Purity analysis of sgRNA by IP-RP LC-UV: an Analytical Quality by Design approach

Single guide RNA (sgRNA) is a key component of the clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing technology, playing a crucial role in sequence-specific DNA targeting. With its growing therapeutic potential, sgRNA has emerged as a promising candidate for drug development. 

As such, robust analytical characterisation of sgRNA is essential to ensure its quality, safety and efficacy in compliance with regulatory requirements for release testing. While ion-pair reversed-phase (IP-RP) chromatography is the gold standard for short oligonucleotides, its application to longer RNA sequences such as sgRNA remains limited. The sgRNA used in this study was synthetised by Kaneka Eurogentec and features phosphorothioate linkages on the three first and three last nucleotides alongside with 2’-O-methyl modifications. These modifications are essential for enhancing resistance to nuclease degradation and increasing overall sgRNA stability. This study presents the development of an IP-RP method for sgRNA purity determination using an Analytical Quality by Design (AQbD) approach aligned with ICH Q14 guidelines. 

The initial step of the AQbD approach involved defining the Analytical Target Profile (ATP), which describes the expected performance characteristics of the method in terms of accuracy and precision. This definition was established with consideration of a typical purity specification of not less than 80%.