In this work, we present on the development of a new integrated workflow to support manufacturing of synthetic peptides. Following a convergent strategy, multiple synthetically manufactured peptide fragments combine to form the pharmaceutically active peptide. For example, a 40-amino acid therapeutic peptide would comprise three to four synthetic peptide fragments, with each fragment manufactured via solid phase peptide synthesis (SPPS).The SPPS process consists of three synthetic steps per amino acid coupling: 1) resin-bound amino acid deprotection, 2) activation of incoming free amino acid, 3) coupling activated amino acid to deptrotected resin-bound peptide. Incomplete deprotection of the resin bound peptide or incomplete coupling of the activated amino acid can occur during each amino acid addition leading to the formation of several difficult to remove single amino acid addition or deletion impurities. These impurities directly affect drug-substance quality and yield.<br/>To assure each reaction is complete prior to forward processing we developed a workflow to sample SPPS-resin from the fragment synthesis reactor after each coupling/deprotection step and analyze via online liquid chromatography coupled to mass spectrometry. First, SPPS resin is sampled; next, trifluoroactic acid cleaves the peptide from the resin and globally deprotects the fragment. After a dilution step, a 15-minute reversed-phase gradient separates the mixture. Mass spectra are collected from 100 to 3000 Da. Extracted ion chromatograms for starting material and product peaks provide completion information for each amino acid step of the linear build. To-date we have evaluated this workflow on a series of synthetic peptide assets under development to ensure quality of the fragment for convergent coupling and realize efficiency gains in terms of fragment build time and reagent equivalents used.

Design and development of an online-liquid chromatography mass spectrometry workflow to enable manufacturing of synthetic peptides

2019 Spring National Meeting

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