Overcoming the Challenges of Antisense Oligonucleotide Quantitation by LC-MS/MS

Oligonucleotide therapeutics have recently gained popularity as noted by recent increases in regulatory approvals of oligonucleotides as drugs, novel liquid nanoparticle delivery approaches, and on-target specificity. In the development of bioanalytical assays, there are many challenging aspects to consider when quantitating oligonucleotides, such as non-specific binding, stability, efficient ionization, sequence modifications, ion-pair chromatography, and complex extractions. At KCAS Bio, we have several strategies to overcome these challenges.

Challenge #1: Chromatographic Retention and Ionization of the Oligonucleotide for Detection on an LCMS System

There are two approaches that have been historically used—hydrophilic interaction liquid chromatography (HILIC) and ion pairing reverse phase chromatography (IP-RP). At KCAS Bio, IP-RP is the typical approach, as it allows for the best resolution and highest response. However, IP-RP comes with its own set of challenges, including the selection and concentration of the ion pairing reagent. The reagent must be volatile and mass spectrometry compatible, in addition to being compatible with the oligonucleotide in length and charge. Typically, fluorinated alcohols, such as HFIP, and alkylamines, such as TEA, are chosen. After the ion pairing reagent has been selected, it is important to determine the minimum effective concentration to reduce potential ion suppression while still achieving good separation and ion pairing.

Challenge #2: The Extraction Process From Biological Matrices

Dissociation and/or enzymatic digestion is typically required to release the oligonucleotide from the protein, lipid, or other chemical group it is conjugated to and ensures the accurate identification and quantification of the specific oligonucleotide. This process involves optimizing the pH, temperature, and specific reagents needed to break the conjugation. To disrupt protein binding and denature the structure of the conjugated oligonucleotide, KCAS Bio has developed a good set of default conditions that work with many oligonucleotides. 

Oligo stability is a key parameter to keep in mind during extraction optimization. Typically, we have the option to extract on ice, under yellow light, and/or in a cold room set at 4°C to mitigate any instability. The main challenge is the extraction technique itself, as oligonucleotides typically require a mixed mode solid phase extraction (SPE) or liquid/liquid extraction followed by an anion exchange solid phase extraction. Both extraction techniques can require extensive optimization during method development.

KCAS Bio Has the Tools & Expertise to Solve These Challenges

KCAS Bio has developed an ample understanding of developing a robust and optimized LC-MS/MS method for quantitation of oligonucleotides. The team has had success with optimization of LC-MS/MS conditions, such as, changing ion pairing reagents and needle rinse, analytical column dimensions, stationary phase, and particle size. Along with enhancing the quality of chromatography through LC-MS/MS conditions, changes in extraction procedure steps and details showed considerable improvement in chromatography sensitivity with reduced background. There are various digestion techniques and reagents used depending on the oligonucleotide sequence. We have experience with multiple solid phase extractions and have identified good extraction solvents that further improve oligonucleotide chromatography.

While oligonucleotides can be challenging to quantitate by LC-MS/MS, KCAS Bio has developed several approaches to successfully extract and quantitate oligonucleotides from a variety of different biological matrices, including plasma and tissues from several species using traditional LC-MS/MS techniques. Furthermore, hybridization LCMS can also be employed as a more sensitive extraction strategy and offers the ability to differentiate oligos from their metabolites (as compared to hybridization ELISA).