Senior Director Waters Corporation Milford, Massachusetts
To date, LNP mRNA samples have mostly been analyzed using surfactants for LNP disruption combined with CGE and sometimes reversed phase liquid chromatography. This approach requires offline sample preparation and can sometimes exhibit problematic repeatability and reproducibility. With this work, we have developed a hydrophilic interaction chromatography (HILIC) method that allows for the direct injection and on-column deformulation of LNPs. This novel approach utilizes an initial mobile phase containing 60-80% acetonitrile at an elevated column temperature with a simple gradient and aqueous ammonium salt eluent. This technique rapidly disrupts lipid components which are not retained under HILIC conditions, and facilitates the subsequent elution, integrity checks and quantitation of mRNA. Use of an amide-bonded 1.7 µm stationary phase column constructed from vapor deposited hardware has facilitated high recovery measurements and facile implementation of the method. Linearity of detection and the repeatability of these analyses with simple UV detection is being explored.
Learning Objectives:
Understand the Limitations of Traditional LNP mRNA Analysis Methods. Recognize the challenges associated with surfactant-based LNP disruption followed by CGE or reversed-phase liquid chromatography, including the need for offline sample preparation and issues with repeatability and reproducibility.
Learn the Principles and Benefits of the HILIC Method for LNP Analysis. Explore how hydrophilic interaction chromatography (HILIC) enables direct injection and on-column deformulation of LNPs, leveraging a mobile phase with high acetonitrile content, elevated temperatures, and ammonium salt gradients for efficient lipid disruption and mRNA quantitation.
Consider the Advantages of Advanced Stationary Phase Technology in HILIC. Understand how an amide-bonded 1.7 µm stationary phase with vapor-deposited hardware facilitates high recovery, reproducibility, and straightforward implementation in mRNA integrity and quantitation workflows.
