Microfluidics for throughput scalable formulation of mRNA lipid nanoparticle technology

Lipid nanoparticles (LNPs) are a potent delivery technology that encapsulate nucleic acids, such as messenger RNA (mRNA), and deliver them intracellularly for therapeutic and vaccine applications. However, a challenge towards the broad application of LNPs for mRNA therapeutics and vaccines is the development of formulation strategies that accommodate the 4-log order range of throughputs needed from early development to clinical translation. While microfluidic processes can improve LNP performance by increasing reproducibility and enhancing LNP physical properties, these production methods are typically not employed commercially due to their limited throughput of <0.1 L/hr. Currently, LNPs are formulated by either bulk mixing or T-junction mixing for clinical applications, but these methods have higher batch to batch variability due to turbulent mixing and they cannot scale down to the small volumes needed for rapid LNP screening. To address this challenge, we engineered a microfluidic platform that incorporates up to 256 mixing devices operating in parallel on a single microfluidic chip for throughput scalable LNP production. These chips achieve >250x production rates compared to standard microfluidic devices, with total flow rates up to 20 L/hr. We fabricated these devices in polydimethylsiloxane (PDMS) as a proof-of-concept chip and in silicon and glass substrates to demonstrate compatibility with pharmaceutical manufacturing. To demonstrate the power of this approach, we show that our microfluidic-formulated LNPs are 4-5 times more potent than LNPs produced by traditional methods. Further, we formulated SARS-CoV-2 mRNA LNP vaccines using our microfluidic platform and induced robust immune responses in mice. Then, we engineered a microfluidic chip to leverage low LNP production rates to precisely mix reagents on-chip to formulate LNP libraries for rapid screening. Finally, we use advanced characterization methods, such as small-angle x-ray scattering (SAXS), to elucidate differences in LNP molecular packing between microfluidic-formulated LNPs and bulk mixed LNPs. In sum, our platform enables throughput-scalable LNP production while maintaining ideal LNP properties for potent mRNA delivery in vivo for emerging RNA-based therapeutics and vaccines.