New Focus on Biomolecules
Peptides and oligonucleotides are becoming increasingly important as biopharmaceuticals. The broad functional spectrum of these molecules offers a wide range of pharmaceutical applications. On the other hand, their variety in chemical and physical properties as well as impurities with similar characteristics complicate the reliable and safe production. For their purification, (ion-pair-) reversed phase (IP-RP) and ion exchange (IEX) chromatography are ideal techniques. The charged oligonucleotide backbone is excellently suited for IEX but on the other hand cannot interact sufficiently with the reversed stationary phase. Therefore ion-pair reagents with a hydrophobic group are required. They act as mediators between the charged target and the hydrophobic stationary phase. This leads to an effective retention of the oligonucleotide on the RP phase. These separation modes are based on different chemical interactions, but both offer high purity levels with increased productivity.
Complete screening set-up with YMC-Triart Prep
For IP-RP YMC-Triart Prep is the ideal choice. The material provides high resolution to achieve the required purity. Since selectivity has the greatest influence on the resolution it is recommended to screen different stationary phases. It often makes sense to consider other modifications in addition to the standard C18 phase. Therefore, the YMC-Triart Prep family offers a wide range of different selectivities for the purification of peptides and oligonucleotides. A phenyl phase can be beneficial for the purification of biomolecules containing aromatic structures. Due to the additional π-π-interactions, this stationary phase offers a unique selectivity. However, for larger peptides and oligonucleotides, a larger pore size is advantageous. YMC-Triart Prep Bio200 C8 was developed specifically for this purpose. Choosing the right stationary phase can determine the productivity and efficiency of the entire process. YMC-Triart Prep is fully scalable from analytical to preparative scale. This allows easy method development and customised solutions.
Extraordinary stability: chemical and mechanical
In general, changing the pH value to extreme conditions can be helpful for difficult purification tasks. Alkaline buffers are particularly useful for effective binding and separation when purifying oligonucleotides and basic peptides. But high pH cannot be used with silica-based phases. For this reason, the YMC-Triart Prep family is based on a hybrid silica material. Crosslinking silica with ethylene bridges enables exceptional chemical stability. Therefore, YMC-Triart Prep is stable even at high pH values. Another advantage of the robustness of YMC-Triart Prep stationary phases are the reproducible results when using 100% aqueous conditions. This is particularly useful for polar peptides.
To regenerate the column and restore chromatographic performance, common CIP (cleaning-in-place) procedures with 0.1 M NaOH can be used. The YMC-Triart Prep stationary phases are stable for more than 300 CIP cycles. Thanks to its robustness and long lifetime, this packing material reduces process costs enormously. In combination with the excellent chromatographic performance and high loadability, YMC-Triart Prep can contribute to higher productivity of the overall process.
Another possibility for the purification of oligonucleotides and peptides: IEX
In addition to RP chromatography, oligonucleotides and peptides can also be purified using ion exchange chromatography. Even if the separation mode is different, the challenges are the same. The ion exchange resins from YMC are based on polymethacrylate beads and are available as a strong anion exchanger (BioPro IEX Q) and a strong cation exchanger (BioPro IEX S). For initial product capture IEX resins with a high dynamic binding capacity (DBC) such as BioPro IEX 75 are advantageous. The high loadability results in higher productivity. For the following intermediate purification and polishing steps smaller particle sizes are typically used to improve the resolution.
BioPro IEX SmartSep with particle sizes of 10, 20 and 30 µm is ideally suited for the purification of oligonucleotides and even allows the separation of shortmers (n-1) and longmers (n+1). In addition, an increased temperature can also be used to further optimise the resolution. The BioPro IEX SmartSep resins are specified up to a temperature of 60 °C. This opens more possibilities for method development. Due to the high dynamic binding capacity, high loadings can be used which improves the overall productivity of the purification process. Another advantage are the outstanding pressure-flow properties. They allow high flow rates and lead to high throughput and an effective purification. In addition, the resins can be cleaned with 1 M NaOH, which drastically extends their lifetime. The BioPro IEX resins are scalable across all particle sizes, which gives more flexibility in method development.
Conclusion
The purification of oligonucleotides and peptides is an exciting challenge. With innovative IEX and IP-RP methods, the high requirements can be mastered effectively and efficiently. It is important that, regardless of the separation mode, you choose the right stationary phase. This ensures a productive and effective process.
Further information on peptide and oligonucleotide purification can be found in our informative Whitepapers or on www.ymc.eu.
