The use of small-interfering RNA (siRNA) oligonucleotides for post-transcriptional gene silencing has proven to be an effective and powerful tool in the discovery of gene function. Of particular interest to drug discovery is the application of siRNA as a biotherapeutic agent (1–5). To evaluate the suitability and effectiveness of siRNA as a biotherapeutic product, it is of interest to generate multiple targets and screen for the desired function. The generation of many variants as candidates requires robust processes for screening to aid in selecting the optimum siRNA candidate. Automation of the production and screening processes has the inherent advantage of rapidly and systematically analyzing large sample quantities. A team of scientists from GE Healthcare collaborated with Merck scientists on a project to transfer a conventional manual concentration/diafiltration process for siRNA production using three filtration systems to an optimized, automated process using two ÄKTAcrossflow filtration platforms. The project output based on automated process hardware and software configurations significantly reduced operator requirements while increasing throughput of siRNA samples by 40% (from 6 per day to 10 per day).
The ssRNA product is purified using an anion-exchange chromatography run on a preparative scale LC system with an automated fraction collector, allowing the purification of multiple samples. After screening of collected fractions, the final pool is analyzed for purity by RP-HPLC and quantified using optical density at 260 nm. Typical results for a 200 μmol synthesis are volumes of 0.6–2 L with concentration ranging 0.5–1 mg/mL in the final samples.
The purified ssRNA product is then transferred for duplexing of complementary strands to create siRNA product. Once the duplex is confirmed to have minimal excess of either single strand, the siRNA product requires diafiltration and concentration using ultrafiltration methods. These methods remove residual salt and reduce the volume adequately for lyophilization. In this process, a 200 μmol RNA synthesis reaction and subsequent downstream purification can produce product in the range of 150–1000 mg of final lyophilized siRNA product.