By Dr. Kurt Sales, CSO
Oligonucleotides, including small interfering RNA (siRNA) and antisense oligonucleotides (ASO), have gained significant attention in recent years as promising therapeutic agents. With their potential to regulate gene expression, they hold promise for treating a variety of diseases, from cancer to genetic disorders. However, successful development of oligonucleotide-based therapeutics requires precise and reliable bioanalytical methods to quantify and characterize these molecules, ensuring drug quality and therapeutic efficacy.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as one of the most powerful techniques for quantitative analysis of oligonucleotides such as siRNA/ASO due to its sensitivity and specificity and ability to multiplex. However, the analysis of oligonucleotides by LC-MS/MS presents a unique set of challenges due to their complex structure and inherent chemical properties.
In this blog, we explore some of the challenges associated with setting up and optimizing LC-MS/MS for oligonucleotide analysis and provide some generally used solutions to overcome these difficulties. Finally, using a case study, we discuss how we overcame the challenges of developing and validating a method to detect an ASO in plasma at Agilex to support a clinical study.
Key Challenges in Oligonucleotide and siRNA Analysis by LC-MS/MS
1. Complexity of Oligonucleotide Structure
Oligonucleotides, including siRNAs, are long, negatively charged molecules composed of nucleotides linked by phosphodiester bonds. This complex structure can pose a challenge for both chromatographic separation and ionization in mass spectrometry.
Solution:
To address the complexity inherent within this class of molecule, optimizing the chromatography conditions is essential. Reversed-phase liquid chromatography (RP-LC) is often used for oligonucleotide separation, but due to the high polarity and negative charge of oligonucleotides, the use of ion-pairing reagents or more specialized stationary phases (e.g., or hydrophilic interaction liquid chromatography (HILIC)) may be necessary to achieve efficient separation. Ion-pairing reagents (for example: TEA (triethylamine) or DIEA (diisopropylethylamine) can help neutralize the negative charge of the oligonucleotides and improve their retention on the chromatographic column.
2. Ionization Efficiency
Ionization efficiency is one of the most critical aspects of successful mass spectrometry analysis. Oligonucleotides, with their high negative charge, typically do not ionize well. Poor ionization can lead to low sensitivity and unreliable quantification, which is especially problematic when analyzing low-abundance siRNA/ASO drugs.
Solution:
Several strategies can be employed to improve ionization efficiency. This is generally iterative during method development. Firstly, adjusting the mobile phase composition by including organic solvents and optimizing the ion pair and buffer concentrations is an essential first step. This balances the need for retention with the suppression that comes from the concentration of the ion pair reagent. Secondly the use of equipment often dictates outcome – for example some researchers have explored the use of matrix-assisted laser desorption/ionization (MALDI) or other ionization techniques to improve efficiency and outcome. In general, electrospray ionization (ESI) remains the most common choice for LC-MS/MS analysis of oligonucleotides. Finally, optimizing the source conditions (e.g., ion spray voltage, gas flow, and capillary temperature) can help increase the number of ions detected to improve ionization efficiency.
3. Sensitivity and Detection Limits
Oligonucleotides and siRNAs are typically present in very low concentrations in plasma/blood, often requiring highly sensitive methods to detect oligonucleotides present at nanomolar or even picomolar concentrations.
Solution:
To address sensitivity issues, a few approaches can be employed:
- Triple Quadrupole (QQQ) Mass Spectrometers: These instruments, capable of multiple reaction monitoring (MRM), can provide excellent sensitivity and specificity, which is ideal for quantifying oligonucleotides in complex matrices.
- High Sensitivity LC-MS/MS Configurations: Instruments with higher sensitivity detectors and optimized ionization sources (e.g., ion funnels, heated capillaries) can improve the detection limits of oligonucleotides.
- Sample Enrichment and Pre-concentration: Sample preparation techniques such as solid-phase extraction (SPE), or immunoaffinity capture can help increase the concentration of siRNA or oligonucleotide targets in the sample, improving the signal-to-noise ratio.
4. Chromatographic Challenges: Column Selection
Choosing the right chromatography column is essential for achieving adequate separation of oligonucleotide species. Oligonucleotides tend to have a broad range of polarities due to variations in their base composition, chain length, and chemical modifications. This complexity can lead to poor resolution during chromatographic separation.
Solution:
Several strategies can help overcome chromatographic challenges:
- Column Chemistry: For reverse phased ion pair chromatography (RP-IP), columns typically consist of a polymeric particle, to resist the high pH conditions necessary for ion pairing, with a bonded hydrocarbon, typically C18. Weak anion exchange (WAX) or HILIC columns are often for separating oligonucleotides due to their unique interactions with the analytes. WAX columns, in particular, can effectively separate oligonucleotides based on their charge and size.
Optimization of Mobile Phases: The use of mobile phases with varying pH, ionic strength, and organic solvent content can help achieve better separation.
5. Fragmentation, instability and non-specific binding
Oligonucleotides and siRNAs are fragile molecules, and under certain MS conditions, they may undergo fragmentation making intact analysis challenging. Moreover, because of their charged state, they are prone to non-specific binding creating challenges at the outset with regards to sample processing and carryover during the analytical run.
Solution:
To minimize fragmentation, optimizing the collision energy in the collision cell is key. Some labs have used low-energy MS/MS scans and high-resolution mass spectrometry (HRMS) to help with the identification of specific modifications without causing excessive fragmentation. HRMS is also a popular platform for metabolite identification – one of the advantages of mass spectrometry is the ability to multiplex and quantify the parent and metabolite in the same analytical run.
Oligonucleotides are highly charged and prone to non-specific binding and thus care really needs to be paid to the sample collecting conditions at the clinic and the sample storage and extraction conditions and reagents, plasticware etc. at the processing lab to ensure that integrity of the molecule is optimized firstly ahead of optimizing the extraction and chromatography and then assessment of carryover during chromatographic runs to ensure that there are no artifacts introduced into the data because of stickiness associated with the molecule as a consequence of its highly charged state.
6. Matrix Effects in Biological Samples
In clinical or preclinical samples, analyzing oligonucleotides in biological matrices such as plasma, serum, or tissues presents the additional challenge of matrix effects, where co-eluting components can interfere with detection and quantification. These interferences can result in ion suppression or enhancement, leading to under or over-estimation of concentration and inaccurate results.
Solution:
To mitigate matrix effects, careful sample preparation is an essential first step to a successful assay. Traditional methods such as solid-phase extraction (SPE), liquid-liquid extraction (LLE), can be used to clean up biological samples and remove potential interferents. Additionally, the use of stable labeled internal standards (SIL-IS) or structurally similar analogue standards will help correct for any variations in sample preparation and ion suppression. These can be challenging to manufacture, especially if the oligonucleotide drug is highly modified, posing an additional challenge to the assay.
7. Regulatory Considerations and Method Validation
For biotech companies developing oligonucleotide-based therapeutics, meeting regulatory requirements is paramount and having a fully validated assay to demonstrate that the LC-MS/MS method is both reproducible and accurate for oligonucleotide quantification is essential. Regulatory bodies such as the FDA and EMA require validation of bioanalytical methods, including specificity, sensitivity, accuracy, precision, and linearity to prove robustness.
It is therefore imperative that a comprehensive method validation process should be employed to meet regulatory standards. This involves assessing parameters such as limit of quantification (LOQ), recovery, precision & accuracy, and stability of the oligonucleotide or siRNA in biological matrices and solvent. Additionally, ensuring robustness across different instrument platforms for multicenter studies and batch-to-batch reproducibility for longer phase studies is crucial for regulatory acceptance. At Agilex Biolabs, our Scientists have extensive experience in oligonucleotide bioanalysis, including troubleshooting methods from third party labs, transfer of the method and optimization to meet the phase of the clinical study and phase-specific validation to meet the requirements of the clinical trial as well as the regulatory agency reviewing the data.
The following case study provides a snapshot of how we overcame the challenges of an ASO to develop and validate an assay to support a clinical study.
Case Study: Analysis of a 21 base-pair oligonucleotide therapeutic by reverse-phase ion pair LC-MS/MS
Here, we present the analysis of a 21-base pair, single stranded oligonucleotide sequence. The method was developed for the therapeutic to support a clinical study in Australia. The molecule was entirely phosphorothioated, had 3 locked nucleic acid (LNA) base pairs at the 5’ and 3’ ends of the sequence in a 3-15-3 format and a mass of ~7000 Da. We used a 20-base pair sequence, also phosphorothioated, and in a 3-14-3 format as a close analogue internal standard for the assay.
As mentioned above, ionization efficiency is a particular challenge with oligonucleotides. Initial method development started by infusion of the therapeutic in solutions containing ion pair reagents, to facilitate the formation of the multiple charge states typical of these molecules. An a priori prediction of charge states was used to identify prospective charge states from background peaks and impurities.
Ionisation was achieved using negative electrospray ionisation (ESI) on a SCIEX 6500+ MS, and multiple charge states were observed and optimized before settling on the [M-8H]-8, with the commonly used phosphorothioate ion of 94.9 taken forward for qualitative work (this fragmentation product is particularly useful as it is highly selective and sensitive for therapeutic oligonucleotides). Having optimized the initial ionization conditions, the MS source conditions were then optimized under mobile phase flow to refine this.
Liquid chromatography conditions were optimized around the use of water, acetonitrile, triethylamine (TEA) and hexafluroisopropylalcohol (HFIP), as well as an ethylene-bridged hybrid C18 column chosen for its high chromatographic efficiency and pH robustness. While methanol is also a viable mobile phase constituent and can offer greater retention under RP-IP conditions, this is also typically accompanied by higher column back pressures and wider peaks. We found that sufficient retention was achieved with acetonitrile leading to it’s use here for the increased chromatographic efficiency. HFIP and TEA varying concentrations of TEA and HFIP were assessed before settling upon an optimum ratio of 5:1, which was found to give a good balance of retention and response.
The primary concern when analyzing oligonucleotides in plasma samples is to reverse the strong protein binding that these molecules display, usually requiring a chaotropic agent to disrupt this interaction. For our study, we found that a mixture of phenol, chloroform and isoamyl alcohol proved effective at this task, with the phenol acting as a chaotropic agent, the chloroform to facilitate formation of distinct liquid layers, which solubilized a large portion of the phenol to give a liquid-liquid extraction (LLE). Moreover, we used isoamylalcohol as a surfactant to control foaming of the resulting crashed proteins into an intermediate layer. This combination allowed easy drawing of the resulting aqueous layer.
A drawback of using phenol is that it is reasonably water soluble allowing some partitioning into the aqueous layer, which needs to be removed before LCMS analysis. In this particular project, we utilized dichloromethane (DCM) in a second LLE procedure, again drawing the aqueous layer. The use of DCM represented good cost savings when compared to solid phase-based alternatives and gave the desired result. The final aqueous layer was then transferred, evaporated and reconstituted in a mixture of the mobile phase solvents for injection on the Sciex 6500+. The assay, now optimized for LC-MS/MS gave strong accuracy and precision data over a range of 0.500 to 50.0 nM, using an injection volume of only 5 µL.
Conclusion
The analysis of oligonucleotides and siRNA by LC-MS/MS presents a unique set of challenges, from complex molecular structures and poor ionization to the need for ultra-sensitive detection and accurate quantification in biological matrices. However, with careful optimization of chromatographic conditions, sample preparation, and MS settings, many of these challenges as outlined in our case study above can be overcome. By understanding these pitfalls and applying the appropriate strategies, biotech companies and bioanalytical labs can work together to effectively use LC-MS/MS to support the development and regulatory approval of siRNA-based therapeutics.
Agilex Biolabs is Australia’s largest Toxicology and Bioanalytical Laboratory. With nearly 3 decades of experience in supporting new drug modalities in first in human studies, our Scientists have extensive experience in oligonucleotide method development, validation, and sample analysis in plasma and urine, in compliance with the latest ICH M10 guidelines and to meet GLP/GCP requirements. To hear more about how Agilex can support your oligonucleotide molecule development requirements please speak to one of our scientific experts or reach out to BD@agilexbiolabs.com
