Liquid phase peptide synthesis via nanostar sieving
File(s)
Author(s)
Yeo, Jet Ing Ngie
Type
Thesis
Abstract
The global demand on peptide drugs is increasing and this has created an imperative for continuing innovation in peptide synthesis method in anticipation of future growing demand. This thesis presents a membrane-based liquid phase peptide synthesis method – Liquid Phase Peptide Synthesis via One-pot Nanostar Sieving (PEPSTAR). PEPSTAR features a one-pot iterative addition of amino acids (AA) to a ‘nanostar’ support, with organic solvent nanofiltration (OSN) for the separation of growing peptide-nanostar from reaction debris after each synthesis cycle. A synthesis cycle consists of coupling of amino acids to the chain termini of the nanostar, removing Fmoc protecting group, then ‘sieving’ out of the reaction debris through a robust OSN membrane. The cycles are repeated in a single reactor-separator, or synthesiser, without any phase or material transfers in between cycles until the target peptide length is reached. A three-arm, star-shaped, and monodisperse nanostar was chosen to facilitate efficient nanofiltration through a three-fold increase of amino acid every cycle and allow precise reaction monitoring to be undertaken with liquid chromatography and mass spectrometry. A one-pot approach synthesis strategy was devised to enable sequential coupling and Fmoc removal to be carried out safely without a separation step in between by quenching the excess amino acids and removing Fmoc simultaneously. A polybenzimidazole-based (PBI) polymer membrane grafted with Jeffamine M2005 polymer brush with high selectivity and permeance was selected for nanofiltration. Different types of peptides, including enkephalin-like model peptides (5 and 10 AA), linear octreotate amide, linear octreotate acid, and fully protected glucagon fragment, were successfully synthesised using PEPSTAR platform. The crude purities were comparable to vendor produced peptides on solid phase, with the purity of 10 AA enkephalin-like model peptide was 84% compared to solid phase’s 35% when similar equivalents of amino acids were used. PEPSTAR method reduced the process mass intensity (PMI) three-fold than that of its predecessor, membrane-enhanced peptide synthesis (MEPS), and is close to that of solid phase peptide synthesis (SPPS). The cost of materials was estimated to be 41% lower than that of SPPS. This thesis also demonstrates the adoption of Fourier transforms infrared (FTIR) absorption spectroscopy, equipped with state-of-the-art signal processing software, to monitor the PEPSTAR process in-situ over real-time. The FTIR results agreed well with offline lab-based analysis.
Version
Open Access
Date Issued
2021-01
Date Awarded
2021-04
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Livingston, Andrew
Sponsor
Eli Lilly and Company
Engineering and Physical Sciences Research Council
Grant Number
Lilly Research Award Programme Grant
EPSRC Programme Grant M01486/X
Publisher Department
Chemical Engineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)