Oligonucleotide progression into early phase studies

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The quality control (QC) testing of oligonucleotide formulations is crucial for their use in early phase clinical trials. Although oligonucleotides are not specifically covered under ICH Q3A and Q6A guidelines, they are subject to certain aspects of the EU GMP Annex 1 (Manufacture of Sterile Medicinal Products) and sections of ICH Q6A pertaining to parenteral drug products. Moreover, the draft EMA Guideline on the Development and Manufacture of Oligonucleotides outlines requirements for both active substances and finished products, which will be important for future regulatory compliance.

Sequence of active substance

To ensure the correct sequence of nucleobases in oligonucleotides, thorough analysis must be conducted during both the manufacturing/synthesis process and after completion of synthesis. This process control is essential for confirming the sequence of the active substance.

Mass Spectrometry (MS) is the primary technique for sequence verification. After synthesis, oligonucleotides are fragmented into smaller parts (one or more nucleotides), which can be achieved by direct or indirect fragmentation techniques.

Direct fragmentation

Direct fragmentation is performed using mass spectrometry in the gas phase via methods such as collision-induced dissociation, laser ionization, or thermal decomposition. These fragments are then analysed with LC-MS/MS (Liquid Chromatography-Mass Spectrometry/Mass Spectrometry). For shorter oligonucleotides, MALDI-MS (Matrix-Assisted Laser Desorption/Ionization) or ESI-MS (Electrospray Ionization) can suffice. For longer strands or duplex DNA/RNA, Triple Quadrupole MS (MS/MS) provides additional advantages by enabling fragmentation through collision-induced dissociation and more detailed fragment analysis.

Indirect degradation/digestion

Indirect methods involve enzymatic digestion or chemical degradation to break specific bonds between nucleic acids. The resulting fragments are analyzed by MS or electrophoresis. However, oligonucleotides often undergo modifications to reduce susceptibility to enzymatic degradation, which can limit the effectiveness of this approach.

Sequence analysis

Once the data from fragmentation is collected, the sequence can be reconstructed. The longer the oligonucleotide, the more complex this process becomes. For this reason, in-process analysis during synthesis is critical for accurate sequence verification.

Identity testing of the product

Confirming the identity of oligonucleotides in their final drug product is essential for regulatory compliance and clinical trial progression. Mass Spectrometry (MS) is the gold standard for identity testing. Both Electrospray Ionization (ESI) and MALDI are commonly used ionization techniques, with negative ionization preferred due to the polybasic nature of oligonucleotides.

However, MS presents several challenges:

Multiple charging during ionization can lead to a mix of charged species, complicating data interpretation.
The presence of sodium (Na+) or potassium (K+) ions can reduce sensitivity and mass accuracy due to adduct formation. Volatile counterions like ammonium (NH4+) can be used to mitigate these issues.
Fragmentation during ionization may yield multiple ion fragments, which could both aid in sequence analysis and complicate identity confirmation.

Other techniques, such as High-Performance Liquid Chromatography (HPLC) coupled with Ultraviolet (UV) detection, can be employed as secondary methods to confirm identity. The retention time in HPLC can provide additional verification, although this is less specific than MS. A UV spectrum of the oligonucleotide is often recorded and compared to a reference spectrum as another identity confirmation step.

Assay and impurity analysis of drug products

To ensure the safety, efficacy, and stability of oligonucleotide drug products, assay and impurity analysis are vital. HPLC is commonly employed for both assay and purity profiling. The draft EMA guideline specifies thresholds for degradation and impurities:

Reporting threshold: 0.2%
Identification threshold: 1.0%
Qualification threshold: 1.5%

Anion exchange chromatography

Anion exchange chromatography remains one of the most traditional techniques for separating negatively charged molecules such as oligonucleotides. In this method, oligonucleotides interact with a positively charged resin in the column, and their elution is controlled by varying salt concentration. The stronger the negative charge on the oligonucleotide, the stronger its interaction with the stationary phase.

However, anion exchange chromatography has limitations, including incompatibility with MS due to ion interference. Using special columns and extended equilibration times may mitigate some issues, but it is generally less efficient and more time-consuming compared to newer techniques.

Reversed phase chromatography with ion pair reagents

Reversed-phase chromatography (RPC), using ion pair reagents, offers a more effective separation method for oligonucleotides based on hydrophobic interactions. By adding ion-pairing agents (such as surfactants or salts) to the mobile phase, oligonucleotides form neutral complexes and are separated based on hydrophobicity.

This method is compatible with MS when volatile counterions like Hexafluoroisopropanol (HFIP) are used. RPC offers significant advantages over anion exchange chromatography, such as broader applicability and better compatibility with MS. The use of MS in conjunction with RPC can provide greater insight into impurities and degradation products, enabling both identification and structural characterization.

Size exclusion chromatography (SEC)

Size exclusion chromatography (SEC), or gel filtration, separates molecules based on size. This method is valuable for analyzing the molecular weight and purity of oligonucleotides. While SEC may not be as effective for precise content analysis, it is especially useful for assessing single-strand content in duplex oligonucleotide formulations.

SEC provides limited information about the identity of impurities or degradation products. Separation between smaller molecular weight impurities can also be challenging, which may affect quantitative analysis.

Conclusion

For oligonucleotide formulations entering early phase clinical studies, quality control through accurate sequence identification, identity testing, and impurity analysis is paramount. Mass spectrometry remains the cornerstone of sequence verification and identity testing, with HPLC serving as a valuable complementary technique. Impurity analysis using various chromatography techniques ensures that both the efficacy and safety of oligonucleotide-based drug products are maintained throughout their development.

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