(T1330-11-61) System Optimization to Mitigate Signal Suppression While Autonomously Running Multiple LC/MS/MS Methods in for SiRNAs, Peptides and Small Molecules in Sequence

Purpose: SiRNA (small interfering RNA) is a novel class of “therapeutics” which silences gene expression during translation/transcription via the formation of the RNA-induced silencing complex (RISC) followed by cleavage of the targeted mRNA. Analysis of siRNA drugs can include multiple “analytes”: intact strand with or without linkers, single strand, double stranded or complexed to proteins in the cells (RISC loaded). Renal clearance is the predominant route of clearance for siRNA molecules, excretion of largely intact strands, provided they have modifications that prevent the enzymatic digestion pathway used on endogenous siRNAs. Quantification of siRNA (anti sense strand) in urine has been successfully and routinely executed using a simple dilution of the sample in mobile phase containing the ion pairing agents HFIP and TEA. A major concern while performing analysis of siRNA therapeutics is the suppression effects of HFIP/TEA mobile phases on other LC/MS/MS methods. A successful workflow model was determined to enable separate methods to be run on the same LC/MS/MS system by implementing an optimized workflow using a multi-system LC. Alternative analysis of peptides and small molecules were successfully performed on the same LC/MS/MS system continuously while mitigating the usual suppression effects of the HFIP/TEA mobile phase additives required for optimal siRNA chromatographic conditions.

Methods: Analysis of the siRNA therapeutics in urine was accomplished using a dilution of 50µL human urine sample in 450µL of mobile phase A followed by direct injection into the LC/MS/MS system. Plasma siRNA methods for comparative analysis utilize SPE cleanup were also demonstrated using the same LC/MS/MS gradient method. A Sciex API 5000 mass spectrometer was used for all methods; siRNA and alternate peptide and small molecules running in sequence. A Shimadzu Nexera LC system with solvent switching capacity and a column oven with six LC column switching capacity. Mobile phase system A consisted of 1000/1 Water/formic acid and 1000/1 acetonitrile/formic using a linear gradient on a Waters BEH C18 LC column for the analysis of peptides and small molecules in positive ionization mode. System B consisted of 1000/10/1 Water/HFIP/TEA and 1000/10/1 methanol/HFIP/TEA mobile phases run in linear gradient using a Waters Oligo C18 for the analysis of various siRNA molecules in negative ionization mode. An intermediate method was used on a separate line with no column attached for both LC systems for the purpose of switch, purge, and equilibration. Analyst 1.6.2 software control was used to change between all methods autonomously. The intermediate method increased flow rate off-column after system toggle to 2ml/min for total of 20min at a 50% isocratic gradient to completely purge the LC system from any residual ion suppression agents without impacting the alternate column equilibration for the next analytical run. After system toggle and flush was completed, a pre-analytical lead method was then used to equilibrate the LC column using the respective analytical method for 5 gradient cycles to equilibrate the analytical column prior to analysis.

Results: A fit-for-purpose validation was performed to quantify a siRNA therapeutic in human urine using LC/MS/MS detection in support of a renal clearance study. The lower limit of quantification was 100 ng/mL, and the upper limit of quantification was 25,000 ng/mL. The inter-assay accuracy (%RE) during validation ranged from -5.0% to 3.7%. The inter-assay precision (%RSD) during validation for was ≤4.1%. During the time this validation took place, alternate small molecule validation assays and peptide analyses were interspersed to continue progress on those studies. Utilization of the optimized multiplexed system showed no significant impact on signal strength and chromatography. All the diverse methods run in tandem on this system performed ruggedly and met all pre-defined acceptance criteria established in those validations.

Conclusion: Using LC/MS/MS for siRNA is feasible by using the high charge states of the molecule to enable quantification within the mass ranges of triple quadrupole instruments. Negative ion pairing agents need to be used to have acceptable chromatography and can be useful for signal enhancement, however these are detrimental to other methods and positive ionization on the instrument. LC systems using the TEA and HFIP additives must be purged extensively or designated to prevent impact other methods on the same system. The total time for switching and equilibrate was less than one hour and is performed overnight entirely autonomously. The result was significantly increase in instrument utilization and throughput of the multiple methods either running in sequence overnight or by optimization of instrument and operation time by autonomously equilibrating the instrument for use at the start of the following workday. By utilizing this multiplex capability analysis of oligonucleotides incorporating the use of ion pairing agents can be included in a workflow on a single LC-MS/MS system, in concert with traditional methods.