Bioanalytics – Chemical
Bioanalytical analysis of RNAi therapeutics is challenging, especially when stabilizing nucleotides are used in siRNA design or the desire to quantitate siRNAs in unusual biological matrices. The design of an siRNA can dictate the method needed, but sensitivity and selectivity must also be considered when developing a bioanalytical method. An added challenge of the bioanalysis of siRNAs is the quantitation of active metabolites, e.g. the N-1 antisense strand. Balancing the fit-for-purpose needs, for example, data use, level of validation/qualification, matrix complexity, or turnaround time, helps define the method needed for a particular analysis. The highly selective method of LC/MS can quantitate full-length siRNA and its metabolites simultaneously, but sensitivity can be sacrificed. By using methods like qPCR, very low levels (pg/mL) can be measured, but the primer annealing aspects of qPCR offer challenges for low affinity binding moieties, like GNAs, used in siRNA design. However, qPCR provides flexibility when quantitating siRNA in unusual matrices. The low sensitivity of qPCR can also facilitate understanding the mechanism of action of RNAi therapeutics. An assay designed to quantitate siRNA RISC loading provides insight into long pharmacologic effects of siRNA takes advantage of the qPCR method’s low sensitivity. Full understanding of the bioanalysis of an RNAi therapeutic uses multiple methods through discovery and development.