An Optimized Scalable Fully Automated Solid-Phase Microwave-Assisted cGMP-Ready Process for the Preparation of Eptifibatide

Abstract

We investigated several strategies, based on the use of microwave-assisted solid-phase peptide synthesis (MW-SPPS) and scalable to kilogram-scale manufacturing, for the preparation of Eptifibatide, a disulfide-bridged cyclo-heptapeptide drug approved as an antithrombotic agent. Following the very fast microwave-assisted Fmoc/tBu synthesis of the linear precursor, we explored both the solution (off-resin) and the solid-phase (on-resin) disulfide formation. In order to optimize the oxidation in solution, we focused our attention on the mild disulfide formation procedure based on the use of air, observing some drawbacks, such as the formation of unwanted oxidation byproducts, such as dimers, or the use of large volumes of an environmentally unfriendly solvent (CH 3 CN). In order to overcome these difficulties, we studied four different on-resin strategies, with the final aim to develop a fully automated, single reactor procedure, exploring different strategies to protect the thiol side-chain functional group on the C-terminal Cys residue and to form the Eptifibatide ring. The main difference among these strategies is represented by the final cyclization mode that was obtained either by direct formation of an S−S disulfide bridge or by head to MPA on cysteine sidechain amide bond formation. In conclusion, the optimization of the latter strategy enabled us to devise an optimized scalable fully automated solid-phase microwave-assisted cGMP-ready process to prepare Eptifibatide.

De Novo Development of Mitochondria-Targeted Molecular Probes Targeting Pink1

“…Coupling was done using the DIC (carbodiimide)/Oxyma (ethyl 2-cyano-2-(hydroxyimino)acetate) approach, as it is a base-free condition known to reduce epimerization [ 43 ]. Additionally, DIC is stable at high temperature (90 °C) [ 44 ]. The cyclization step has been done via an amide bond to a dicarboxylic acid spacer attached to the N-terminus of the peptide by SPPS methodology, by means of “on-resin cyclization”.…”

An Optimized Safe Process from Bench to Pilot cGMP Production of API Eptifibatide Using a Multigram-Scale Microwave-Assisted Solid-Phase Peptide Synthesizer

“…This solid-phase strategy had the advantage of synthesizing and cyclizing eptifibatide in the same reactor. 15 However, in the multigram-scale process developed herein, the use of the less expensive building-block Fmoc-Cys(Trt)-OH (compared to Fmoc-Cys(StBu)-OH used in the previously reported strategy) and the increased HPLC purity yield of eptifibatide crude obtained by air oxidation in solution (65% vs 40.9%) let us further investigate solution disulfide bond formation. Therefore, several oxidants and reaction conditions were tested, 36−40 in order to obtain the highest eptifibatide HPLC crude purity (data not shown).…”

“…Our results definitely demonstrate that the strong collaboration between an academic facility and a contract development manufacturing organization, for small molecule APIs and intermediate industrial GMP production, is a powerful example of a joint laboratory satisfying the needs of a market entry strategy into peptide API production. 15 Cleavage Step. The cleavage, with concomitant deprotection of acid sensitive amino-acid side chains was performed by treating MPA(Trt)-Har(Pbf)-Gly-Asp(OtBu)-Trp(Boc)-Pro-Cys(Trt)-Rink Amide AM resin (15.2 g, 5 mmol) with the cocktail TFA/TIS/H 2 O/DODT 107 mL, 72:7:7:14 (v/v/ v/v) for 30 min at room temperature under mechanical stirring (150 rpm).…”

“…CEM Corporation (Charlotte, NC, U.S.A.) provides three microwave-assisted solid-phase synthesizers allowing the scale-up of a MW-SPPS optimized process. 15 These instruments can perform syntheses from 0.005 to 5 mmol scale (Liberty Prime and Liberty Blue systems) and from 5 to 800 mmol scale (Liberty Pro system). The latter was used within the work reported herein for a cGMP compliant industrial production of a 70 mmol pilotscale peptide active pharmaceutical ingredient to be commercialized as a generic drug.…”

“…As recently described, automated MW-assisted solid-phase synthesis significantly decreased production times and waste volumes to obtain up to 5 mmol eptifibatide (Figure 1). 15…”