Formulation of Nucleic Acids by Encapsulation in Lipid Nanoparticles for Continuous Production of mRNA

Abstract

The development and optimization of lipid nanoparticle (LNP) formulations through hydrodynamic mixing is critical for ensuring the efficient and cost-effective supply of vaccines. Continuous LNP formation through microfluidic mixing can overcome manufacturing bottlenecks and enable the production of nucleic acid vaccines and therapeutics. Predictive process models developed within a QbD Biopharma 4.0 approach can ensure the quality and consistency of the manufacturing process. This study highlights the importance of continuous LNP formation through microfluidic mixing in ensuring high-quality, in-specification production. Both empty and nucleic acid-loaded LNPs are characterized, followed by a TFF/buffer exchange to obtain process parameters for the envisioned continuous SPTFF. It is shown that LNP generation by pipetting leads to a less preferable product when compared to continuous mixing due to the heterogeneity and large particle size of the resulting LNPs (86–104 nm). Particle size by continuous formation (71 nm) and the achieved encapsulation efficiency (EE) of 88% is close to the targeted parameters for Pfizer’s mRNA vaccine (66–93 nm, 88%EE). With the continuous encapsulation of nucleic acids in LNPs and the continuous production of mRNA in in vitro transcription, the basis for the holistic continuous production of mRNA is now established. We already showed that a fully autonomous process requires the incorporation of digital twins and a control strategy, with predictive process models and state-of-the-art PAT enabling real-time-release testing. This autonomous control can considerably improve productivity by about 15–20% and personnel as well as chemical reduction of about 30%. The results of this work complement this, laying the basis for fully continuous, bottleneck-free production of mRNA and other cell- and gene-therapeutic drug/vaccine candidates in a GMP- and QbD-compliant Biopharma 4.0 facilities on a flexible scale.

Digital Twin Fundamentals of mRNA In Vitro Transcription in Variable Scale Toward Autonomous Operation

“…The results published here are the basis for a fully continuous, bottleneck-free production process of mRNA, including HTS, which can in the future be adapted to other drugs/vaccine candidates. For this, a predictive process model and process analytical technologies, as well as the continuous formulation of mRNA into lipid nanoparticles as described in detail already, 105 will be needed. Potential drug candidates can be screened in the PFR by generating a segmented flow in a high-throughput approach and then manufactured from 1,000 clinical doses to the 10 million manufacturing scale doses in one GMP- and QbD-compliant Biopharma 4.0 facility, enabled by the integration of state-of-the-art PAT and predictive validated process models.…”

Digital Twin for Continuous Production of Virus-like Particles toward Autonomous Operation

“…For this purpose, the software determines the diffusion coefficient from the time-dependent fluctuations in the scattered light by applying a second-order correlation function. Based on this, the hydrodynamic diameter was calculated, taking into account the eluent properties. , The particle concentration is based on the light scattering, which was recorded by the MALS detector. To calculate the particle concentration, information about the refractive index of the particles and the solvent, as well as the particle volume, is required.…”

“…Based on this, the hydrodynamic diameter was calculated, taking into account the eluent properties. 105,106 The particle concentration is based on the light scattering, which was recorded by the MALS detector. To calculate the particle concentration, information about the refractive index of the particles and the solvent, as well as the particle volume, is required.…”

CMC Strategies and Advanced Technologies for Vaccine Development to Boost Acceleration and Pandemic Preparedness

“…The continuous LNP generation process can further be connected to the continuous formulation and filling process. One example of such a process is discussed by Aline Hengelbrock [ 77 ] where different methods of LNP generation and ethanol removal are presented.…”