SEC-MALS analysis of polydeoxyribonucleotides: Revealing product heterogeneity and enabling measurement standardization

The accurate analysis of complex and high molecular weight biomaterials is often difficult. Polydeoxyribonucleotide (PDRN), a biomaterial extensively employed in tissue repair, exhibits a close correlation between its physiological activity and weight-average molar mass (Mw). However, its complex macromolecular nature (high viscosity, low solubility, and broad molecular weight distribution) makes accurate analysis challenging. Conventional analytical methods, such as mass spectrometry and gel electrophoresis, face significant limitations in this regard. Although size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) is the prevailing analytical method, it often yields unreliable and highly variable Mw data in practice. Consequently, there is a clear need to develop an analytical method capable of delivering accurate and repeatable results. In this study, an optimized analytical method was established through the systematic optimization of multiple SEC-MALS parameters. This method was rigorously evaluated, demonstrating reliable accuracy, reproducibility, and a broad Mw detection range of 100-8500 kDa. Subsequently, based on this method, suitable sample preparation conditions for PDRN were established via experimentation, ensuring complete dissolution while preventing sample aggregation. Finally, this method was applied to eight different PDRN samples, producing stable and consistent results. Our analysis revealed previously overlooked but significant discrepancies in Mw properties across different PDRN samples, which were also reflected in the dispersity (Ð) and the proportion of low-molecular-weight fragments. These variations highlight critical challenges in quality control and product consistency. The comprehensive analysis of the experimental data advances our understanding of PDRN. For manufacturers, accurate Mw determination must be accompanied by the analysis of the Ð, a critical parameter for sample characterization. The broad analytical range of this method ensures accurate and comparable analysis for samples within its scope, providing a reliable analytical tool for achieving batch-to-batch consistency.