Quality Control at the Source of Oligonucleotide Manufacturing: Phosphoramidite Monomer Purity Analysis Using Welch Columns
Introduction
Oligonucleotide therapeutics are short, synthetic strands of DNA or RNA. With advantages including high specificity, potent activity, and long-lasting effects, oligonucleotide drugs have attracted significant attention for the treatment of rare diseases, genetic disorders, chronic diseases, among other conditions.
The synthesis of oligonucleotide drugs depends on high-purity phosphoramidite monomers, because phosphoramidite impurities can be carried through subsequent synthesis cycles, affecting final drug product.
In this application note, we evaluated Advanchrom C18 and Xtimate C18, two hybrid silica HPLC columns, for phosphoramidite monomer purity analysis.
Solid-Phase Synthesis of Oligonucleotide Drugs
Currently, most oligonucleotide drugs are manufactured by solid-phase synthesis using phosphoramidite monomers as building blocks. During synthesis, phosphoramidites are sequentially incorporated into the growing oligonucleotide chain through repeated cycles of deprotection, coupling, oxidation, and capping, conventionally progressing from the 3' end towards the 5' end.
Each phosphoramidite contains a protected nucleoside, with protecting groups such as dimethoxytrityl (DMT) used to control the reactivity of functional groups during synthesis. Additional base or sugar modifications may also be included to improve stability and nuclease resistance.
Because phosphoramidite-related impurities may be carried through multiple synthesis cycles and ultimately affect the impurity profile of the finished oligonucleotide, strict control of phosphoramidite purity is essential for oligonucleotide manufacturing.
Phosphoramidite Monomer Purity Analysis Using Welch Columns
In this application note, Welch Materials evaluated Advanchrom C18 and Xtimate C18, two hybrid silica HPLC columns with a broad pH tolerance of 1.0 – 12.5, for the analysis of phosphoramidite monomer purity.
Both columns provided excellent performance with high column efficiency and high resolution, with purities of 98.19% and 98.12%, resolutions of 3.73 and 4.33, and theoretical plate numbers ≥ 62,000.
Chromatographic Conditions
- Column: Advanchrom C18, Xtimate C18 (4.6×250 mm, 5 µm)
- Mobile Phase: A) 10 mmol/L ammonium bicarbonate (NH4HCO3), pH 9.0; B) Acetonitrile
- Flow Rate: 1.0 mL/min
- Detector Wavelength: 237 nm
- Column Temperature: 25 °C
- Injection Volume: 10 µL
- Sample: DMT-dA(Bz) Phosphoramidite (1 mg/mL)
- Diluent: Acetonitrile
- Gradient Profile:
Time (min) A (%) B (%) 0 35 65 20 10 90 25 10 90 25.1 35 65 40 35 65
| # | Ret. Time (min) | Cont. (%) | Plates (USP) | Tailing (USP) | Res. (USP) |
|---|---|---|---|---|---|
| 1 | 15.715 | 52.92 | 56308 | 0.93 | / |
| 2 | 16.895 | 45.27 | 62892 | 0.93 | 4.33 |
| # | Ret. Time (min) | Cont. (%) | Plates (USP) | Tailing (USP) | Res. (USP) |
|---|---|---|---|---|---|
| 1 | 16.150 | 52.90 | 56626 | 0.94 | / |
| 2 | 17.187 | 45.22 | 62750 | 0.94 | 3.73 |
Why Do Two Main Peaks Appear
In both chromatograms, two phosphoramidite peaks appear. Both peaks represent the desired phosphoramidite monomer. This is because, in this monomer, the phosphorus atom is bonded to 3′-O-deoxyadenosine, a diisopropylamino group, and a β-cyanoethoxy group, with a lone pair of electrons. Together, they create a chiral center at phosphorus, giving R and S configurations. Because the deoxyribose and base moieties already contain fixed chiral centers, the molecule forms two diastereomers. These diastereomers have different retention times in HPLC and therefore appear as two main peaks.
Conclusion
Phosphoramidite monomers are critical raw materials in oligonucleotide synthesis, making reliable purity analysis essential for controlling quality from the beginning of the manufacturing process.
In this application, Advanchrom C18 and Xtimate C18 demonstrated excellent chromatographic performance for phosphoramidite analysis, providing high resolution, high column efficiency, and excellent purity.
