Outsourcing Oligonucleotide Testing: Lessons and Strategies

The benefits to progressing oligonucleotide therapies far outweigh the complexities of their analytical testing methods. Here’s why.

Oligonucleotides, also known as oligos, are strands of artificially created DNA or RNA that are instrumental in numerous molecular and synthetic biology applications. These applications range from genetic testing and forensic research to next-generation sequencing (NGS). Synthetic oligonucleotides come in various forms: antisense single stranded products, siRNA double stranded products and aptamers, along with various oligonucleotide conjugates, including antibody conjugates. They have the potential to treat a wide range of diseases, including many undruggable diseases that have serious or potentially life-threating implications, such as neurodegenerative diseases, cancer, high blood pressure, hemophilia and a variety of rare genetic/metabolic diseases. Each type of oligonucleotide drug substance or product requires suitable testing to ensure its purity and safety.

While oligonucleotide R&D has been ongoing for decades, there is no specific guideline yet from the International Council for Harmonisation (ICH), European Medicines Agency (EMA) or U.S. Food and Drug Administration (FDA) for the development and manufacturing of synthetically manufactured oligonucleotides. Recently published draft guidelines from the EMA have outlined that “specific analytical procedures to control the identity, purity and assay of the oligonucleotide should be developed” which include identification by mass spectrometry, high-performance liquid chromatography-ultraviolet (HPLC-UV) and ID by sequence analysis. Oligonucleotides are explicitly excluded from ICH guideline Q3A/VICH GL10, but the goal and principal of the guideline still apply — known and unknown impurities must be quantified. The impurity profile of oligonucleotides results in impurities that are very similar to each other, and so it is commonly acceptable to group these together based on their relative retention time. A combination of stability indicating and, if needed, orthogonal methods should be employed to identify impurities that are difficult to detect. An assay can be determined by UV absorption; lyophilized oligonucleotides are hydroscopic, so those performing the assay should take water content into consideration.

This range of testing is wide and requires many different techniques and because of this, equipment and experience with oligonucleotide testing is essential when looking for a external partner to perform development, verification, validation and release/stability testing. The wide range of testing required for drug substances and drug products includes, but is not limited to, the following:

• Physical characterization testing
  • Water content
  • Residual solvents
  • pH
  • Elemental analysis by atomic absorption spectroscopy
  • Appearance
  • Osmolality
  • Particulate matter
• Identity
  • Sequence confirmation by HRMS
  • Identification by MS
  • Sequence confirmation by melting point
• Assay, purity and impurities
  • Orthogonal HPLC methods, including purity by denaturing IPRP, purity by denaturing anion exchange
  • Purity and impurities by HPLC-MS
  • Assay by UV spectrometry
  • Elemental impurity analysis by ICP-OES or ICP-MS
• Endotoxin
• Sterility

Oligonucleotide methods for purity and impurity by HPLC-MS, Denaturing IPRP-HPLC-UV, while robust, are also sensitive to contamination from other compounds. It is important that the chosen outsourcing partner not only has the high-tech equipment required, but also the dedicated equipment for oligonucleotide testing, or an understanding of the cleaning and HPLC set up requirements for this type of testing. Bio Inert HPLCs and UPLCs can also be used for testing oligonucleotides but are not specifically necessary. These factors are important to consider when contract research organization laboratories or contract manufacturing organizations share equipment, such as HPLCs with other drug substances and products.

The complexity of processing HPLC data for double stranded oligonucleotides, or impurities by mass spectrometry, not only requires analysts with established practical knowledge on how to troubleshoot methods during validation, but also during routine release and stability analysis. Profiling impurities is essential to ensure that results are analyzed for trends or possible difficulties pertaining to method execution.

There is a trend toward moving parenteral oligonucleotide drug products from pre-filled syringes to pre-filled syringes with a safety device and auto-injectors. Device functional capabilities, like activation force, delivered volume and break loose/glide force is also a factor to consider when outsourcing your oligonucleotide drug product testing.

Aside from the technical aspects associated with method development, it is also important to consider equipment, expertise and knowledge. Other factors to consider are quality (regulatory inspection history, and internal GMP quality controls), project management, adequate stability storage, a proven track record with oligonucleotide testing and open communication lines. A flexible clinical research organization (CRO) laboratory, like PPD™ Laboratory services, enables success in solid-phase synthesis by expertly navigating the multiple cycles of deprotection, coupling, oxidation/sulfurization and capping steps involved in the addition of each base in the oligonucleotide. The complexity of this process can lead to delays in delivery of drug substances and drug products, or in some cases failures of drug substances to meet release specifications. A CRO laboratory that understands the delays and can adapt release timelines, and stability set down timetables is necessary.

Other important studies for oligonucleotides to consider during early and late phase development are forced degradation studies, temperature cycling studies, and photostability studies. Photostability studies being primary among these as oligonucleotide drug products and substances can be susceptible to degradation from UV light.

The benefits to progressing and pursuing oligonucleotide therapies far outweigh the complexity and technologies associated with development and execution of analytical test methods for these drug products. Our laboratory services team works closely with sponsors to build an open and honest partnership, where knowledge and learnings can be shared to progress the development and implementation of sometimes difficult testing methods. Our experts have the same focus in mind as our partners – to work as one team to get drug products to the market faster and improve the quality of life for affected patients.