Among genomic medicine modalities, RNA-based therapeutics are a rapidly expanding class of drugs thanks to the demonstrated clinical success of the messenger RNA (mRNA) COVID-19 vaccines against SARS-CoV-2 virus. The underlying mRNA and LNP delivery technology is characterized by a great flexibility and development speed with respect to production and application, which has far-reaching potential to change the current industry approach to prophylactic vaccines and enable new treatments for cancers and beyond.  

With industry investment and interest at an all-time high, scalable manufacturing processes are needed to support the growing number of investigational RNA-LNP programs ranging from novel personalized therapies to rapidly deployable large-scale pandemic responses. However, as a relatively new technology, there remain barriers to successful industrialized manufacture of LNP-encapsulated RNAs. 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, requiring precise control over the mixing of two liquid streams, one containing lipids dissolved in an organic solvent (i.e., ethanol) and another with RNA in an acidic buffer, to induce spontaneous self-assembly. Microfluidic mixing offers controlled and rapid non-turbulent mixing of the LNP and RNA species and is the predominant method to formulate lipid nanoparticles (LNPs) because of its scalability and reproducibility^1-4. Enabling technology platforms such as Precision NanoSystems’ NanoAssemblr® instruments equipped with NxGen™ microfluidic technology can produce batches of RNA-LNPs from µL to liter volumes across a wide range of flow rates to ensure robust and reproducible particle production at both small and large scale. In particular, the NanoAssemblr Ignite™, Ignite+™, Blaze™ and Blaze+™ are easy-to-use platforms that facilitate a risk-based, cost-efficient approach to rapidly explore 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 equally 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 includes limit size behavior, in-line dilution and downstream tangential flow filtration (TFF) (Figure 1).  
  


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

Limit Size

The prevailing tenet of process development is to maintain critical quality attributes (CQAs), such as size, polydispersity (PDI) and morphology, while critical process parameters (CPPs), such as total flow rate (TFR), increase with production scale to achieve higher throughput. The limit size is a parameter of RNA-LNP formulations, defined as the minimum nanoparticle size achievable for a specific LNP composition and RNA payload, typically ranging from 40 to 120 nm, which is influenced by microfluidic process parameters^5-7. 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 particles⁷. This limit-size information can be applied to select flow rates, define operational limits and optimize throughput to produce nanoparticles with consistent CQAs that can withstand minor CPP fluctuations.

The new benchtop NanoAssemblr Ignite+ 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), limit size characterization is fast and economical to perform. Extending the capabilities beyond the Ignite 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, the next phase is to translate that behavior to larger scale systems, such as Blaze/Blaze+ (up to 115 mL/min and 10 L). Using Blaze+ reduces 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. Material produced at this scale can support further analytical characterization of lead RNA-LNP drug candidates, initiation of formulation stability studies, evaluation of upstream and downstream procedures including TFF, and enable larger cohort in vivo animal studies during preclinical evaluation⁷.

In-Line Dilution

After formulation, the ethanol percentage is typically 20-33% of the total volume which prevents the RNA-LNPs from organizing into a stable state. LNP particles are prone to degradation if left in a high-solvent environment for too long; therefore, after formulation, they need to be diluted and further purified downstream to remove and exchange the organic solvent. Timely dilution of the formulated LNPs is necessary for particle stability, which becomes even more important for large batch volumes because of the increased time required for the formulation process.  

Typically, at research scale, dilution is accomplished by manually pipetting or bulk dilution by pouring the formulation into the diluent buffer. However, at clinical scales, this is neither practical nor scalable. To circumvent this, the NanoAssemblr Ignite+, Blaze, Blaze+ and GMP System 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 vs. bulk dilution can be executed on the Ignite/Ignite+ before moving to larger scale studies on the Blaze/Blaze+. Since the Blaze/Blaze+ enables longer large-scale runs which resemble those performed on the clinical scale GMP System, it provides 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 early on in process development to mitigate potential setbacks later on. Subsequently, in-line dilution parameters and stability studies can be validated at a larger scale on the Blaze/Blaze+ for eventual transfer to the GMP System.

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 need careful optimization of filtration unit operations to achieve high flux and throughput while maintaining nanoparticle size and morphology—physicochemical properties that are intrinsically tied to their biodistribution and in vivo function⁸. Tangential flow filtration (TFF) 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, therefore it is important to understand this early during preclinical development since it can impact the manufacturability of an RNA-LNP drug candidate. This insight could be useful to help select lead candidates during the development process. TFF studies performed at smaller scale on Ignite+ can be easily transitioned to the Blaze/Blaze+ 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 to understand what the process will look like at 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 is also true for nanomedicine production, which present the opportunity for product-agnostic manufacturing platforms because of the modular nature of both the RNA and LNP technologies. Closed system feasibility studies and risk assessments can be performed on the Blaze/Blaze+ instrument with respect to consumables (i.e., bioprocess bags) and unit operations.

Establishing robust manufacturing processes will undoubtedly be critical to unlock the full potential of genomic medicines as industry momentum shifts these new modalities into the mainstream. To that end, employing vertically scalable production platforms moving from the Ignite/Ignite+ to the Blaze/Blaze+ will be invaluable to define and optimize downstream parameters such as limit size, in-line dilution and TFF to support rapid and cost-effective preclinical process development. The Ignite/Ignite+ 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 small scale. The preliminary results can be transferred and adapted to the Blaze/Blaze+ 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 utilize single-use components allowing for quick adaptation to process development changes. As 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, market approval and commercial manufacturing.  

Download the application note Developing a Scalable RNA-LNP Drug Product for Clinical Translation to learn more about essential process development activities during preclinical development.

References

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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.