Characterization of RNA using mass photometry: Method and technical perspectives.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Developing analytical methods for RNA-based products like mRNA vaccines is crucial for quality control.
• Mass photometry (MP) is an emerging label-free, single-molecule technique for biotherapeutic analysis.

Purpose of the Study:

• To establish best practices for using MP in analyzing mRNA vaccine constructs and other RNA samples.
• To evaluate MP's capability in determining mRNA molecular weight and quantifying heterogeneous species.

Main Methods:

• Systematic evaluation of experimental parameters (surface chemistry, buffer composition) for RNA measurement.
• Assessment of low-mass species using kinetic analysis and buffer controls.
• Comparison of MP results with charge detection mass spectrometry and fragment analyzer measurements.

Main Results:

• Cationic-coated sample carriers and salt-containing buffers are essential for reproducible RNA detection and mass resolution.
• MP can quantify heterogeneous mRNA species, including those difficult to resolve by traditional methods.
• MP results showed good agreement with orthogonal techniques, validating its accuracy.

Conclusions:

• MP is a rapid, low-sample consumption technique for comprehensive mRNA characterization and quality assessment.
• MP serves as a valuable standalone method or an orthogonal complement to existing RNA analytical techniques.