Development and optimization of LNP formulations – using the KNAUER NanoScaler

Significance of the Topic

Lipid nanoparticles (LNPs) have emerged as a leading platform for delivery of nucleic-acid therapeutics due to their biocompatibility, biodegradability, high encapsulation efficiency and scalable manufacturing. The successful deployment of mRNA vaccines against SARS-CoV-2 has accelerated interest in LNP systems for applications in infectious diseases, oncology, and genetic disorders. A robust, reproducible and scalable mixing technology is central to the development of clinically relevant LNP formulations.

Objectives and Study Overview

This study presents the use of KNAUER’s NanoScaler, a benchtop Impingement Jets Mixing (IJM) system, to optimize LNP formulation parameters at Curapath. Major objectives included:

• Defining conditions for tunable particle size and low polydispersity index (PdI)
• Maximizing encapsulation efficiency of mRNA and plasmid DNA (pDNA)
• Assessing reproducibility and scalability from 1 mL research batches to multi-liter production
• Comparing performance against a conventional Herringbone microfluidic mixer
• Demonstrating in vitro transfection potency in various cell lines

Methodology and Instrumentation

Curapath optimized LNPs using NanoScaler mixers (IJM 1–5) under varying total flow rates (TFR) and aqueous:organic ratios. Key process steps:

• Injection of lipid-in-ethanol and nucleic-acid-in-buffer streams via precision pumps and a 1 mL sample loop
• Post-mixing dilution in PBS and ethanol removal by centrifugal concentrator or tangential flow filtration
• Particle characterization by dynamic light scattering (Stunner)
• Encapsulation efficiency measured with Quant-iT RiboGreen assay
• Transfection assessed by luciferase expression in HepG2, HEK 293 and HeLa cells
• System cleaning validated by absence of lipid or nucleic-acid residues

Main Results and Discussion

Particle size and PdI:

• mRNA-LNPs: 80–100 nm; pDNA-LNPs: 110–130 nm, PdI <0.2
• Optimal TFR of 2–4 mL/min yielded smallest, most uniform particles
• Increasing TFR beyond 5 mL/min increased size variability

Encapsulation efficiency remained above 90% across flow rates. Initial lipid concentrations above 2.9 mg/mL provided consistent size and PdI; lower concentrations led to larger and variable particles. In-line dilution did not affect LNP characteristics. Scale-up experiments (1 mL to 50 mL) produced comparable size, PdI and encapsulation. Larger IJM mixers enabled multi-liter production within hours. Transfection assays showed mRNA-LNPs delivering superior luciferase expression, particularly in HepG2 cells.

Benefits and Practical Applications

The NanoScaler platform provides:

• High reproducibility and batch‐to‐batch consistency for R&D and production
• Precise control of particle dimensions by adjusting TFR
• Capability to optimize with minimal sample consumption
• Seamless scale‐up using identical mixing technology
• Efficient cleaning protocol compatible with GMP environments

Future Trends and Potential Applications

Advances in continuous manufacturing and automation will further streamline LNP production. Future opportunities include integration of real-time analytics, expansion to diverse nucleic acid payloads (e.g., siRNA, gene editors), development of next-generation vaccines and therapeutics, and alignment with evolving regulatory standards for lipid nanoparticle platforms.

Conclusion

KNAUER’s NanoScaler IJM system demonstrates robust capability for formulating high-quality LNPs encapsulating mRNA and pDNA. Its reproducibility, tunability, and direct scalability support rapid translation from preclinical development to commercial manufacturing, meeting industry demands for efficient and flexible nucleic acid delivery systems.

Instrumentation

• NanoScaler Impingement Jets Mixers (IJM 1–5) with precision pumps and sampling loops
• Vivaspin 500 centrifugal concentrator and TFF hollow fiber filtration units
• Dynamic light scattering analyzer (Stunner)
• Quant-iT RiboGreen fluorescence assay
• Herringbone microfluidic mixer (Darwin Microfluidics) for benchmark comparison

References

1. Anderson EJ et al. Safety and immunogenicity of SARS-CoV-2 mRNA-1273 vaccine in older adults. NEJM 2020;383(25):2427-2438.
2. Polack FP et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. NEJM 2020.
3. Hou X et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater 2021;6(12):1078-1094.
4. KNAUER expands into lipid nanoparticle production equipment. Knauer.net.
5. Biontech investor information on vaccine partnerships. investors.biontech.de.