For the April edition of the BPF Journal Club, I would like to introduce an extremely interesting communication by Collins and co-workers from CEM Corporation (https://doi.org/10.1038/s41467-023-44074-5). The subject is of particular interest to those involved in peptide chemistry, peptide drug discovery, and manufacturing. The work addresses important issues related to solid-phase peptide chemistry (SPPS), in the context of large-scale production.

Since its introduction in the early 1960’s SPPS has revolutionized peptide science, especially the discovery and development of peptide therapeutics. Recent years have witnessed the emergence of incretin-based diabetes/obesity therapeutics which, as a result of unprecedented demand, require large-scale manufacturing. While some, including liraglutide and semaglutide (both from Novo-Nordisk), can be made in part by recombinant expression, others, notably tirzepatide (Eli Lilly & Co.), rely entirely on chemical synthesis. Utilizing SPPS at scales of hundreds of kilograms or more highlights several inherent limitations of the SPPS method. One of these is the poor atom-efficiency resulting from the several-fold stoichiometric excess of Fmoc-protected amino acid and coupling reagent used in each step. The second source of inefficiency involves the high consumption of solvents such as N,N-dimethylformamide (DMF) or N-methyl-pyrrolidone (NMP) used in wash steps between couplings. This produces a large volume of waste and substantially increases disposal costs. Both factors contribute to an unfavorably high process mass intensity (PMI) score (https://doi.org/10.1021/acs.joc.3c01494). This concern is further exacerbated by a regulatory climate that has restricted the use of DMF in the European Union (https://doi.org/10.1002/cssc.202301639).

The authors present an effective solution to the above problem by introducing a novel SPPS protocol that utilizes microwave-assisted evaporation and nitrogen flushing of pyrrolidine following the Fmoc deprotection step. Pyrrolidine is used in place of piperidine due to the former’s lower boiling point (bp 87 degC for pyrrolidine vs. bp 106 degC for piperidine). This modification results in a reduction of solvent and base waste of around 80%. The method is optimized using the Jung Reddman sequence and validated for several peptides including liraglutide and semaglutide, as well as the proteins proinsulin and barstar. The possibility of epimerization was checked by comparing a liraglutide sample made by the new method with a commercial sample noting no significant differences. The new methodology holds promise for minimizing not only production costs but also environmental and regulatory concerns for future large-scale peptide projects.

John P. Mayer, Ph.D.
Member, BPF Scientific Advisory Board
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