The Effect of Cucurbit[7]uril and Solution Conditions on Protein-Protein Interactions and Aggregation Propensity

  • Marcello Martinez Morales

Student thesis: Phd


Protein and peptide drugs have gained increasing importance in the portfolio of pharmaceutical companies reflected by the growing number of approved drug products in the US and Europe. One of the key challenges represents physical instability of the drug substance, in particular processes of self-association. Several excipients are available to modulate the underlying protein-protein interactions responsible for self-association processes. Here, Cucurbit[7]uril, a macrocycle able to bind hydrophobic amino acid residues is examined for its potential to improve physical stability of protein and peptide therapeutics. Studies presented here, compare solution behaviour of proteins and peptides with mutated Cucurbit[7]uril binding sites examining whether addition of Cucurbit[7]uril is capable of mitigating or fully suppressing self-association processes. One study determines the effect of Cucurbit[7]uril on the aggregation propensity of a monoclonal antibody. Results provide evidence of specific interaction between Cucurbit[7]uril and aromatic amino acid residues located on an aggregation-prone region resulting in improved solution behaviour. Another study, determines the effect of Cucurbit[7]uril on the kinetic profile and morphology of Enfuvirtide fibrils, a commercially available peptide drug. Modulation of fibrillation onset via Cucurbit[7]uril is monitored by Thioflavin T assays and circular dichroism spectroscopy. In contrast to previous reports, findings presented here, indicate Cucurbit[7]uril’s potential to delay rather than fully suppress fibrillation onset through binding of a C-terminal aromatic amino acid residue. Furthermore, this work contains a study evaluating a high-throughput PEG solubility assay for its correlation with the diffusion interaction parameter. Conventional methods used in solubility assessment of proteins have high material consumption and are not amenable to high-throughput screening. A set of 8 mAbs formulated at 15 different solution conditions is used to investigate correlation with observations made from DLS measurements. Results highlight correlation of trends observed in PEG-induced phase separation with averaged protein-proteins interactions as described by the diffusion interaction parameter.
Date of Award1 Aug 2021
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorJeremy Derrick (Supervisor), James Warwicker (Supervisor), Robin Curtis (Supervisor) & Alain Pluen (Supervisor)


  • protein solubility
  • protein aggregation
  • formulation development
  • mAbs
  • macrocycles
  • biopharmaceuticals
  • excipients

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