Orthogonal Molecular Weight Analysis of Challenging Macromolecules Using MALS and Mass Photometry: Example of...

The molecular weight of complicated biological macromolecules is typically difficult to determine precisely. As an example, the complicated nature of high viscosity, low solubility, and extremely broad MW distribution of polydeoxyribonucleotide (PDRN), which was extracted from salmon sperm cells and possesses a number of physiological activities related to its weight-average molecular weight (MW), subjected it to challenges for accurate MW determination. The commonly used technologies such as mass spectrometry and gel electrophoresis might not work properly for the accurate determination of PDRN MW; thus, it is necessary to develop effective analytical methods to address this challenge.

Considering PDRN as a polymer sample, multiangle light scattering (MALS) was employed for analysis. Both size-exclusion chromatography-MALS (SEC-MALS) and asymmetric flow field-flow fractionation-MALS (AF4-MALS) obtained consistent MW results. Notably, AF4-MALS has achieved the preliminary separation and quantification of RNA impurities in PDRN samples. Additionally, considering PDRN as a single-particle sample, the mass photometry was employed as an orthogonal technique for MALS measurement. It is demonstrated that the MW results obtained by the three methods were consistent.

It is demonstrated that the developed orthogonal methods can get a consistent MW of PDRN, also providing an example of how these methods can provide both "overall" and "single-particle" data that will be useful in accurate weight-average molecular weight determination of complicated biological samples.