The Benefit of High pH in GLP-1 Agonist Purification 

GLP-1 Agonists are the most important peptide therapeutics nowadays. Their revolutionary potential as antidiabetic and anti-obesity drug led to an extensive demand of the peptide drugs worldwide. But the purification of these lipopeptide is challenging. The latest Application Note from YMC provides a practical and insightful guide for tackling these difficulties. It highlights why working at a high pH can offer clear advantages during the purification process.

Purification Step 1: Removal of Bulk Impurities

The initial purification step was carried out with the crude Tirzepatide material. The crude had a purity level of 20%. The method development was performed at analytical scale screening 5 different stationary phases from the YMC-Triart Prep family, a highly flexible hybrid silica process media. Unlike conventional silica, hybrid silica allows the use of high pH conditions - opening new possibilities for method development. The best results regarding separation efficiency, target purity and recovery rate were obtained with YMC-Triart Prep Bio200 C8, a C8-modified stationary phase with lower hydrophobicity and optimised pore for crude peptide purifications. Subsequently, the loading was increased to improve the productivity of the purification.

The final fractionation was optimised and yielded in a target purity level of 93% starting from a very low purity of initially 20% of the crude. To achieve the required final purity of >99.5%, a polishing step is required. 

Purification Step 2: Final Polishing

The second chromatography step in this purification process was a polishing step. It was carried out with YMC-Triart Prep C4-S which gave the best results in the stationary phase screening in combination with adjusting the method parameters. The purification was performed at pH 8.0 leading to great separation results. This is only possible due to the innovative hybrid silica base of the YMC-Triart Prep stationary phases. The C4 modification seems to be the ideal modification for a polishing purification of Tirzepatide and also other GLP-1 peptides as it provides the ideal hydrophobicity for these challenging peptides.

This second step yielded in a final product purity of 99.7%. Thus, a highly efficient purification process could be established using high pH as a key factor for GLP-1 agonists purification especially for the polishing step. With conventional silica-based RP phases, this is not possible.

Analytical process control: YMC-Triart at small scale

The YMC-Triart series is also available as analytical material leading to full flexibility in method development. Beside the purification process development, the process control can also be done with the highly stable YMC-Triart series. 

Conclusion

This two-step process approach is perfectly suited for an efficient purification of Tirzepatide. YMC-Triart Prep Bio200 C8 is the best stationary phase for the initial purification of crude Tirzepatide with low purity levels. For a polishing step, YMC-Triart Prep C4-S gave the best results leading to a final purity level of >99%. The possibility to increase the pH of the mobile phase is a key factor in the purification of these type of peptides and is possible with the highly stable YMC-Triart series. For more details have a look at our latest Application Note or contact us directly.