Speciality Chemicals Magazine JAN / FEB 2022 | Page 45

PEPTIDES & PROTEINS peptide industry . Foremost were the issues of racemisation during couplings and side-reaction suppression . ¹⁵ As optimised protocols and careful control of the temperature were developed to minimise these issues , application of the microwave method grew in popularity , especially in the CDMOs and R & D labs in pharmaceutical companies involved in lead development , once the HT attachments were added to the Liberty Blue models , which allowed continuous sequential synthesis of up to 12 peptides in a single run at a scale up to 2 mmol . More recently , as more sustainable or greener processes have become important , CEM has developed optimised methods which use lower quantities of reagents , consume less solvents and use more environmentally friendly solvents . ^{16 , 17} In particular , the Liberty Prime machine combines these features to reduce waste generation by nearly 80 % while still speeding up the synthesis . As microwave technology improved over the past decade , development of a larger-scale instrument which could produce GMP material for use in clinical trials and ultimately for commercial production has been realised . Paramount to this instrument was to design the appropriate reactor where optimal mixing is achieved while having a rapid equilibration of temperature without creating local hotspots from the microwave radiation . Additionally , software and system data recording is required to be 21 CFR Part 11-compliant and provide complete audit trail traceability , run history and method reports . The CEM Liberty Pro

Figure 1 - UPLC-MS chromatogram for Liraglutide ( crude ) synthesised at 25 mmol scale on the CEM Liberty Pro . MW = 3751.28

( pictured ) currently has three different reactor sizes ( 2L , 8L , and 15L ) to accommodate differing synthesis scales . By comparison with a conventional 75L reactor , the Liberty Pro fitted with the 15L completed an 800 mmol synthesis of a ten-residue peptide in 7.5 hours , which was five times faster than the conventional SPPS reactor . The Liberty Pro system has recently been added to AmbioPharm ’ s site in North Augusta , South Carolina . This adds a level of US-based synthesis to the AmbioPharm operation for providing cGMP grade peptides for clinical use . The site is equipped for all of the downstream processing activities , quality control and quality assurance to release cGMP peptides in an expedited manner .

Case Study : Liraglutide

Liraglutide ( Sequence : H-His-Ala-Glu- Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser- Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys ( γ-Glupalmitoyl ) -Glu-Phe-Ile-Ala-Trp-Leu-Val- Arg-Gly-Arg-Gly-OH ) was synthesised at a 25 mmol scale using the Liberty Pro microwave peptide synthesiser with optimised cycles . Synthesis was performed on Fmoc-Gly-ProTide resin at 0.24 mmol / g substitution . Fmoc deprotections were conducted at 80 ^o C for four minutes with 16 % piperidine in DMF . Couplings used CEM ’ s patented carbodiimide-oxyma activation chemistry at 80oC for five minutes using a fourfold excess of amino acids ( Fmoc-Lys ( palmitoyl- Glu-OtBu ) -OH was coupled using a twofold excess ). Coupling for histidine was performed at 40 ^o C to minimise racemisation . A total of 13 hours was required to complete the synthesis , which provided an 80 % yield based on resin weight gain . Solvent consumption was 118 L , which was a significant reduction compared to traditional SPPS processes at this scale . A crude purity of approximately 76 % was obtained from UPLC-MS analysis ( Figure 1 ). •

* - Sandeep Singh of CEM also contributed to this article

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