Circular RNA (circRNA) vaccines have attracted increasing attention due to their stable closed-loop structures and persistent protein expression ability. During the synthesis process, nicked circRNAs with similar molecular weights to those of circRNAs are generated. Analytical techniques based on differences in molecular weight, such as capillary electrophoresis, struggle to distinguish between circRNAs and nicked circRNAs. The characteristic degradation products of circRNAs and their biological activities remain unclear. Therefore, developing methods to identify target circRNAs and non-target components and investigating degradation patterns will be beneficial to gaining an in-depth understanding of the properties and quality control of circRNAs vaccines. The reversed-phase HPLC (RP-HPLC) method was established for identification of target circRNAs, product-related substances, and impurities. Subsequently, we investigated the degradation patterns of circRNAs under thermal acceleration conditions and performed biological analysis of degradation products and linear precursors. Here, RP-HPLC method effectively identified circRNAs and nicked circRNAs. With thermal acceleration, circRNAs exhibited a “circular→nicked circRNAs→degradation products” degradation pattern. Biological analysis revealed that the immunogenicity of degradation products significantly decreased, whereas linear precursors did not possess immunogenicity. Thus, our established RP-HPLC method can be used for purity analysis of circRNA vaccines, which contributes to the quality control of circRNA vaccines and promoting the development of circRNA technology.
