This study focused on understanding diketopiperazine impurities formation during solid phase peptide synthesis (SPPS) of tirzepatide (TZP), a 39 amino acid synthetic peptide. During SPPS synthesis of TZP, we found significant desProPro double amino acid deletion impurity. For this reason, we initially conducted root cause analysis and identified the Fmoc- protection group was not stable for TZP API intermediate with proline as penultimate amino acid. During post-coupling hold times, it is found that Fmoc- deprotection can proceed auto-catalytically in various solvents without any base addition. Followed by self-deprotection of Fmoc group, subsequent DKP formation on the deprotected peptide occurred at steady reaction rate. Then we measured the DKP formation under different peptide sequence, solvent composition, temperature and stabilizer additive conditions. Based on those measurement, we proposed the detailed Fmoc- self-deprotection and subsequent DKP formation mechanisms that include intramolecular pathways triggered by the amine group of the penultimate proline and intermolecular pathways catalysed by the deprotected amine group in the peptide and DKP by product. A macro-kinetics model is then built to describe various peptide API intermediates stability under different solvents and temperature conditions.

Understanding diketopiperazine formation mechanism and control strategies during solid phase peptide synthesis of peptide: Impact of solvent, peptide sequence, storage conditions and stabilizer additives

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