Repository logo
 

Investigating the Structure and Physical Stability of Glp1-Like Peptides Using Mass Spectrometry


Type

Thesis

Change log

Authors

Gibson, Katherine 

Abstract

Working with fibrillation prone peptides is an adventure, to say the least, but harnessing their therapeutic potential is a challenge worth facing. On comparison to small molecules, peptides provide improved efficacy coupled with reduced toxicity; the difficulty lies in their pharmaceutical developability, and ensuring their fidelity throughout a defined shelf-life. In addition to chemical degradation, physical aggregation of peptides into oligomers and fibrils can compromise the safety, efficacy and bioavailability of peptide therapeutics. It is imperative that we understand these processes, not only to mitigate for them, but so we can develop a set of measurable criteria which we use to confidently predict fibrillation propensity.

The research conducted here focuses on Glucagon-like Peptide 1 (GLP-1-like) scaffolds. Translation of GLP-1 itself as a therapeutic was burdened with poor bioavailability and metabolic instability. Thus, literature precedent describes the search for alternatives. These peptide agonists are particularly attractive as treatments for Type II Diabetes Mellitus (TIIDM) as they offer glucose dependent signalling with subsequent insulin production and glucose regulation. The majority of GLP-1-like peptides are currently still dosed via liquid injection but explaining the origin of batch-to-batch variation in physical stability of these drug substances is difficult and not yet fully understood

In this work, two model peptides were studied as each showed batch-to-batch variation in their physical stability. The first system looked at two batches of a lipidated GLP-1-like peptide (RS19 and RS57), that had shown batch dependent fibrillation propensity throughout early stage development despite being manufactured with no process changes. The second system used four batches of C-terminally amidated GLP-1(7-37) analogue (GLP-1-Am) that had been synthesised in-house where each batch was a result of a different purification pathway. Each of these batches fibrillated differently to each other, as well as to a commercial batch of comparable purity.

Mass spectrometry (MS) is an increasingly important tool in biopharmaceutical characterisation, with great potential for understanding the conformational bias of inherently dynamic systems like fibrillating peptides. Two complementary structure based MS techniques are travelling wave ion mobility MS (TWIMS) and hydrogen-deuterium exchange MS (HDX-MS), and the two peptide systems in this work provided different case studies with which structural mass spectrometry could be used to investigate the origins of batch variation in physical stability. TWIMS was used to investigate the five GLP-1-Am batches, and the two batches of lipidated GLP-1-like peptide to see if process related changes manifested as differences in conformation and oligomer distribution. Experiments were completed on samples that were freshly prepared or stressed to see if there was a correlation to physical stability. HDX-MS was used to investigate GLP-1-Am batches only. In both systems, differences were observed between batches however issues with reproducibility meant that determining any unambiguous links with fibrillation propensity was not possible.

Two current state-of-the art instruments, cyclic ion mobility spectrometry (cIMS) and millisecond HDX-MS were also used in this work to improve the resolution with which we study therapeutically relevant peptides. cIMS was successfully applied to the lipidated GLP-1-like peptide system to detect and identify the location of isoAsp residues, based on conformational differences when Asp or isoAsp were present. As an example of a dynamic system where fast amide proton exchange was expected, millisecond HDX-MS was used in this work, and showed differences in the deuteration rate of the five GLP-1-Am batches for experiments acquired in the same dataset. However, the reproducibility of results between days remained a significant challenge. Despite this, the analysis presented here highlights the potential of developing these techniques.

Description

Date

2023-06-15

Advisors

Jackson, Sophie

Keywords

Mass Spectrometry, Peptide, Hydrogen-deuterium exchange, Ion mobility

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Sponsorship
EPSRC (2108344)
EPSRC AstraZeneca