Oligonucleotides, short synthetic strands of nucleic acids, are essential in therapeutics, diagnostics, and molecular biology research. Their quality is paramount, as even trace impurities can impact efficacy and safety. Identifying and quantifying impurities in oligonucleotides is a crucial aspect of quality control, ensuring compliance with regulatory guidelines and maintaining consistency in production. Alfa Chemistry provides comprehensive impurity profiling solutions using state-of-the-art analytical techniques to support oligonucleotide development and quality control.
Types of Impurities in Oligonucleotides
Oligonucleotide impurities can affect the stability, efficacy, and safety of oligonucleotide therapeutics. These impurities are mainly:
- Failure Sequences and Truncated Oligonucleotides
During solid-phase synthesis, incomplete coupling reactions can lead to truncated oligonucleotides, also known as n-x sequences. These impurities are generated when nucleotide monomers fail to incorporate at specific positions, leading to shorter sequences that may interfere with therapeutic function. Despite capping strategies to terminate unreacted sequences, these residual failure sequences remain a major problem in the manufacturing process.
- Depurinated and Degraded Products
Oligonucleotides are prone to depurination, a process where purine bases (adenine and guanine) are hydrolyzed, leading to abasic sites that compromise structural integrity. Additionally, exposure to heat, pH variations, or nucleases can cause strand cleavage and degradation. These degraded species must be accurately quantified to ensure oligonucleotide stability.
- Phosphorothioate and Phosphate Impurities
Oligonucleotide modification strategies, such as phosphorothioate backbones used to enhance stability, can lead to diastereomeric impurities. These arise due to incomplete sulfurization or oxidation during synthesis. Similarly, variations in phosphorylation states (mono-, di-, or triphosphorylated species) can introduce heterogeneity in oligonucleotide samples, impacting biological activity.
Chemical protecting groups, oxidizing agents, and reagents used in synthesis can generate unwanted side products. For example, excessive oxidation of guanine residues can lead to the formation of 8-oxo-guanine, which can impact oligonucleotide function. Additionally, alkylation or cross-linking reactions may generate adducts that need to be identified and removed.
- Residual Solvents and Reagents
Manufacturing processes involve organic solvents (e.g., acetonitrile) and reagents such as triethylamine, which may persist in the final product if not efficiently removed. Regulatory guidelines set strict limits on residual solvents and reagents due to their potential toxicity.
Our Analytical Techniques
Accurate identification and quantification of oligonucleotide impurities require advanced analytical techniques. We employ the following methods to ensure high specificity, sensitivity and resolution of impurity analysis.
- High-Performance Liquid Chromatography (HPLC): Reverse-phase (RP-HPLC) and ion-pair reversed-phase HPLC (IP-RP-HPLC) are widely used for separating and quantifying oligonucleotide impurities based on hydrophobic interactions. Strong anion-exchange HPLC (SAX-HPLC) further enables high-resolution separation of failure sequences and modified oligonucleotides.
- Capillary Electrophoresis (CE): CE offers high-resolution separation based on charge-to-mass ratios and is highly effective in detecting truncated impurities.
- Mass Spectrometry (MS): Liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption/ionization (MALDI-MS) provide molecular weight determination and structural elucidation of oligonucleotide impurities.
- Nuclear Magnetic Resonance (NMR) Spectroscopy: NMR can be employed for structural verification and impurity identification at the atomic level.
- UV-Vis Spectroscopy and Fluorescence Detection: These techniques provide complementary data on oligonucleotide purity, though they are less specific than chromatographic and MS-based methods.
Why Choose Us
- Customized Analytical Solutions
Alfa Chemistry provides customized analytical solutions to meet specific client needs, ultimately achieving high-sensitivity impurity identification and quantitation.
- Expert Consultation and Data Interpretation
Beyond generating analytical data, Alfa Chemistry provides expert consultation to help clients interpret results and make informed decisions.
- Reliable and Efficient Service
Alfa Chemistry's efficient workflow and dedicated support team ensures that clients receive accurate impurity analysis reports in a timely manner, allowing for a smooth regulatory submission and product development process.
Our products and services are for research use only and cannot be used for any clinical purposes.
