Automated Concentration and Diafiltration of Multiple siRNA Samples

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.
Feb 01, 2013


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).

Controlled, systematic, quality small-interfering RNA (siRNA) production is the core of any successful RNA therapeutics program. To effectively screen potential targets for in vivo activity, it is necessary to create an optimized workflow at the appropriate scale, capable of delivering 100 unique siRNA duplex targets per month. Creating effective workflow capabilities that produce multitudes of unique compounds at scales ranging from milligrams to grams levels requires efficient and flexible use of equipment and personnel. The synthesis and purification unit operations of the process utilize equipment with programmed methods allowing scientists to complete their respective tasks. Manual crossflow filtration (CFF) steps of the process lacked process robustness and were a source of variation between production runs. The project objective was to streamline the process to improve process efficiency and productivity while improving product quality.


Figure 1: Schematic process workflow for siRNA production.
The production of RNA oligonucleotides in the process workflow begins with chemical synthesis using the ÄKTAoligopilot platform to create single-stranded RNA (ssRNA) products (see Figure 1). Each synthesizer has the capacity to synthesize two oligomers, typically 6500 KD to 7500 KD in size, per 24-hour cycle at scales ranging between 10 μmol to 800 μmol. Following a manual deprotection of the siRNA product, reversed-phase high-performance liquid chromatrography (RP-HPLC) and liquid chromatography–mass spectrometry (LC/MS) are used to analyze the quality of the starting feed material and to assign lot specifications for further downstream purification.

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.