(622d) Liquid-Phase Assembly of Tirzepatide’s C-Terminus (30–39) via Crystalline Pentamer Building Blocks

Synthetic peptides, composed of a series of natural and unnatural amino acids, are promising pharmaceutic modalities for the treatment of a wide variety of indications such as diabetes, obesity, sleep apnea, cardiometabolic diseases and more. Traditional solid-phase peptide synthesis (SPPS) techniques are still the industry gold-standard for manufacturing high-volume peptide drugs, either as a standalone technique to synthesize the full molecule, or as a combination of SPPS fragment synthesis with solution-phase molecule assembly. Nevertheless, SPPS technology draws abundant criticism due to (i) its reliance on specialized resin and manufacturing equipment (burdening supply chain and global manufacturing capacity), (ii) high cost, (iii) high PMI and use of chemicals of concern (impacting environment). As drug demand continues to rise, there is an acute need for alternative peptide synthesis technologies.

Solution/liquid phase peptide synthesis is an excellent alternative technology to make short peptide fragments using greener and easily available reagents in standard small molecule manufacturing equipment. Like SPPS, it involves a sequence of coupling protected amino acids followed by deprotection, removal of excess reagents and proceeding with the next cycle. A commonly used protecting group is fluorenylmethoxycarbonyl (Fmoc), but it requires basic conditions for deprotection which can lead to unwanted side reactions such as deletion impurity formation via the diketopiperazine (DKP) mechanism. Instead, the Benzyloxycarbonyl (Cbz) protecting group affords easy installation and removal under mild conditions while being stable, allowing for peptide chain elongation while protecting the amino group from unwanted reactions.

In this presentation, we will discuss the development of a process to synthesize a crystalline pentamer starting from solution phase coupling of Cbz-protected amino acid derivatives, solvent exchange ahead of liquid-liquid extraction to remove excess reagents, Pd/C mediated hydrogenation to remove the Cbz protecting group, and eventually isolation as an HCl salt via cooling crystallization. Technical challenges encountered during process development (especially around extraction, hydrogenation and crystallization) will be shared, along with process performance results at manufacturing scale.