Determining the gastrointestinal stability of peptide therapeutics via chromatographic methods
File(s)
Author(s)
Bird, Maxim
Type
Thesis
Abstract
Peptide therapeutics normally require parenteral administration, which can cause fear, pain, potential tissue damage and an increased risk of infection. Therefore, extensive research has been conducted into developing strategies which overcome the barriers faced in the oral delivery of peptide therapeutics. These barriers include the susceptibility of peptide structures to denature or aggregate in the gastrointestinal tract as well as their proteolytic degradation via the multiple peptidases present. Traditional cell and animal screening tests are slow and expensive, driving the needs for alternative screening approaches to be evaluated.
Self-interaction chromatography (SIC) represents an automated, high-throughput alternative technique of determining peptide propensity to aggregate through the chromatographic determination of the osmotic second virial coefficient. This thesis describes how SIC has been adapted for studying the stability and aggregation potential of peptide therapeutics under human intestinal conditions, and this data was compared to conventional dynamic light scattering and size exclusion chromatography measurements. The results of investigations with semaglutide and liraglutide showed that the second virial coefficient measurements were able to recognise transitions in glucagon-like-peptide-1 (GLP-1) therapeutics physical behaviour, but required prior understanding of the peptide oligomeric state behaviour in solution. When applied to insulin, it was found that the technique required more adaptations, as insulin is a peptide which readily formed stable oligomers.
This thesis also outlines the development and implementation of a covalently immobilised pepsin reactor. A study using peptide hydrophobicity standards found that these immobilised enzymes maintained their selectivity relative to solution reaction-based assays. However, a greater than 50-fold decrease in activity and 5-fold reduction in substrate affinity was observed, which was compensated for by a 500-fold increase in concentration. Additionally, the immobilised pepsin showed good stability over a 12-week period with an average reduction in activity and substrate-affinity of 5.9% and 4.8%, respectively, between weeks 2 and 8.
Self-interaction chromatography (SIC) represents an automated, high-throughput alternative technique of determining peptide propensity to aggregate through the chromatographic determination of the osmotic second virial coefficient. This thesis describes how SIC has been adapted for studying the stability and aggregation potential of peptide therapeutics under human intestinal conditions, and this data was compared to conventional dynamic light scattering and size exclusion chromatography measurements. The results of investigations with semaglutide and liraglutide showed that the second virial coefficient measurements were able to recognise transitions in glucagon-like-peptide-1 (GLP-1) therapeutics physical behaviour, but required prior understanding of the peptide oligomeric state behaviour in solution. When applied to insulin, it was found that the technique required more adaptations, as insulin is a peptide which readily formed stable oligomers.
This thesis also outlines the development and implementation of a covalently immobilised pepsin reactor. A study using peptide hydrophobicity standards found that these immobilised enzymes maintained their selectivity relative to solution reaction-based assays. However, a greater than 50-fold decrease in activity and 5-fold reduction in substrate affinity was observed, which was compensated for by a 500-fold increase in concentration. Additionally, the immobilised pepsin showed good stability over a 12-week period with an average reduction in activity and substrate-affinity of 5.9% and 4.8%, respectively, between weeks 2 and 8.
Version
Open Access
Date Issued
2025-10-15
Date Awarded
2026-03-01
Copyright Statement
Attribution-NonCommercial-ShareAlike 4.0 International Licence (CC BY NC-SA)
Advisor
Williams, Daryl
Sponsor
Engineering and Physical Sciences Research Council
Eli Lilly and Company (Firm)
Grant Number
EP/T005556/1
EP/T518207/1
Publisher Department
Department of Chemical Engineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)