Babak Basiri

Understanding siRNA fraction unbound (fu) in relevant physiological compartments is critical for establishing pharmacokinetic-pharmacodynamic relationships for this emerging modality. In our attempts to isolate the equilibrium free fraction of GalNAc-conjugated siRNA using classical small molecule in vitro techniques, we observed that hydrodynamic radius was critical in determining the size exclusion limit requirements for fu isolation; largely validating the siRNA 9rigid rod9 hypothesis and providing insight into size-based kidney and lymphatic filtration of these molecules. With this knowledge, we developed an orthogonally-validated 50 kDa MWCO ultrafiltration assay to quantify fu in biological matrices including human, non-human primate, rat, mouse plasma, and human liver homogenate. To enhance understanding of the siRNA-plasma interaction landscape, we examined the effects of various common oligonucleotide therapeutic modifications to the ribose and helix backbone on siRNA fu,plasma and found that chemical modifications can modulate plasma protein binding by at least 20%. Finally, to gain insight into which specific plasma proteins bind to siRNA, we developed a qualitative screen to identify binding "hits" across a panel of select purified human plasma proteins. SIGNIFICANCE STATEMENT N/A

Despite the exquisite potential of siRNA as a therapeutic, the mechanism(s) responsible for the robust indirect exposure-response relationships have not been fully elucidated. To understand the siRNA properties linked to potent activity, requires the disposition of siRNA to be characterized. A technical challenge in the characterization is the detection and quantitation of siRNA from biological samples. Described herein, a Locked Nucleic Acid (LNA) Hybridization-Ligation ECL ELISA was designed for ultra-sensitive quantification of both sense and antisense strands of siRNA independent of structural modifica-tions. This assay was applied to measure siRNA in serum and tissue homogenate in preclinical species. We observed rapid clearance of siRNA from the systemic circulation which contrasted the prolonged accumulation within the tissue. The assay was also able to distinguish and quantify free siRNA from RNA-induced silencing complex (RISC) and Argonaute 2 (Ago2) associated with therapeutic siRNA. We utilized an orthogonal method, LC-MS, to investigate 3′ exonuclease activity toward the antisense strand metabolism. Taken together, we have demonstrated that the LNA Hybridization-Ligation ECL ELISA is arobust analytical method with direct application to measuring the exposure of siRNA therapeutics seamlessly across biological matrices.

Alkylamines are widely used as ion-pairing agents during LC-MS of oligonucleotides. In addition to a better chromatographic separation, they also assist with the desorption of oligonucleotide ions ...

Hexafluoroisopropanol (HFIP) has been widely used as an acidic modifier for mobile phases for liquid chromatography-mass spectrometry (LC-MS) analysis of oligonucleotides ever since the first report of its use for this purpose. This is not surprising, considering the exceptional performance of HFIP compared with carboxylic acids, which cause significant MS signal suppression in electrospray ionization. However, we have found that other fluorinated alcohols can also be utilized for mobile phase preparation and the choice of optimal fluorinated alcohol is determined by the ion-pairing (IP) agent. Although HFIP is a very good choice to be used alongside less hydrophobic IP agents, other fluorinated alcohols such as 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFMIP) can significantly outperform HFIP when used with more hydrophobic IP agents. We also found that more acidic fluorinated alcohols assist with the transfer of oligonucleotides with secondary structure (e.g., folded strands and hairpins) into the gas phase.

Rationale: In recent years, several liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods have been reported for the quantitative determination of drugs and metabolites in cerebral spinal fluid (CSF). Artificial CSF (aCSF) is often used as a surrogate for preparing calibration curves and quality control samples in these methods. However, aCSF does not accurately represent the composition of real CSF because it is missing all of the proteins and lipids, which may alter the electrospray ionization (ESI) response when performing LC/MS/MS analyses. In the current study we compared the mass spectral response of several compounds with a range of physiochemical properties in aCSF (essentially a mixture of salts and buffers), diluted plasma (ranging from 1:5 to 1:200) and real CSF to find the best surrogate for CSF in LC/MS/MS methods. Methods: A number of analytes from polar to non‐polar, high protein binding to low protein binding, employing different sample preparation methods, were prepared in diluted plasma, actual CSF or aCSF and tested using LC/MS/MS. The analytes included cotinine and its metabolites, quetiapine, norquetiapine, chlorpromazine, efavirenz and lamivudine. The similarity of MS responses from these compounds in aCSF and diluted plasma to CSF was assessed by comparing the slopes of the calibration curves generated from using linear regression modeling. Results: For all compounds, the lowest percent difference in response ratio (0 to 17%) was observed from 1:200 diluted plasma. Our results indicated that, irrespective of the inherent physiochemical properties of the analytes or the method of sample preparation, 1:200 diluted plasma performed as the best surrogate for CSF in LC/MS/MS methods. Conclusions: The percent difference in response ratio has been established to demonstrate how different compounds behave between CSF, aCSF and dilute plasma. Although among the compounds tested some of them showed a very similar MS response in actual and aCSF, there were analytes that demonstrated significant differences in ESI‐MS signal when sprayed from these two matrices. However, even in such cases, 1:200 diluted plasma generated results with no significant difference from CSF. Therefore, we recommend that in order to develop robust and dependable bioanalytical LC/MS methods from CSF samples, it is more appropriate to prepare calibration curves and quality control samples in diluted plasma instead of aCSF. Copyright © 2016 John Wiley & Sons, Ltd.

A new method for reversed phase HPLC determination of thiamine and its major in vivo phosphorylation products, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP), was developed using tetrabutylammonium hydroxide as the ion‐pairing agent. The separation was performed on a Phenomenex Kinetex EVO C₁₈ column with a gradient of a phosphate‐buffered aqueous solution of the ion‐pair reagent and methanol. The duty cycle for the assay was 13 min and pyrithiamine was successfully used as the internal standard for the first time in a thiamine HPLC measurement protocol. Detection of the fluorescence derivatives of the analytes as well as the IS allowed for lower detection limits in order to support biological applications in cell culture models. The linearity, sensitivity, specificity, accuracy and precision of the method were evaluated and met the requirements specified by the US Food and Drug Administration. The calibration curves proved to be linear and the method was validated over the range from 1.0–4000 nm for both cells and the media where complete recovery of the analytes was also achieved. Copyright © 2015 John Wiley & Sons, Ltd.

Recent findings have elucidated numerous novel biological functions for oligonucleotides. Current standard methods for the study of oligonucleotides (i.e., hybridization and PCR) are not fully equipped to deal with the experimental needs arising from these new discoveries. More importantly, as the intracellular capacity of oligonucleotides is being harnessed for biomedical applications, alternative bioanalytical techniques become indispensable in order to comply with ever-increasing regulatory requirements. Owing to its ability to detect oligonucleotides independent of their sequence, LC-MS is emerging as the analytical method of choice for oligonucleotides. In this article, the current applications of LC-MS in the analysis of oligonucleotides, with an emphasis on RNA therapeutics and biomarkers, will be examined. In addition, the theoretical framework of oligonucleotide ESI is carefully inspected with the purpose of identifying the contributing factors to MS signal intensity.