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Minimising impurity formation in oligonucleotide synthesis

Strategies for high quality drug products

Blog 1 of 3

 

At ICON we recognise the critical role that impurity control plays in the successful development of oligonucleotide (ON) therapeutics. Through our Accelerated Pharmaceutical Solutions and integrated CMC support, we assist clients in optimising their drug product development strategies, with a particular focus on ensuring product purity and overall composition in accordance with regulatory guidelines.

In the first installment of our three-part series, we provide an introduction to oligonucleotide therapeutics and examine how API synthesis strategies impact impurity formation, highlighting best practices for risk mitigation.

Oligonucleotide therapeutics

Oligonucleotide therapeutics are opening up new treatment possibilities for a broad spectrum of diseases, including rare genetic disorders and conditions previously deemed undruggable. These advanced therapies are built from synthetic RNA or RNA-DNA hybrids, precisely designed to bind to target RNA sequences. Rather than merely inhibiting proteins, ONs modulate gene expression, enabling highly targeted interventions across new therapeutic frontiers.

Recent years have seen accelerated investment in the development of platform technologies to streamline ON discovery and development. One leading force in this progress is the European Pharma Oligonucleotide Consortium (EPOC), formed in 2017. EPOC brings together industry experts to define best practices and practical solutions that support faster development timelines, while maintaining the highest safety and quality standards. Through its publications and recommendations, EPOC continues to play a pivotal role in advancing ON manufacturing practices and innovation across the sector.

Oligonucleotides – API synthesis considerations

The synthesis of ONs is a sophisticated, stepwise process distinct from traditional small-molecule chemistry. These molecules are typically short, ranging from 20 to 40 nucleotides, and incorporate a ribose-phosphate backbone, along with modified nucleobases to improve metabolic stability, binding specificity, and delivery.

API synthesis relies on solid-phase synthesis (SPS), which enables precise, controlled construction of sequences one nucleotide at a time. Each synthesis cycle includes four core chemical reactions and must be tightly controlled to ensure efficiency and minimise impurities. High coupling efficiency, ideally exceeding 99+%, is vital, especially for longer sequences, as even small per-step losses can result in poor yields and ultimately translated to significant accumulation of truncated products.

Impurity formation increases with sequence length and complexity. This risk is further influenced by the nucleotide sequence itself, the choice of chemical modifications, and the consistency of reaction conditions. From the initial selection of solid supports and reagents to optimisation of protection and deprotection strategies, every decision can impact product quality. Even seemingly minor changes can introduce hard-to-remove contaminants that persist through purification steps.

Achieving a high-purity API requires more than just precise synthesis, it demands a deep understanding of the chemistry involved and a commitment to continuous process optimisation. Robust purification strategies, combined with proactive impurity control from the earliest stages, are key to delivering high quality ON therapeutics.

Critical Quality Attributes (CQAs)

Ensuring the quality, safety, and efficacy of ON drug products depends on a well-defined set of Critical Quality Attributes (CQAs). While the mode of action of ON therapeutics has similarities with biologicals in the way this type of API is synthesised, we should consider the guidance outlined in ICH Q6A, which provides specifications for new drug substances and products.

These parameters guide development, manufacturing, and regulatory approval, and collectively form the backbone of a robust drug development strategy.

  • Purity: A non-negotiable standard, purity ensures the product is free from contaminants such as truncated sequences and residual solvents. Even trace impurities can compromise therapeutic performance or safety.
  • Identity: Confirms the correct nucleotide sequence and structural integrity. 
  • Impurity profile: Includes all process- and product-related byproducts (e.g., N-1 sequences), which must be quantified and tightly controlled to avoid toxicity or loss of potency.
  • Potency: Reflects the biological activity and concentration of the ON, ensuring consistent dosing and therapeutic outcomes across batches.
  • Stability: Determines shelf life and performance over time. Storage conditions, packaging, and degradation risks must be assessed early.
  • Residual solvents and water content: Must be minimised in line with ICH Q3C guidelines. Excess solvents or moisture can degrade product quality, particularly in lyophilized forms.
  • Stereochemical purity:  Especially relevant for ONs with modified backbones like phosphorothioates, where stereochemistry affects binding affinity and pharmacological activity. Controlling isomer ratios supports better targeting and reduces off-target effects.

Each of these attributes is interconnected, and together, they ensure that ON drug products are not only effective but also safe and reliable for patients. Then based on the predominantly parenteral administration of ON drug products, the relevant compendials for a sterile drug product would apply.

As the field of oligonucleotide therapeutics evolves, maintaining control over synthesis and impurity formation becomes increasingly critical. At ICON, we offer the integrated CMC expertise and regulatory insight needed to help sponsors navigate this complexity and bring quality-purity ON drug products to market faster.

Stay tuned for part two of our series, where we’ll explore drug product process development and quality control strategies.

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Learn more about our comprehensive Accelerated Pharmaceutical Solutions and how ICON’s experts can support the development of optimal formulations for your clinical trial.

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