(T1130-02-08) End-To-End Process for Improved Purity, Combined with Innovative Analytics of LNP-Based Therapeutics

Purpose: Lipid nanoparticles (LNPs) are at the forefront of nucleic acid delivery technologies, playing a crucial role in the advancement of these therapies. There are, however, several challenges that need to be addressed regarding their manufacturing and characterization: correct formation and integrity of the drug product, sufficient recovery during purification while maintaining functionality, and producing a well-purified and thoroughly characterized product. To introduce fine-tuned separation and obtain uniform and functional product, purification on CIM monolithic columns was developed.

Methods: RNA was transcribed in vitro from pDNA and both were purified using CIM monolithic columns, enhancing the quality of encapsulation process [1]. The purification of the encapsulated RNA-LNP product is currently streamlined at large scales with TFF. To enhance separation capabilities, a chromatography purification method for the RNA-LNP product was developed using CIM monolithic columns. Throughout this process, PATfix analytics monitored all quality attributes. For RNA-LNP characterization, the PATfix LNP Switcher Platform was employed to assess product quality, including encapsulation efficiency, encapsulated RNA quantity and purity, and the presence of RNA-lipid adducts. Additionally, lipid ratios, cell-based assay for RNA-LNP activity and size measurements (NTA, DLS) were determined.

Results: Produced RNA molecules of different size and type, were purified using CIM monolithic column process. In this stage, payloads were characterized and encapsulated with high efficiency into LNPs. Mobile phases for RNA-LNP purification were fine-tuned to achieve high recovery and product stability using high-throughput techniques (CIM 96-well monolithic plates). Under optimized conditions, LNPs were loaded onto CIMmultus columns immediately after encapsulation and subsequent neutralization. The purification process effectively removed ethanol and unencapsulated RNA. Compared to standard purification process, the results demonstrate higher recoveries, more uniform particles due to lower size distribution and elevated activity in terms of protein production, assessed by cell-based assay measuring luciferase reporter protein. Total mRNA recovery and LNP activity were significantly improved using this novel process compared to existing filtration methods.

Conclusion: Novel chromatographic process using CIM demonstrates superior activity and uniformity of the RNA-LNP particles compared to traditional methods. By integrating monolithic technology at each stage from RNA production to LNP integrity assessment, we ensure consistent product quality and overall process performance and efficiency, supporting the development of safe and effective LNP-based therapies.

References: [1] Krušič et al, Molecular Therapy: Nucleic Acid, 2025