Investigation of the factors contributing to the physical stability of biological therapeutics

Biological therapeutics, primarily peptides and proteins, are becoming increasingly commonly used due to their specificity and efficacy. Unfortunately, they can suffer from aggregation, resulting in immunogenicity and poor bioavailability. The mechanisms behind such losses in physical stability, which is itself sensitive to a wide range of factors, can result in batches of the same therapeutic displaying different aggregation behaviour. Glucagon-like peptide 1 (GLP-1) is 31-residue peptide that, along with its analogues, is used in the treatment of diabetes and obesity. Four batches of GLP-1 produced by a single supplier using solid-state synthesis were found to exhibit batch-to-batch variation in their physical stability. These were characterised by a myriad of techniques to measure properties ranging from secondary structure and alterations in chemical composition, to the presence of different oligomeric states. This was to attempt to determine potential origins of the batch-to-batch variation observed. Pertinent factors that may influence physical stability were examined more extensively to aid in this goal. Cu²⁺, Fe³⁺ and Zn²⁺ ions were found to be able to influence the physical stability of GLP-1 at concentrations as low as 1 μM. The metal ions affected either the compactness of the monomeric or other oligomeric states, or increased the population of oligomers, some of which were known to be off-pathway and inhibit aggregation. However, it was determined that in the GLP-1 batches studied, the amount of these metal ions was insufficient to account for the differences in physical stability observed. Varying water content in different samples of lyophilised protein and peptide samples has also been proposed to affect physical stability and lead to batch-to-batch variation. Here, a non-destructive method of measuring water content in pharmaceutical preparations using time-domain NMR was developed in collaboration with AstraZeneca. It was then used to investigate whether water content can alter the physical stability of a therapeutic peptide, however, results were somewhat inconclusive. Lastly, a method of producing GLP-1 by recombinant means was adapted to allow for a comparison of the physical stability of peptide produced recombinantly with the GLP-1 batches produced by synthetic means.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Barber, Jack

Advisor dc:contributor.advisor

• Jackson, Sophie

• Aggregation
• Amyloid
• Batch-to-batch variation
• Characterisation
• GLP-1
• Peptide
• Protein
• Protein purification
• TD-NMR
• Trace metals

Licence

• https://www.repository.cam.ac.uk/bitstreams/4b689e18-97f7-4112-a82c-6a86ff89457e/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0003-2993-1397

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/375627