Purity of Nucleic Acid
It is essential to characterize the diverse impurities and degradation products present in chemically synthesized therapeutic oligonucleotides. These impurities include:
- The n–x shortmers: Common impurities arise from failed base coupling at the 5’ end and incomplete capping, leading to n–1 impurities characterized by various single deletions.
- The longmers: These primarily consist of n+1 or n+2 species.
- The modified full-length species: These impurities refer to the full-length product with alterations on its nucleobases or phosphorothioate linkages.
- End-modification impurities: Impurities arising from incorrect or incomplete modifications at the 5’ or 3’ ends of the oligonucleotide.
- Modification impurities: These include any unintended chemical modifications to the oligonucleotide, such as changes to the nucleobases, sugar moieties, or phosphate backbone.
Impurity detection priorities may vary across different types of oligos in vitro production processes, contingent upon synthesis methods, reaction conditions, and reagents utilized. Creative Biolabs has devised comprehensive testing methods, as outlined in Tab.1, to identify impurities as a crucial quality parameter. These methods encompass all potential impurities that may arise during nucleic acid production while adhering to relevant regulatory quality control standards. The use of diverse analytical techniques has facilitated process optimization, ensuring the safety and efficacy of the nucleic acid.
Tab.1 The nucleic acid impurity test methods.
| Items | Decriperition | Test Methods |
|---|---|---|
| Synthetic impurities | These impurities include deletion sequences ('shortmers'), addition sequences ('longmers'), modified full-length species, End-modification impurities, and Modification impurities. | High-Performance Liquid Chromatography (HPLC), capillary electrophoresis (CE), or mass spectrometry |
| Ions and salts | Ensure that the concentrations of ions and salts in solvents, reagents, and buffers used in the synthesis reaction meet specifications. | Ion chromatography (IC) or atomic absorption spectroscopy (AAS). |
| Solvent residues | Detect solvent residues used during synthesis to ensure the product does not contain them. | Gas chromatography (GC) and liquid chromatography (LC). |
| Exogenous DNA contamination | Detecting the presence of exogenous DNA or RNA contamination, originating from the laboratory environment, instruments, reagents, or operators, is crucial when synthetic oligos are employed for diverse applications. | Polymerase chain reaction (PCR) or colorimetric assays. |
| Exogenous RNA contamination | Reverse Transcription Polymerase Chain Reaction (RT-PCR) or Southern Blot Assays. | |
| Exogenous Protein contamination | The primary routes of external protein contamination during nucleic acid production involve the introduction of enzymes (such as nucleases and proteases) and the presence of residual intracellular proteins. | Nano Orange Assay. |
SERVICES
The purity of nucleic acids is essential for ensuring their effectiveness in a wide range of applications. Choosing appropriate sample preparation methods and purity detection techniques, as well as rigorously controlling impurity levels, are of paramount importance for ensuring the accuracy and reliability of experimental results. Creative Biolabs is committed to offering high-quality purity test services for worldwide customers. For more detailed information, please feel free to contact us and get a quote. We'll reach out to you within 24 hours and formulate the best method for your project.
