The synthesis of oligonucleotides comes with a complex impurity profile which creates challenges in their analytical separation with product impurities being similar to the parental sequence. In addition, the oligo backbones can be highly modified via thiolation which creates diastereoisomers.
Many of the available ICH quality guidelines are applicable for oligos, with a few exceptions. For example, the guideline for method validation applies for oligos but with some adjustments. Focusing on guidelines for impurity of drug substance and drug product ICH Q3 A and B but explicitly do not cover oligonucleotides but the principles can be applied to process and product related impurities.
However, this field is developing, and researchers are awaiting updates regarding quality guidelines. The EMA recently drafted guidelines relating to development and manufacture of oligos, and the Chinese Center for Drug Evaluation (CDE) is preparing guidelines for their chemical synthesis. Furthermore, the upcoming ICH Q6 update also promises to cover oligonucleotides.
This talk focused on the product related impurities of oligonucleotides, of which the toxicological qualification is vital. This typically involves a combination of analytical techniques to identify and quantify the various impurities that can be present in chemically synthesized oligonucleotides. Your choice of method can depend on type of impurities, required sensitivity of detection, and available equipment.
Liquid Chromatography with UV detection (LC-UV) Methods
Evaluating the need for orthogonal methods is essential in LC-UV analysis. One must also assess the peak purity of the main UV peak and the impurity UV peaks, which helps in controlling co-eluting impurities, such as those identified by HR-MS. Using MS-compatible buffer and solvent systems can facilitate the eventual hyphenation for better process control and impurity identification. Employing 2D-LC technologies, such as salt swapping and additional characterisation, can be beneficial. Attention must be paid to retention time shifts, and peak alignment strategies should be employed accordingly.
Liquid Chromatography with UV detection and Mass Spectrometry (LC-UV-MS) Methods
For LC-UV-MS methods, low-resolution analysis enables the quantitation and identification of impurities within a single method, while controlling co-eluting impurities and avoiding false positive detection. It is crucial to aim for low noise and adduct levels in MS, as leaching of metal ions from buffer instrument components can affect results. Implementing such methods in a QC environment can be challenging, necessitating evaluations of long-term strategies either in-house or externally.
High-resolution LC-UV-MS methods provide a deeper understanding of the impurity profile through supportive information and isotopically resolved MS spectra. Optimising source settings for the most informative charge states involves balancing signal intensity, adduct presence, and in-source fragmentation. It requires intensive data processing to handle the resultant information effectively.