Characterizing protein-excipient interactions for improved biopharmaceutical formulation

  • Sophia Marzouk

Student thesis: Phd


Formulation studies are performed when a new pharmaceutically active ingredient (API) of interest, such as a protein, is under development in order to find conditions for maintaining the chemical and physical stability of the drug to ensure efficacy and safety for the patient (i.e. prevent any adverse effects). A commonly used approach for controlling physical stability involves the use of excipients also known as co-solvents, which are small molecules added in solution to stabilise the protein against various stresses and in particular minimize any potential aggregation events (Mahler et al., 2009). Excipients are carefully chosen to not interfere with the activity of the protein, to maintain protein chemical stability, and to assure that they are safe to administer to patients (Canchi & García, 2013). Protein-excipient interactions are especially weak and transient and controlled by multiple mechanisms with the different chemical groups exposed on protein surfaces. There is currently a lack of systematic studies which are needed to disentangle the different types of excipient interactions. It is crucial to understand these interactions to improve biopharmaceutical formulations in the future. The protein-excipient interactions determine how changing excipient concentration alters protein conformational and colloidal stability, both of which are key indicators of critical formulation attributes, such as the aggregation propensity and the concentrated solution rheological and phase separation behaviour. Here, a novel size-exclusion chromatography coupled with a multi-angle light scattering detection (SEC-MALS) technique is developed to measure preferential interaction parameters, which quantify the accumulation or exclusion of a co-solvent around the surface of a protein relative to the bulk solution. The method relies on using static light scattering as an indirect approach for measuring the refractive index increments of proteins at constant solvent and co-solvent chemical potential from which the preferential interaction parameters can be derived. In order to benchmark the method, it has been applied to five proteins (BSA, ovalbumin, lysozyme, chymotrypsinogen A and ribonuclease A) in solutions containing either sodium chloride or arginine chloride at two pH conditions. Both salts are found to be preferentially excluded from the surface of proteins, with a significantly greater exclusion for sodium chloride compared to arginine chloride. We then show the method can be applied to obtain preferential interaction parameters of denaturants with the folded and the unfolded state of proteins under the same solution conditions, which has never been done before. Interestingly, except for ribonuclease A, the folded state of the protein interacts more favourably with urea than guanidine hydrochloride. On the other hand, the opposite trend was observed for the unfolded state. The preferential accumulation of guanidine hydrochloride around proteins was reported to be significantly greater than in the corresponding concentration of urea. Such results indicate that urea and guanidine hydrochloride denature proteins through a different mechanism of action. The developed SEC-MALS constitutes a promising tool for probing protein-excipient interactions in formulations. It has been shown to provide a more rapid, consistent and accurate quantification of preferential interaction parameters than previously established methods (densimetric and vapor pressure osmometry measurements). Because SEC-MALS is already common to many research laboratories and formulation development groups, we expect the approach to be straightforward to implement. In the last part of the dissertation, the focus is on understanding how changing the solvent from regular to deuterated water alters the protein colloidal stability characterized in terms of protein-protein interaction measurements. The isotropic solvent substitution effect was investigated on the NIST (Nation
Date of Award1 Aug 2023
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorPaul Popelier (Supervisor), James Warwicker (Supervisor) & Robin Curtis (Supervisor)


  • co-solvent
  • SLS
  • DLS
  • Light scattering
  • solvent isotope effect
  • preferential interaction parameter
  • protein
  • biopharmaceutical formulation
  • protein-excipient interactions
  • protein-excipient
  • protein-protein interactions
  • excipient
  • formulation
  • denaturant

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