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 the complete YMC-Triart Prep set-up. The best results regarding separation efficiency and 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. During method development the chromatographic parameters like mobile phase composition and organic modifier were adapted to improve the overall separation results. Subsequently, the loading was increased to improve the productivity of the purification. This chromatographic purification step with YMC-Triart Prep Bio200 C8 leads to a massive improvement of the purity level of Tirzepatide.

The second chromatography step in this purification process was a polishing step via RP. This step 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 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.