Oligonucleotide therapeutics (oligos) have emerged as a groundbreaking class of drugs with the potential to treat previously untreatable diseases. By targeting RNA or modulating protein function, therapies such as antisense oligonucleotides (ASOs), small interfering RNA (siRNA), and aptamers offer new avenues for precision medicine. However, their development is not without hurdles, particularly in bioanalysis. Their unique properties demand advanced analytical methods to ensure accuracy, stability, and regulatory compliance.
Navigating Bioanalytical Challenges: Tools & Strategies
Bioanalysis of oligos is uniquely complex due to their susceptibility to degradation, non-specific binding, and structural similarities between parent compounds and metabolites. Several validated approaches are used to address these challenges. With the right lab testing partner, sponsors can overcome these hurdles more efficiently and with greater confidence.
- Instability & Degradation in Biological Systems. The intrinsic instability of native oligonucleotides—driven by susceptibility to enzymatic degradation in vivo—must be addressed by the drug developer through backbone modifications. However, after sample collection, bioanalytical workflows face their own set of stability challenges. During storage, handling, and processing, enzymatical degradation can occur, affecting analytical accuracy. Specialized stabilizers can be added to preserve sample integrity during the bioanalysis process for Hybrid-ELISA-, PCR-, and LC-MS-based methods.
- Non-Specific Binding & Matrix Effects. Oligonucleotides, particularly those with phosphorothioate (PS) modifications, tend to bind plasma proteins and other biological components, introducing variability and complicating quantification. Extraction techniques like solid-phase extraction (SPE) and liquid-liquid extraction (LLE) help reduce these matrix effects by isolating the analyte from interfering substances and increase the specificity for LC-MS-based bioanalysis. While ion-pairing reagents are not used to reduce non-specific binding directly, they can enhance the overall LC-MS resolution to increase the specificity, eliminating matrix effect.
- Difficulty in Distinguishing Parent Compounds from Metabolites. Oligo metabolites often exhibit similar structures and molecular weights to their parent compounds, making differentiation a core challenge in bioanalysis. High-resolution mass spectrometry (LC-HRMS) offers the resolution and precision to confidently identify and quantify these components, ensuring robust metabolite profiling.
- Regulatory & Standardization Challenges. As oligonucleotide therapies evolve, regulatory frameworks continue to adapt. Drug developers face the challenge of aligning with global standards while validating novel analytical techniques. WuXi AppTec supports clients with deep regulatory expertise and validated platforms to ensure compliance with U.S. regulatory bodies, EMA, and ICH guidance.
Navigating these challenges requires expertise, specialized equipment, and a commitment to rigorous validation. Drug developers navigating complex workstreams and limited resources have options to streamline preclinical research. A trusted lab testing partner can offer:
- Advanced analytical capabilities: Access to platforms like LC-MS/MS, LC-FLD, HRMS, Hybrid-ELISA, PCR, and ddPCR tailored to oligo properties.
- Regulatory expertise: Support for method validation and evolving agency expectations.
- Customized solutions: Adaptive strategies based on the unique characteristics of each therapeutic candidate and its development stage.
- Automation and high-throughput screening: Enhanced consistency and throughput in sample preparation and analysis.
Navigating Evaluating Immunogenicity & Immunotoxicology
Although oligonucleotide therapeutics generally present lower immunogenic risk than biologics, delivery systems and chemical modifications may still trigger immune responses. Regulatory authorities recommend a tiered approach to safety assessment that involved immunogenicity testing and immunotoxicity evaluation. This can involved detecting anti-drug antibodies (ADAs) using bridge or direct ELISA formats, followed by confirmatory assays and titer evaluations. Drug developers must also monitor cytokine release, complement activation, and innate immune activation using multiplex immunoassays and flow cytometry.
Navigating Innovation & Emerging Trends in Oligo Bioanalysis
New bioanalytical technologies continue to expand the capabilities of oligonucleotide testing. Key trends shaping the future include:
- High-Resolution Mass Spectrometry (HRMS): Enabling detailed metabolite profiling and improved analytical selectivity.
- Droplet Digital PCR (ddPCR): Delivering enhanced sensitivity for low-abundance analytes, critical in biodistribution and late-phase studies.
- Automation and High-Throughput Workflows: WuXi AppTec integrates automation in sample extraction and LC-MS/MS processes to improve consistency, reduce labor, and accelerate timelines.
Choosing the appropriate platform depends on study objectives. Some methods offer higher precision (e.g., LC-HRMS), while others provide greater sensitivity (e.g., qPCR and ddPCR). Bioanalytical labs typically guide sponsors to select the approach that best meets their program’s goals rather than combining multiple platforms, which can introduce reporting complexity and cost.
A final word on Oligo Analysis
Bioanalysis is foundational to the success of oligonucleotide therapeutics. With continued innovation in stabilizers, detection technologies, and automated workflows, the field is rapidly advancing. Looking forward, expect to see broader adoption of HRMS, ddPCR, and technology-assisted data analysis as the industry addresses increasingly complex therapeutic modalities, including exosome-delivered oligos. By staying ahead of regulatory and analytical advancements, drug developers can unlock the full potential of these transformative therapies.
About WuXi AppTec
As a global company with operations across Asia, Europe, and North America, WuXi AppTec provides a broad portfolio of R&D and manufacturing services that enable the global pharmaceutical and life sciences industry to advance discoveries and deliver groundbreaking treatments to patients. Through its unique business models, WuXi AppTec’s integrated, end-to-end services include chemistry drug CRDMO (Contract Research, Development and Manufacturing Organization), biology discovery, preclinical testing and clinical research services, helping customers improve the productivity of advancing healthcare products through cost-effective and efficient solutions. WuXi AppTec received an AA ESG rating from MSCI for the fourth consecutive year in 2024 and its open-access platform is enabling more than 6,000 customers from over 30 countries to improve the health of those in need – and to realize the vision that “every drug can be made and every disease can be treated.”
