How NxGen Microfluidic Devices Enable Large Scale Production of Nanoparticles

“Microfluidic technology is the state of the art in making Lipid Nanoparticles that are crucial to the safety and efficacy of genomic medicines”

Figure 1. Microfluidic mixing for self-assembly of lipid nanoparticles

The production of RNA lipid nanoparticles (LNPs) such as those used in COVID-19 vaccines involves mixing RNA in an aqueous buffer with lipids dissolved in ethanol to trigger the self-assembly of LNPs. The mixing conditions of the RNA and lipids are crucial to drug product quality. During fluid mixing, fluid flow occurs within a continuum from turbulent to laminar conditions. Turbulent mixing is familiar in our day-to-day lives, for example, when we stir our tea with a spoon. At the molecular level, turbulent conditions are highly random and difficult to control. The effects of this may not be very consequential in the kitchen, but sophisticated and innovative drug delivery nanoparticles, subject to regulatory scrutiny, require more sophisticated production controls.

The microfluidic mixing process involves complex intermolecular interactions between molecules of solvent, buffer, and at least five chemical types that affect the resulting particle's physical characteristics. These characteristics, such as the particle size, affect their pathway in the body and the resulting immune response. Therefore, well-defined particle characteristics are critical to ensure the safety and efficacy of the drug product. Microfluidic mixing provides access to non-turbulent conditions that enable rapid, controlled, and reproducible mixing of fluids in milliseconds to enable continuous self-assembly of drug delivery nanoparticles in a controlled environment. The fluids are manipulated at the micro-scale and often flow through channels of different lengths and geometries.

Several advantages of microfluidic mixing have established it as the go-to technology for the development of RNA vaccines⁴, and other drug delivery technologies:

• - Reduced reagent consumption                                                    
• - Controlled non-turbulent mixing 
• - Tunable process parameters to optimize particle characteristics 
• - Small footprint to minimize cleanroom space requirements
• - Batch-to-batch reproducibility

Benefits of Microfluidics for Nanoparticle Production Over Traditional Methods

Both process and chemistry influence the fabrication of nanoparticles. When lipid-based nanoparticles were first used for small molecule drug delivery, such as the liposomal cancer drug Doxil™ doxcorubicin, they were created through a complex multi-step process that included thin-film hydration followed by high-pressure homogenization and sonication or extrusion through a nanoporous membrane. The resulting liposomes consist of an aqueous compartment within a lipid bilayer. Liposomes were also used in preclinical research to deliver genes by first forming liposomes containing cationic lipids and then mixing them with nucleic acids to allow electrostatic complexation. After discovering that cationic lipids are cytotoxic, researchers began using pH-sensitive ionizable lipids that are not charged at physiological pH to improve tolerability. At the same time, a second-generation bottom-up self-assembly method was developed, in which lipids dissolved in ethanol were combined with nucleic acids dissolved in an aqueous buffer. Particle formation and nucleic acid complexation were accomplished in a single step, simplifying production and allowing for a more scalable continuous flow format. By encapsulating the nucleic acid inside a condensed-core particle, the resulting lipid nanoparticles differed in structure from liposomes⁵. LNPs were discovered to outperform first-generation liposomes⁶, however, this method is dependent on the creation of turbulence, which is accomplished by combining streams of aqueous and ethanol reagents at high speeds, such as in a T-junction mixer. Random molecular collisions govern mixing, which necessitates large volumes and high flow rates. Therefore, scaling the process up or down is challenging and requires considerable process redevelopment.

However, the third-generation technique that includes a microfluidic Staggered Herringbone Micromixer (SHM) mixer maintains the advantages of continuous flow manufacturing and additionally allows greater control over the mixing environment. Non-turbulent fluid mixing ensures consistent conditions for each volume of liquid passing through the mixer that imparts reproducibility within a batch and between batches. In addition, process parameters such as the flow rate ratio (FRR) and total flow rate (TFR)², can be tuned to dial in the physicochemical characteristics of the produced particles, influencing the performance of LNP drug products. There is a range of microfluidic channel geometries and architectures that have been employed in nanomedicine production. Two of the most successful are listed below:

NanoAssemblr® Classic Mixer - This design produces size-controlled nanoparticles by altering flow rate and total flow rate ratios. The Classic Mixer is very established in the field of preclinical development and has been cited in >500 peer reviewed publications. However, it has some limitations. It needs consistent fabricating of herringbone structures at the microscale, which leads to complicated and expensive processes³. The original Staggered Herringbone Micromixer mixers published by Belliveau et al. were designed to operate optimally at 5 mL/min, already an order of magnitude faster than other microfluidic designs⁷. Precision NanoSystems improved the design of SHM with the Classic Mixers that operate up to 20 mL/min, the highest throughput that can be possibly achieved in an SHM while maintaining flow characteristics. This capacity is enough to encapsulate ~ 850 mg (gross) of mRNA – enough for over 28,000 vaccine doses -- in a 4-hour run.

NanoAssemblr® NxGen™ Mixer - Precision NanoSystems has developed this new mixer design that enables simplified single-mixer scale-up, a direct replacement for Classic Mixer. The NxGen™ design features unique circular structures within the flow route resulting in exceptional mixing efficiency under non-turbulent conditions and a higher single-mixer flow rate. This scalable design has been made to smaller dimensions to limit consumption of reagents for applications in screening, more sensitivity, lower cost, rapid analysis, precise control, and reproducibility. NxGen™ produces particles with the same critical quality attributes as the Classic Mixer³ while enabling single-mixer flow rates from 1 to 200 mL/min, which is higher than flow rates frequently reported for T-junction mixers^8. At 200 mL/min, a single NxGen™ mixer can nominally encapsulate 8.5g of mRNA (> 283,000 vaccine doses) in a 4h run – 10 times the capacity of the Classic Mixer. For additional perspective, Pfizer-BioNTech’s phase 3 study for their COVID-19 vaccine required 44,000 doses – or ~1.32g of encapsulated. Therefore, the wide range of flow rates⁹ available with NxGen™ is well suited for preclinical development and translating to clinical studies without changing technologies, which minimizes process redevelopment and risk.

Table 1. Comparison of the NanoAssemblr ®Classic Mixer (SHM), NxGen™ Mixer (Toroidal mixer (TrM)), T-Junction Mixer * Based on typical mRNA formulation parameters and vaccine dose of 30 µg mRNA per dose. Does not account for process yield.

NanoAssemblr® Classic Mixer	NanoAssemblr NxGen™ Mixer	T-Junction Mixer
Flow rate capacity 1 mL/min to 20mL/min	Flow rate capacity 1 mL/min to > 200 mL/min	Flow rate capacity 40 mL/min to 60 mL/min10
Nominal gross product capacity: > 850mg (> 28,000 vaccine doses) per mixer per 4h shift*	Nominal gross product capacity: 8.5g (> 283,000 vaccine doses) per mixer per 4h shift*	Nominal gross product capacity: 2.6g (85,000 vaccine doses) per mixer per 4h shift*
Platform used: NanoAssemblr® Classic Mixer	Platform used: Spark™ Ignite™ & Ignite+™ Blaze™ GMP system

Non-turbulent mixing is more attractive than traditional formulation methods due to the physics happening at the micro-scale that promotes:

1. Uniform sized nanoparticles: With non-turbulent mixing, nanoparticles can be synthesized with high uniformity and a suitable size for nano applications (around 50–300 nm), making it possible to control the flow and mixing conditions.¹
2. Better drug-loading efficiencies and storage: Although hydrophobic drugs can be entrapped in liposomes, the encapsulation efficiency is typically low because of entrapment at the bilayer interface. In this regard, Kastner et al.¹ created liposomes using a chaotic advection micromixer device to solubilize propofol, a poorly water-soluble drug. 

   The result:

   • - Liposomes encapsulated significantly more drugs than sonicated liposomes
   • - Furthermore, liposomes demonstrated good stability and remained unaffected after eight weeks of storage at 4 °C and 25 °C 
   •  
3. Promising clinical application delivery results: Microfluidics provides better protection of nucleic acid and stability. In a study using microfluidic-based core-shell nanoparticles, researchers created a novel lipid/polymer hybrid nano assembly composed of siRNAs complexed in the inner hydrophilic core of reverse poly ɛ-caprolactone-polyethyleneimine (PCL-PEI) micelles before coating a neutral lipid membrane^1. Compared to bulk-mixed lipid/micelle/siRNA nanoparticles, the core-shell nanostructure produced by microfluidics provided more robust protection of siRNA locked in the core and better stability in circulation. Furthermore, these nanoparticles inhibited tumor growth by significantly downregulating EGFR (epidermal growth factor receptor) mRNA and protein expression levels in vitro and in vivo.
4. Translating liposomes from the bench to the clinic: With the aim of scalability, Forbes et al.³ used a NanoAssemblr® Classic Mixer to produce liposomes while incorporating insulin, bovine serum albumin (BSA), or ovalbumin (OVA) along with SHM device in-line purification and in-line particle size monitoring for in-process control.

The result:

• The liposome formulations offered a high protein loading (20–35%) compared to sonication or extrusion methods (<5%).
• Optimization was easy for producing highly loaded liposomes with size control (60–100 nm) and a PDI < 0.2.

Scaling Nanoparticle Production for Clinical or Industrial Use

RNA-based therapeutics demand the move beyond the laboratory for large-scale manufacturing of genomic medicines. Microfluidic technology allows production to be scaled from batch sizes suitable for pre-clinical studies to clinical and commercial production using the same process with minimal redevelopment. As detailed above, a single NanoAssemblr® Classic mixer operating at 20 mL/min can produce 28000 vaccine doses in a 4h shift – enough for a phase I/II study. For larger batches, scaling out is performed with the same unit operation by employing multiple identical microfluidic mixers in parallel. Additionally, NxGen™ technology allows facile scale-up as well, where mixer dimensions are enlarged to allow an order of magnitude higher flow rates through a single mixer while preserving the underlying physics of mixing that ensures well-controlled, reproducible conditions. Therefore, NxGen™ allows the scale-up of nanoparticle production from pre-clinical to industrial scales using the same technology, thus minimizing process redevelopment and mitigating risk.

Scalable Microfluidic Method for Production of Ionizable Lipid Nanoparticle (iLNP)² - NxGen^™ can provide scale-independent production because the systems can be run from small laboratory-scale to continuous production via scale-up rather than scale-out. In a study by Roces et al., Precision NanoSystems GenVoy-ILM™  lipid mix composition, which contains an ionizable lipid similar to MC3, with an apparent pKa value 6.0 was chosen. The NanoAssemblr® Classic Mixer and NxGen™ were then used to generate blank and polyadenylic acid (PolyA)-loaded GenVoy-ILM™ iLNP at 12 (Classic and NxGen), 60 (NxGen only), and 200 mL/min TFR (NxGen GMP). The blank formulation was significantly (p < 0.05) smaller in size (55 nm) than the PolyA-loaded counterpart (78 nm). At all speeds tested, both LNPs had comparable sizes. In addition, the nanoparticles showed equivalent sizes with high polyA encapsulation efficiencies (larger than 95%).²

The new NxGen™  microfluidic mixer design allows seamless scale-up production from bench-scale (12 mL/min) to GMP production requirements of over 12 L/h.³ Furthermore, with tangential flow filtration, it is possible to achieve scalable downstream processing to support the microfluidic production of nanomedicines with a high yield. Finally, the study confirmed the manufacturing possibility of the nanoparticles quickly and reproducibly using a scale-independent manufacturing process, reducing the risk of the journey from the bench to the approved product.

Microfluidic platforms with high throughput and flow rate capacity, such as the NanoAssemblr® (Precision NanoSystems, Vancouver, BC, Canada)², are already available on the market. The NanoAssemblr® manufacturing platform allows the incorporation of nucleic acid and lipid formulations for mRNA drugs into one end of a device small enough to fit on a lab workbench. A well-formed, stable LNP is just a button-press away. LNPs made with the NanoAssemblr® Platform have a unique homogeneous structure with exceptionally uniform. Furthermore, because NxGen™ technology is scalable from lab scale to commercial batch sizes with same mixer design, LNP production volumes can be easily increased, saving several months on the development time of every drug candidate in the pipeline resulting in cost savings. And it dramatically simplifies mRNA-LNP development, putting it within reach of researchers who understand the disease well, enabling them to explore the clinical potential of genetic drugs.

For more information on microfluidics, please click on the link below.
https://www.precisionnanosystems.com/platform-technologies/nxgen

References:

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