In recent years, there has been growing interest in therapeutic peptides within the pharmaceutical industry with more than 50 peptide-based therapies currently on the market, 170 in clinical trials and &gt;200 in preclinical development. The classical approach to synthetic peptide manufacture relies on solid-phase peptide synthesis (SPPS). SPPS is an approach developed nearly 60 years ago by Merrifield<sub>,</sub> utilizing a ca. 100 µm d<sub>p</sub> resin support covalently bound to the C-terminal end of an amino acid. With this resin-bound, Fmoc-protected amino acid the desired peptide is synthesized by a stepwise process using selective protecting group chemistry and filtration of spent reagents to obtain the desired peptide. Using this peptide synthesis cycle, linearly built peptides in excess of 50 amino acids in length can be synthesized. Unfortunately, as peptide length grows overall yield and purity generally decrease due to incomplete reactions generating large numbers of closely related, difficult to purify single amino acid addition and deletion impurities. In this work, we present on the development and application of an online-LC-MS based process analytical technology (PAT) platform to support development and inform manufacture of linearly built synthetic peptides. Applications focus on monitoring amino acid couplings and deprotection reactions to assure reaction completion, elucidating coupling/deprotection kinetics, and identifying and tracking critical impurities. Further, we have leveraged this new PAT tool to assess resin washing online (resulting in significant reductions in solvent consumption) as well as assure finished materials meet desired critical quality attributes.

Online liquid chromatography mass spectrometry to improve process development of solid-phase peptide syntheses

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