King, Toby
(2025)
Improving therapeutic protein formulation stability by
optimising excipient properties.
PhD thesis, University of Nottingham.
Abstract
Excipients are included within protein biotherapeutic solution formulations to improve colloidal and conformational stability but are generally not designed for the specific purpose of preventing aggregation and improving cryoprotection in solution. In this work, we have explored the relationship between the structure and antiaggregation activity of excipients by utilising coarse-grained molecular dynamics modelling of protein–excipient interaction, and applied the resulting information in the design and synthesis of novel excipients. Using human serum albumin (HSA) and insulin as model proteins, the interaction of 41 excipients (polysorbates, fatty alcohol ethoxylates, fatty acid ethoxylates, phospholipids, glucosides, amino acids, and others) is reported in terms of the reduction of solvent accessible surface area (SASA) of aggregation-prone regions (APRs), proposed as a mechanism of aggregation prevention. Polyoxyethylene sorbitan had the greatest degree of interaction with aggregation-prone regions, decreasing the solvent accessible surface area of APRs by 20.7 nm2 (40.1%). Multiple molecules of insulin were also modelled via the same approach, facilitating the additional exploration of the relationship between APR interaction and aggregation reduction. In insulin, a greater reduction in SASA of APRs was additionally observed in sytems with glucosides, ceramides and phosphatidylinositols. Physicochemical descriptors generated by Mordred are employed to probe the relationship between chemical properties and interaction with APRs using partial least-squares regression. A leave-oneout cross-validated model to predict excipient interaction with HSA had a root-mean-square error of prediction of 4.1 nm2 and a mean relative error of prediction of 0.077. In both HSA and insulin, longer molecules with a large number of alcohol-terminated PEG units tended to interact more, with qualitatively different protein interactions, wrapping around the protein. Shorter or less ethoxylated compounds tend to form hemimicellar clusters at the protein surface. Using these data to form hypotheses on the improved design of excipients, compounds with many short chains of five to ten PEG units in many distinct branches and at least some hydrophobic content in the form of medium-length or greater aliphatic chains (i.e., six or more carbon atoms) were proposed. As a result, the synthesis of a library of novel excipients, consisting of a tyrosine central scaffold with multiple short ethylene glycol trimers and decyl moieties, is reported. The combination of molecular dynamics simulation and quantitative modelling is an important first step in an all-purpose protein-independent model for the computer-aided design and synthesis of stabilising excipients, and evidence is presented in support of the APR-shielding hypothesis of aggregation prevention.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Thomas, Neil R.
Laughton, Charles A.
Hirst, Jonathan D
Humphrey, James R. |
| Keywords: |
molecular dynamics, tyrosine, surfactant, excipient, protein stability, structure-activity relationship, protein aggregation, aggregation prone region, aggrescan, protein–excipient interaction |
| Subjects: |
R Medicine > RS Pharmacy and materia medica |
| Faculties/Schools: |
UK Campuses > Faculty of Science > School of Pharmacy |
| Item ID: |
81204 |
| Depositing User: |
King, Toby
|
| Date Deposited: |
30 Jul 2025 04:40 |
| Last Modified: |
30 Jul 2025 04:40 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/81204 |
Actions (Archive Staff Only)
 |
Edit View |
Tools
Tools
![[thumbnail of Thesis final version with corrections incorporated]](https://eprints.nottingham.ac.uk/style/images/fileicons/application_pdf.png "Thesis final version with corrections incorporated")