We present a comprehensive study of ion-pair reverse-phase (IP-RP) chromatography for mRNA polishing, demonstrating its ability to remove double-stranded RNA (dsRNA), RNA fragments and residual DNA template.
We developed a step elution IP-RP strategy to simplify the purification process, with consistent recovery rates and purity across different column scales (1 – 80 mL bed volume). Column sanitization with 75% acetonitrile was replaced with NaOH cleaning methodology, thus removing the need for concentrated acetonitrile. We show how IP-RP utilizing up to 20% acetonitrile (ACN) removes double-stranded RNA hybrids. When mRNA is not treated with DNase, IP-RP separates two forms of DNA from full length mRNA, reaching residual DNA levels comparable to DNase treated mRNA. Reverse-phase polishing was validated with different RNA constructs with nominal length from 900 – 9000 nucleotides, highlighting the necessity of temperature control for effective dsRNA removal, especially for longer RNA sequences, such as self-amplifying RNA (saRNA).
Our findings highlight the importance of IP-RP while simplifying the technological requirements for its adoption in clinical manufacturing processes.
