Advancing RNA-Based Therapeutics: IQ Consortium Experts on Optimizing Chromatographic Bioanalysis

Nucleic acid (NA)-based therapies such as small interfering RNA (siRNA) and antisense oligonucleotides (ASOs) enable precise modulation of gene expression, opening new possibilities for treating diseases once considered undruggable. The successful development of these therapies depends on optimizing bioconjugation, bioanalysis, biotransformation, and tissue distribution—particularly understanding tissue pharmacokinetics through computational modeling to inform dosing.

Chromatographic techniques, such as liquid chromatography coupled with mass spectrometry (LC–MS), play a vital role in the bioanalysis of these oligonucleotide drugs. Separation science is used to quantify parent compounds, identify metabolites, and characterize biotransformation pathways, ensuring accurate assessment of stability and tissue exposure.

Although oligonucleotides generally present fewer safety concerns than small molecules, regulatory guidelines for characterization and analysis are still evolving. A recent white paper from the IQ Consortium Nucleic Acid Working Group (1) compiles industry insights and best practices, emphasizing analytical and chromatographic strategies to advance regulatory understanding and accelerate patient access to RNA-based therapeutics. LCGC International spoke to three members of the group, Vibha Jawa of EpiVax (Providence, Rhode Island), Shirin Hooshfar of Eli Lilly and Company, (Indianapolis, Indiana), and Wenying Jian of Johnson & Johnson Innovative Medicine (Spring House, Pennsylvania) about their work.

What was the inspiration behind this paper? What did you see that made you and your co-authors believe that there was a need for it?

Oligonucleotides represent a promising therapeutic modality capable of targeting previously undruggable pathways. The field has been advancing rapidly, with 18 ASO and siRNA therapies approved by regulatory authorities for the treatment of various diseases to date and many more in development. Successful development of oligonucleotide therapeutics hinges on several critical factors, including delivery platforms, absorption, distribution, metabolism, and excretion (ADME) properties, and clinical pharmacology characteristics, as well as the specialized assays and studies used to evaluate these parameters.

Given the fast-paced growth of the field and the diverse approaches being explored to address these key challenges, there is a clear need to establish best practices. This includes recommending appropriate assay platforms, identifying relevant studies, and developing de-risking strategies to support both preclinical and clinical development. Recognizing this gap, the IQ Consortium team which is comprised of experts from multiple companies with extensive experience in preclinical and clinical development of oligonucleotide therapeutics, have collaborated to summarize the current landscape and proposed recommendations. This white paper summarized the recommendations to inform best practices and regulatory guidelines in ADME/drug metabolism and pharmacokinetics (DMPK) and clinical pharmacology strategies for nucleic acid-based therapeutics.

What are the key chromatographic challenges associated with analyzing oligonucleotides by LC-MS, and how do ion-pair reagents like triethylamine (TEA) and hexafluoroisopropanol (HFIP) help overcome them?

Oligonucleotides are highly charged, polar molecules, which make them difficult to retain on conventional reversed-phase columns during LC-MS analysis. Their poor retention and broad peak shapes pose significant chromatographic challenges. To address this, ion-pairing reagents such as TEA and HFIP are commonly used.

Organic bases like TEA form ion pairs with the negatively charged phosphate backbone of oligonucleotides. This interaction increases the overall hydrophobicity of the molecule and reduces charge heterogeneity, thereby improving retention and peak shape on reversed-phase columns. HFIP, a weak organic acid, acts as a counter-ion and pH modifier. It helps neutralize excess base, stabilize the ion-pairing environment, and optimize chromatographic behavior.

However, the use of these reagents introduces additional challenges. They can be unstable and require frequent fresh preparation. Moreover, they tend to suppress ionization in the mass spectrometer, leading to reduced sensitivity on the mass spec. Instruments often need to be dedicated specifically for oligonucleotide analysis, which can be a resource burden for laboratories.

How does temperature influence chromatographic separation and peak shape in oligonucleotide LC analysis, and what trade-offs does this introduce in method robustness and column longevity?

Temperature plays a critical role in the chromatographic separation of oligonucleotides by influencing their interaction with the stationary phase. Elevated column temperatures can enhance mass transfer and reduce viscosity, which improves peak shape and resolution. For double-stranded oligonucleotides such as siRNA, higher temperatures can promote strand dissociation, enabling better separation and more specific detection of the individual sense and antisense strands.

However, these benefits come with trade-offs. Prolonged exposure to high temperatures can accelerate the degradation of column packing materials, leading to reduced column performance, deteriorated peak shapes, and compromised assay robustness over time. This degradation may necessitate more frequent column replacement compared to typical small molecule LC-MS assays, increasing operational costs and resource demands.

Why might hybridization-based sample extraction using complementary chain probes on magnetic beads offer improved sensitivity over traditional solid-phase extraction (SPE) in LC-MS assays?

SPE for oligonucleotide is a broadly applied generic sample preparation technique that relies on the physicochemical properties of analytes and matrix components for separation. However, SPE can co-extract endogenous biomolecules with similar properties as analytes which may interfere with detection or cause ion suppression, ultimately reducing assay sensitivity and specificity.

In contrast, hybridization-based sample extraction leverages the sequence specificity of complementary oligonucleotide probes immobilized on magnetic beads. These probes selectively bind to the target analyte such as antisense oligonucleotides or individual strands of siRNA, based on Watson-Crick base pairing. This targeted capture allows for extensive washing steps to remove non-specific matrix components, resulting in cleaner extracts with significantly reduced background interference.

The enhanced selectivity and minimized matrix effects contribute to improved sensitivity and robustness in LC-MS assays.

How do liquid chromatography–tandem mass spectrometry (LC-MS/MS) using a triple quadrupole and liquid chromatography–high resolution mass spectrometry (LC-HRMS) compare for oligonucleotide bioanalysis, and what are the advantages and limitations of each for quantitation and metabolite identification?

LC-MS/MS is commonly used for targeted quantitation. It operates in multiple reaction monitoring (MRM) mode by selecting a specific precursor ion in Q1, fragmenting it in Q2 (collision cell), and detecting a specific product ion in Q3. This approach offers high selectivity and sensitivity, often yielding better signal-to-noise ratios than detection based solely on molecular ions. However, method development requires tuning the analyte to identify optimal precursor-product ion pairs and instrument parameters. Additionally, this technique is inherently targeted by only monitoring predefined transitions. One cannot retrospectively analyze the data for metabolites or unexpected species unless they were included in the original method setup.

For oligonucleotides, LC-MS/MS presents challenges. These molecules ionize into multiple charge states and overlapping ions from different charge states of analytes and metabolites may increase interference. In addition, their fragmentation is often inefficient or yields common fragment ions, which can compromise selectivity.

In contrast, LC-HRMS typically uses full-scan acquisition of intact molecular ions. Multiply charged ions and their isotope patterns are visible, allowing direct inspection and selection of interference-free ions for quantitation. HRMS is an untargeted approach, enabling retrospective data mining for metabolite identification. Hybrid HRMS instruments can also perform high-resolution MS/MS, allowing selective fragmentation of molecular ions and detection of specific fragments with high mass accuracy. This dual capability supports both sensitive quantitation and comprehensive metabolite profiling in a single run.

What role does deconvolution play in high-resolution mass spectrometry of oligonucleotides, and why is specialized software critical for accurate interpretation?

In HRMS of oligonucleotides, deconvolution plays a crucial role in interpreting complex spectra. Oligonucleotides typically ionize into multiple charge states, each containing a series of isotope peaks. For quantitation, it is often sufficient to sum selected ions from one or more charge states to reconstruct a chromatographic peak, making deconvolution optional in this context.

However, when investigating biotransformation, deconvolution becomes essential. It allows the conversion of multiply charged spectra into a single, neutral mass representation, enabling clear visualization of the parent molecule and identification of metabolites based on molecular weight differences. Specialized software is critical for accurate deconvolution, as it ensures precise mass assignment and reliable interpretation of complex oligonucleotide spectra.

Why are stable isotope-labeled analogs less commonly used for siRNAs and ASOs?

In LC-MS analysis, using an appropriate internal standard (IS) is essential for ensuring assay accuracy and consistency. An ideal IS closely mimics the analyte’s physicochemical properties but differs in molecular weight, allowing it to co-extract, co-elute, and co-ionize with the analyte. This enables compensation for variability in sample recovery, injection volume, and ion suppression or enhancement, with the IS-to-analyte signal ratio used for quantitation.

Stable isotope-labeled analogs are generally preferred as ISs in LC-MS due to their structural similarity and minimal impact on chromatographic behavior. However, for oligonucleotides, producing stable isotope-labeled ISs is challenging and costly. Oligonucleotides ionize into multiple charge states, requiring significant mass shifts to avoid overlap with the analyte during detection. Achieving this separation demands extensive labeling, which complicates synthesis and increases cost.

As a practical alternative, many assays use oligonucleotides with the same sequence as the analyte but modified by adding extra nucleotides to the end of the sequence. This approach maintains similar behavior during analysis while providing sufficient mass difference for clear distinction and has proven effective in practice.

How does LC-fluorescence detection (LC-FD) differ from LC-MS?

LC-FD differs from LC-MS in both detection principle and assay design for oligonucleotide quantitation. In LC-FD, the target oligonucleotide (such as ASO or antisense of siRNA) hybridizes with a fluorescently labeled complementary probe, often based on peptide nucleic acid (PNA) technology. In PNA probes, the phosphate backbone is replaced with peptide bonds, reducing charge repulsion and enhancing duplex stability. The resulting duplex is separated via ion-exchange chromatography and detected by fluorescence.

LC-FD typically offers higher sensitivity than conventional LC-MS due to signal amplification from fluorescence. However, it lacks the mass-based selectivity of LC-MS. Shortmer metabolites or other interfering species that hybridize or bind with the probe can also generate signals, potentially affecting specificity. In terms of assay design, LC-MS generally does not require sequence-specific reagents unless hybridization-based extraction is used, whereas LC-FD requires custom probe design and synthesis tailored to the target sequence.

In LC-FD assays, why is extensive chromatographic separation needed to distinguish parent oligonucleotides from active metabolites, and how does this affect sensitivity and throughput?

In LC-FD assays, extensive chromatographic separation is essential to distinguish parent oligonucleotides from metabolites. Unlike LC-MS, which differentiates analytes based on mass-to-charge ratios, LC-FD relies solely on retention time for identification. This is because both the parent oligonucleotide and its metabolites can hybridize with the same fluorescent probe, forming similar duplexes that are indistinguishable by fluorescence alone.

As a result, extensive chromatographic separation is required to resolve these species. If metabolites co-elute with the parent compound, they can contribute to the fluorescence signal and lead to inaccurate quantitation. To achieve sufficient separation, longer chromatographic runs are often necessary, which can reduce assay throughput and efficiency. This trade-off between resolution and speed is a key consideration in LC-FD method development for RNA-based therapeutics.

How can advances in chromatographic techniques support standardization and regulatory acceptance of RNA-based therapeutic bioanalysis?

As RNA-based therapeutics continue to evolve, there is a growing need for standardized and robust bioanalytical methods to support regulatory acceptance. Advancements in chromatographic techniques are central to this effort. One promising direction is the development of ion-pairing-free chromatography, such as hydrophilic interaction chromatography (HILIC), which eliminates the need for ion-pairing reagents. While HILIC has shown success in pharmaceutical analysis, especially in cleaner matrices, its application in bioanalysis of oligonucleotides faces challenges such as limited column capacity, retention variability, and reduced ruggedness. These limitations can affect assay reproducibility and sensitivity, which are critical for regulatory compliance. Nonetheless, ongoing improvements in column technology, mobile phase optimization, and integration with high-resolution mass spectrometry are helping overcome these barriers. By enhancing chromatographic performance and reliability, these innovations contribute to better data quality, improved assay robustness, and alignment with regulatory expectations for method validation and product characterization. This, in turn, facilitates regulatory acceptance of RNA-based therapeutics by ensuring consistent, high-quality bioanalytical data throughout development.

References

1. Christensen, J. K.; Colletti, N.; Hooshfar, S. et al. Translational and Clinical Development of Therapeutic siRNA and ASOs: Current Industry Practices, Perspectives, and Recommendations. Nucleic Acids Res. 2025, 53 (18), gkaf778. DOI: 10.1093/nar/gkaf778