(M0930-06-36) Elements of Robust Modified Oligonucleotide Extraction through SPE

Purpose: Successful bioanalysis and understanding of the DMPK properties of oligonucleotide therapeutics (OTx) require efficient isolation and purification from their biological matrices. This is essential for proper identification, profiling and quantitation of the therapeutic oligonucleotides and their metabolites. Such RNA or DNA analog drugs block the activity of specific genetic elements or sequences to facilitate the treatment of diseases such as macular degeneration, cholesterol control or viral diseases. Reliable analytical methods are not only critical to understand their impurity and degradant profiles during synthesis and development, but also their metabolic conversions during treatment regime. To improve the transport and uptake of OTx drugs across membranes, additional structural features are incorporated to facilitate high affinity binding to the target site, and resistance to nucleases. However, these features are non-natural, therefore, monitoring their degradation products and their potential toxicity is a regulatory requirement to understand the drug stability, efficacy, safety, and metabolic lifetime. Clear documentation of these features necessitates efficient and selective separation of OTx drugs and their products from other components of cellular fluids for subsequent detection. Here, we report elements of robust extraction of modified oligonucleotides through an optimized sample preparation method and carefully chosen ion exchange sorbent for selective binding and purification of OTx and their conjugates such as lipids. Such purification is also accompanied with advanced LC-MS method development to enable lower detection limits and excellent recoveries over a broad sample amounts including molecules with higher hydrophobicity.

Methods: Biological fluids are rich in proteins, and they interfere with OTx extraction. Therefore, removal of protein can be one important element involved in reproducible OTx recovery. We tested enzymatic protein digestion as a way to achieve this goal. Proteinase-mediated digestion time and concentration were tested under denaturing conditions for removing the interfering ability of resident protein but without affecting the OTx integrity. An ion exchange resin for efficient binding and elution of oligonucleotide and its conjugates was tested for highly reproducible and repeatable recoveries. To ensure selectivity, these nucleic acid conjugates are also mixed with charged non-nucleic acid molecules and SPE was performed at various conditions to understand the optimal binding and elution conditions of oligonucleotides from the ion exchange sorbent. Weakly bound non-nucleic acid compounds were washed away with carefully selected wash solution regime and eluted with an ion-pairing solvent containing elution solution for excellent recovery of bound nucleic acid and their conjugates.

Results: Our studies identified optimal protein digestion conditions so that the interference of resident protein during OTx recovery from biological matrix is eliminated. They also revealed the requirement of additional conditions such as organic solvent addition to maintain the solubility of lipid-conjugated nucleic acids in aqueous solutions. However, both unconjugated and conjugated nucleic acids were able to bind to this novel ion exchange sorbent efficiently for selective purification and subsequent analysis. This sorbent could accommodate broad range of analyte quantities to provide the corresponding recovery amounts. One novelty of this elution solution is that no further sample preparation is required for subsequent LC-MS analysis. Such a ready to inject form of elution saves time and avoid sample losses during drying and reconstitution. The eluted nucleic acid components can readily be analyzed by regular ion-pairing reversed phase chromatography and UV/MS-based detection and quantitation. Utility of this sorbent in a variety of device formats (microelution and cartridge format) and their compatibility to accommodate varying sample amounts is also demonstrated. This type of SPE analysis was repeatable and highly reproducible across multiple batches providing confidence in the analytical tools and methodology.

Conclusion: Optimal digestion conditions for protein removal, excellent and selective recovery of oligonucleotide conjugates through novel ion exchange sorbent based SPE were identified. Sample preparation for SPE was streamlined and the identified format of SPE elution required no drying and reconstitution as it could directly be subjected to LC-UV/MS based detection. Selective binding and elution were highly consistent across multiple batches of resin indicating the robust chemical properties of the sorbent. Such a consistency can facilitate confident DMPK analysis of therapeutic oligonucleotides for monitoring the efficacy, safety, metabolic conversions besides understanding their functional lifetime in biological environment in a timely fashion.