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

Annunziata D’Ercole,Anna Maria Papini,Alfredo Paio,Matteo Zini,Paolo Rovero,Lorenzo Pacini,Giuseppina Sabatino,Francesca Nuti,Arianna Ribecai

doi:10.1021/acs.oprd.1c00368

Abstract

A growing industrial interest toward the peptide drug market fueled the need for the development of effective and cGMP compliant manufacturing methods for these complex molecules. Solid-phase strategies are considered methods of election for medium-length peptide syntheses not only on the research scale but for multigram-scale production, as well. The possibility to use microwave-assisted technology on the multigram scale, recently introduced, prompted us to evaluate the possibility to conveniently set up a safe and fully cGMP-compliant pilot process to produce eptifibatide, a generic peptide active pharmaceutical ingredient. Accordingly, we developed an optimized process on the laboratory scale (1–5 mmol), which was subsequently successfully scaled up to 70 mmol, obtaining all the information required by regulatory agencies to validate the process and qualify the pilot scale plant. The process consists of 5 steps: (1) automated microwave-assisted solid-phase synthesis of eptifibatide linear precursor; (2) cleavage from the resin with concomitant amino acid side-chains deprotection; (3) disulfide-bond formation in solution; (4) purification by flash column chromatography; (5) ion-exchange solid-phase extraction. Since the direct scale-up of a multigram-scale cGMP compliant peptide API production procedure is a challenge that requires an accurate understanding of each involved step, we initially performed a quality management risk assessment, which enabled a smooth and effective achievement of a successful final result.

Highlights

According to the latest “Transparency Market Research” report, the global peptide therapeutics market was valued in 2018 at ca. 25 billion USD, and it is anticipated to expand until 2027 at a Compound Annual Growth Rate (CAGR) of 7.9%.1 a recent report by Roots Analysis, Business Research and Consulting, expects that the peptide therapeutics market following industry trends and global forecast 2021− 2030, will be directly associated with increasing investments in R&D activities, in search of new drug substances for treating infectious diseases, metabolic disorders, diabetes, cancer, and other diseases.[1]
Final aim of the present work is to provide a proof-of-concept that industrial manufacturing of peptide active pharmaceutical ingredient (API) can take advantage from the performances of MW-assisted solid-phase synthesizers available on the market. This approach can be strategic for the scale-up of the synthetic process, for those manufacturers entering the market of peptide generic drugs and requiring scientific knowledge transfer and support in overcoming possible unforeseen events in peptide chemistry
We report the laboratory process optimization (1−5 mmol) of the heterodetic cyclopeptide API eptifibatide acetate (Figure 1) in five steps: (1) eptifibatide linear precursor automated MW-assisted solid-phase synthesis (Liberty Blue, CEM, Charlotte, NC, U.S.A, step 1); (2) cleavage from the resin and amino acid side-chain deprotection; (3) in solution disulfide-bond formation; (4) purification by flash column chromatography, followed by (5) ionexchange solid-phase extraction

Summary

■ INTRODUCTION

According to the latest “Transparency Market Research” report, the global peptide therapeutics market was valued in 2018 at ca. 25 billion USD, and it is anticipated to expand until 2027 at a Compound Annual Growth Rate (CAGR) of 7.9%.1 a recent report by Roots Analysis, Business Research and Consulting, expects that the peptide therapeutics market following industry trends and global forecast 2021− 2030, will be directly associated with increasing investments in R&D activities, in search of new drug substances for treating infectious diseases, metabolic disorders, diabetes, cancer, and other diseases.[1]. Albericio and co-workers proposed as green solvents 2-MeTHF and Gamma-Valerolactone (GVL), demonstrating good swelling capacity of the solid support (2chlorotrityl chloride and Wang resins), solubilization capability, and reactivity of amino acid building blocks and coupling reagents, even if demonstrated only on the small scale.[6−8] Another aspect investigated to improve the efficiency of solid-phase peptide synthesis (SPPS), from the instrument engineering perspective, is temperature control of the reaction. 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 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]

■ RESULTS AND DISCUSSION

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES