Validation of mRNA concentration determination by slope spectroscopy technology: a matrixed approach

Cell & Gene Therapy Insights 2023; 9(5), 611–624

DOI: 10.18609/cgti.2023.084

Published: 14 June 2023
Research Article
Travis Alvine, Natalie Unsinn, John J Long, Joseph Ferraiolo

mRNA-based therapeutics are different from small molecules and other biologics that represent significant analytical challenges. mRNA characterization for pre-clinical/clinical testing and lot release are required to compete in the competitive marketplace and align with regulatory standards. Faster and more reliable results require innovative solutions to meet these analytical challenges. Nucleic acid concentration determination is measured by determining the ultraviolet (UV) absorbances at an analytical wavelength of 260 nm. These absorbance measurements allow scientists to measure nucleic acid concentration based on the known extinction coefficient for RNA. The spectral signature of their maximum absorbance peak at 260 nm is proportional to nucleic acid concentration. The advantages of this UV nucleic acid quantitation method are that it is simple, direct, and requires just a small volume of your sample for measurement. One challenge, however, that the analytical labs run into is its limitation for specificity, as matrix components that absorb similar wavelengths can lead to inaccuracies in the consequent nucleic acid concentration determination. We have observed that the standard fixed-pathlength UV in current traditional cuvette-based UV solutions using a 1 cm cuvette and/or smaller fixed pathlengths still does not resolve the quality of the given measurement and lead to hours of required investigation time. The use of dilution factors, which increase prep time and variability, and fixed-pathlength measurements in determining the concentration of a UV chromophore in solution does not provide an easily transferable and robust method that can be platformed within a company or process. Today, researchers can selectively quantify nucleic acid absorbance in the presence of chemical and nucleic acid impurities, notably DNA and dsRNA. Analytical software uses full-spectrum data and advanced algorithms to identify nucleic acid impurities and provide corrected nucleic acid concentrations.