Solid phase peptide synthesis (SPPS) is generally the method of choice for the chemical synthesis of peptides, allowing routine synthesis of virtually any type of peptide sequence, including complex or cyclic peptide products. In large‐scale peptide manufacturing, resin substitution is a critical quality attribute which strongly impacts the financial viability of the synthesis and potentially the drug substance quality. Solid phase peptide synthesis (SPPS) is governed by the resin substitution which is of utmost importance for calculating the required input of amino acids and reagents which in turn impacts the associated yield of the synthesis. 2-chlorotrityl chloride (2-CTC) is one such resin that offers several advantages in SPPS with multiple applications.

Although literature [1,2] provides several methods to determine such substitution, for highly moisture sensitive resins like 2-CTC resin, the accurate substitution determination becomes very difficult. In this case study, the determination of substitution of 2-CTC resin by the process of loading Fmoc-amino acid to the 2-CTC resin and the subsequent Ultra High Performance Liquid Chromatography (UHPLC) measurement of the liberated Fmoc-amino acid after cleaving the loaded resin is discussed. A multi-point standardisation method that involves the standard solutions of the corresponding Fmoc-amino acid to be quantified was used.