Two quality and stability indicating imaged CIEF methods for mRNA vaccines

Area of Science:

• Analytical Chemistry
• Biotechnology
• Pharmaceutical Sciences

Background:

• Messenger ribonucleic acid (mRNA) vaccines encapsulated in lipid nanoparticles (LNPs) require robust quality control to ensure stability and efficacy.
• Existing methods may not fully capture the complex interplay of factors affecting mRNA-LNP integrity.
• Developing advanced analytical techniques is crucial for characterizing these sophisticated drug delivery systems.

Purpose of the Study:

• To develop and validate two novel imaged capillary isoelectric focusing (icIEF) methods for assessing mRNA-LNP quality and stability.
• To investigate the impact of stress conditions and lipid composition on mRNA-LNP characteristics.
• To establish methods capable of detecting batch-to-batch variability and monitoring stability over time.

Main Methods:

• Development of two icIEF methods utilizing UV absorption detection, with one employing 9 M urea and the other a combination of 5.5 M urea and 2 M N-ethylurea.
• Analysis of stressed and lipid composition-modified mRNA-LNP samples.
• Enhanced sensitivity achieved through fluorescence detection with an added dye; results corroborated by encapsulation assays and particle sizing.

Main Results:

• The 9 M urea method provided detailed information via broad, jagged peaks, with peak area showing dependence on LNP size (RSD 2-8%).
• The 5.5 M urea/2 M N-ethylurea method demonstrated high isoelectric point (pI) repeatability (<0.5%) and linear correlation between peak area and mRNA content (RSD 2-7%).
• Both methods detected stress-induced changes in pI and peak area, and fluorescence detection significantly improved sensitivity.

Conclusions:

• The developed icIEF methods are effective tools for characterizing the stability and quality of mRNA-loaded LNPs.
• These methods can be applied to evaluate the impact of storage conditions (temperature, time, freeze-thaw cycles) and distinguish between different lipid compositions.
• The techniques offer valuable insights into batch-to-batch variability, supporting the development and quality control of mRNA vaccines.