Category: Formulation and Quality
Purpose: The analytical challenge of the siRNA molecules is the resolution of the modified oligoribonucleotide strands from the ubiquitous synthetic impurities, such as “shortmers” or less than full length species (N-1 species and full-length degradation products such as defluorination species). Currently, siRNA analytical method development is a labor intensive “trial-and-error” process. Chromatographic parameters such as column choice and temperature are clear variables in the separation. Most importantly, concentration ranges of the critical ion pairing (IP) reagent and gradient can be difficult to optimize as even a slight change in concentration can affect miscibility of solvents in addition to peak elution.
Optimization of strand separation was accomplished using Fusion AE software (S Matrix, Inc.). This software provides analytical statistical software package to model the resolution trend. It provides fine tuning of method robustness with respect to the pH, temperature and gradient, and also allows expansion of the assessment to other less impactful operating parameters such as column batch variation, operator to operator, etc. A design of experiments (DOE) study has been implemented to accelerate the method development of the purity assessment and identification of impurities for this product. This approach will serve as a strategy for future siRNA method development optimization.
Methods: Denatured Ion-Pairing Reversed Phase Ultra Pressure Liquid Chromatography (IP-RP UPLC) is customarily used for duplex purity analysis focusing on single strand separation and resolution of impurities. An in-house provided highly modified oligonucleotide duplex and corresponding single strands were used as the analyte. The fixed parameters were column chemistry and diameter, ion pairing reagent and organic solvent, and detector conditions. A Waters Acquity H class UPLC with quaternary pump, Acquity Oligonucleotide C-18 1.7µm 2.1mm diameter column, Hexafluoro-2-propanol (HFIP)/ Triethylamine (TEA) and methanol mobile phase composition, and 254nm wavelength were used in the experiment.
The variable parameters tested were as follows (Table 1):
Results: Initial review of the first round of tests resulted in the following findings: