Developing novel low concentration excipients to suppress protein aggregation

  • Nikita Vekaria

Student thesis: Phd


When developing a new monoclonal antibody, the formulation is key in improving the stability of the concentrated therapeutic. The formulation design ensures the therapeutic protein is conformationally and colloidally stable and maintains a long shelf life. Excipients are added to improve the stability of proteins and are usually added at high concentrations to exert excipient effects. The aim of this thesis is to explore the effects of low concentration additives to improve conformational and colloidal stability of model proteins. Several parameters were used to measure conformational and colloidal stability. The melting temperature (T_m), midpoint of denaturation (C_m) and change in free energy between the native and unfolded state (DeltaG) measurements were conducted to measure conformational stability. Measuring colloidal stability involved static light scattering (SLS) measurements to obtain second virial coefficient (B_{22}) measurements and onset temperature of aggregation (T_{agg}) from SLS and dynamic light scattering (DLS) thermal ramps. DLVO model was applied to obtain the Fuchs stability ratio, which describes the probability of two aggregates sticking together following collision, giving another parameter to assess colloidal stability of different solution conditions. Chapter 3 highlights the effects of low concentration of tripolyphsophate (TPP) and adenosine 5'-triphosphate (ATP) on aggregation propensity. The binding of 5 mM TPP and ATP to an acidic protein, recombinant human serum albumin (rHSA), increased repulsive protein-protein interactions (PPI). This in turn significantly reduced aggregation propensity, which we show was due to an increase in the repulsive barrier upon the approach of two rHSA molecules, as quantified by the Fuchs stability ratio. Chapter 4 explores the effects of low concentrations of arginine and glutamate containing dipeptides on the aggregation behaviour of three model proteins. The effects exhibited by the dipeptides could be explained by electrostatics, the addition of the dipeptides to rHSA and ovalbumin, the most repulsive proteins, increased aggregation propensity. On the other hand, addition of the dipeptides to alpha-chymotrypsinogen, which exhibited attractive PPI, improved colloidal stability. The final chapter uses chemical denaturants to observe excipient effect on conformational and colloidal stability of partially and fully unfolded protein. Colloidal stability of unfolded protein is also defined by the size of aggregates formed following dilution refolding. These experiments provide a new method to predict aggregation propensity. Low concentration excipients are developed in this thesis to provide a novel way to reduce protein aggregation. Excipients are also used to determine effects on colloidal stability of partially unfolded protein.
Date of Award31 Dec 2021
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorJames Warwicker (Supervisor) & Robin Curtis (Supervisor)


  • Arginine
  • ATP
  • Protein aggregation
  • Excipients

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