP being prone to degradation if left in a high-solvent environment for too long. Therefore, after formulation, dilution and downstream purification are required to remove and exchange the organic solvent. Timely dilution of the formulated LNP is necessary for particle stability, particularly with large batch volumes, because of the increased time required for the formulation process.

Typically, at the research scale, dilution is accomplished by manual pipetting or through bulk dilution by pouring the formulation into the diluent buffer. However, at clinical scales, this approach is neither practical nor scalable. The NanoAssemblr™ Ignite+™, Blaze™/Blaze+™, and GMP system’s options incorporate in-line dilution post-formulation with dilution cartridges available for each system. In addition, in-line dilution ensures consistency since volumes are diluted at the same ratio compared to manual or bulk dilution.

Initial feasibility of in-line dilution versus bulk dilution can be executed on the NanoAssemblr™ Ignite+™/ Ignite+™ systems before moving to larger-scale studies on the NanoAssemblr™ Blaze™/Blaze+™ systems. Since the NanoAssemblr™ Blaze™/Blaze+™ systems enable longer large-scale runs that resemble those performed on the clinical scale GMP system, they provide valuable insight into formulation stability and particle behavior after prolonged formulation time. Typically, dilutions of up to 30:1 are required to evaluate long-term formulation stability.

It is advantageous to model in-line dilution during preclinical development at clinically relevant flow rates to uncover potential dilution sensitivities and lead LNP-drug-candidate stability issues in an economical and timely manner. Taking this step early on in process development can help mitigate potential setbacks later. Subsequently, in-line dilution parameters and stability studies can be validated at a larger scale on the NanoAssemblr™Blaze™/Blaze+™ systems for eventual transfer to the GMP system.

RNA-LNP downstream purification considerations

Following in-line dilution, ultrafiltration/diafiltration (UF/DF) is used to remove residual solvents and concentrate the RNA-LNP drug product to the final formulation in the desired buffer. LNP formulations can be shear sensitive and filtration unit operations need to be carefully optimized to achieve high flux and throughput while maintaining nanoparticle size and morphology — physicochemical properties that are intrinsically tied to their biodistribution and in vivo function (8).

Tangential flow (TFF) filtration is a scalable approach for UF/DF but migrating from less scalable purification methods such as dialysis and centrifugal filtration during scale-up is not without cost and risk. CPPs for the TFF process, such as the type of filtration cartridge, membrane material, shear rate, molecular weight size cut-off (MWCO), and transmembrane pressure (TMP) must all be appropriately characterized to prevent unwanted alterations to the size and morphology of the nanoparticles (4,8).

TFF varies at different scales and not all RNA-LNP formulations are inherently compatible with TFF systems, which is important to consider during early preclinical development as it can impact the manufacturability of an RNA-LNP drug candidate. This insight could be useful in selecting lead candidates during the development process. TFF studies performed at a smaller scale on an NanoAssemblr™ Ignite+™ system can be easily transitioned to the NanoAssemblr™ Blaze™/Blaze+™ instrument to scale up filter sizes and pumping systems. In addition, larger batch volumes are important to generate specific data for larger/long-term stability studies and gain understanding of what the process will look like at a clinical scale.

The current trend in biomanufacturing for protein therapeutics is towards creating more flexible, multi-product facilities through the adoption of single-use technologies, closed systems, and continuous processing. This shift is also true for nanomedicine production, which presents the opportunity for product-agnostic manufacturing platforms because of the modular nature of both the RNA and LNP technologies. Studies to assess the manufacturing process in a closed system environment can be performed on the NanoAssemblr™ Blaze+™ system, including consumables (i.e., bioprocess bags) and unit operations.

Establishing robust manufacturing processes will be critical to unlocking the full potential of genomic medicines as industry momentum shifts these new modalities into the mainstream. Employing vertically scalable production platforms moving from the NanoAssemblr™ Ignite™/Ignite+™ systems to the NanoAssemblr™ Blaze™/Blaze+™ systems will be invaluable to defining and optimizing downstream parameters, such as limit size, in-line dilution, and TFF, while supporting rapid and cost-effective preclinical process development.

The NanoAssemblr™ Ignite™/Ignite+™ system can first be used to effectively determine the feasibility of scalable processes, like in-line dilution, compared to manual processes for a given formulation at a small scale. The preliminary results can be transferred and adapted to the NanoAssemblr™ Blaze™/Blaze+™ system to evaluate process parameters at scale, which is predictive of clinical volumes, to de-risk technology transfer to cGMP production. In addition, the suite of NanoAssemblr™ instruments utilizes single-use components, allowing for quick adaptation to process development changes.

As the industry continues to evolve, these technology tools are key for acquiring extensive process knowledge in the process development phase, accelerating timelines, and providing a direct route to clinical development and, eventually, to market approval and commercial manufacturing.

 

References

1. Daniel S, Kis Z, Kontoravdi C, Shah N. Quality by Design for enabling RNA platform production processes [published online ahead of print, 2022 Apr 28]. Trends Biotechnol. 2022;S0167-7799(22)00080-4. doi:10.1016/j.tibtech.2022.03.012

2. Reichmuth AM, Oberli MA, Jaklenec A, Langer R, Blankschtein D. mRNA vaccine delivery using lipid nanoparticles [published correction appears in Ther Deliv. 2016 Jun;7(6):411]. Ther Deliv. 2016;7(5):319-334. doi:10.4155/tde-2016-0006

3. Whitley J, Zwolinski C, Denis C, et al. Development of mRNA manufacturing for vaccines and therapeutics: mRNA platform requirements and development of a scalable production process to support early phase clinical trials. Transl Res. 2022;242:38-55. doi:10.1016/j.trsl.2021.11.009

4. Roces CB, Lou G, Jain N, et al. Manufacturing Considerations for the Development of Lipid Nanoparticles Using Microfluidics. Pharmaceutics. 2020;12(11):1095. Published 2020 Nov 15. doi:10.3390/pharmaceutics12111095

5. Belliveau NM, Huft J, Lin PJ, et al. Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA. Mol Ther Nucleic Acids. 2012;1(8):e37. Published 2012 Aug 14. doi:10.1038/mtna.2012.28

6. Zhigaltsev IV, Belliveau N, Hafez I, et al. Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing. Langmuir. 2012;28(7):3633-3640. doi:10.1021/la204833h

7. Precision NanoSystems. Application Note: Developing a Scalable RNA-LNP Drug Product for Clinical Translation. Document ID: ignite+-AN-0622

8. Precision NanoSystems. Accelerating The Development And Scale-Up Of mRNA Vaccines. Cell & Gene. Published March 2, 2022. https://www.cellandgene.com/doc/accelerating-the-development-and-scale-up-of-mrna-vaccines-0001 Accessed August 9, 2022.