Jeluk, Aimée
(2025)
Impacts of Temperature-Induced Change of Formulation Buffers on the Stability of Proteins in Solution.
PhD thesis, University of Nottingham.
Abstract
Therapeutic proteins are most commonly formulated as liquid dosage forms. Buffers are fundamental components of liquid protein formulations incorporated with the purpose to maintain protein stability on changes, particularly in the formulation pH, arising from processing or storage. However, the properties of a buffering system, its pH, ionic strength and composition, are dependent on environment temperature and pressure. Consequently, the buffer per se can be the source of pH variations in liquid protein formulations exposed to temperature changes (temperature excursions) and in that way affect stability of proteins in solution. The aim of this thesis was to assess the effect of temperature-induced pH changes in different buffers on the conformational and colloidal stability of proteins in solution. It further considered if buffers can be designed such to increase stability of proteins in solution on potential changes in temperature.
The initial studies demonstrated temperature dependent pH changes of typically used buffer systems (Tris-HCl, Na-ACES, Na-HEPES, Na-Phosphate, Histidine-HCl, Na-MES and Na-Citrate), as their ∆pKa/°C values differ. Based on that, the buffers were then designed to have a desired pH and ionic strength at specific temperatures in 5 - 80 °C range and effects of designed buffers on the protein stability was compared. Bovine Serum Albumin (BSA) and Immunoglobulin G (IgG) were used as representative examples of proteins commonly used in pharmaceutical formulations.
The results obtained from a range of experimental analyses demonstrate that temperature-induced pH shift of a buffer, dependent on its dissociation constant (∆pKa/°C), influences both the conformational and colloidal stability of the proteins in their solutions on changes in solution temperature. Solution stability of proteins was found to correlate to the direction of the buffer pH shift in relation to the protein isoelectric point (pI); the thermal (conformational) stability increased as the buffer pH shifts towards the pI upon the temperature change, while conversely, the stability towards thermal aggregation (colloidal stability) decreased when the buffer pH shifts closer to the pI due to the temperature change.
Importantly, protein solutions with specifically designed, optimised buffering system for certain temperature showed increased stability relative to protein solutions where buffers are not specifically designed. The latter can be attributed to the protein destabilization due to not only temperature-induced stress but, in addition, by the temperature-induced shift in pH of the buffering system.
The observed significance of the buffer design for certain temperature is particularly relevant in situations where the dissociation constant (pKa) of the buffer is temperature dependent (∆pKa/°C). Consequently, the results in this thesis demonstrate that the stability of a protein in solution should be considered in the context of how the buffer system used in the formulation changes with temperature. This is particularly important in protein stress tests, accelerated stability studies, protein processing and storage, cold chain interruptions, and different temperature excursions.
The buffer design approach could be further applied in buffer screening studies to assess the influence of the buffer species on protein stability independently of the buffer ∆pKa/°C. If stability tests are repeated for a series of buffers with different ∆pKa/°C, the effect of buffer species (nature of ions) on the protein stability can be decoupled from the effects produced by changes in pH of the buffer due to different temperatures.
The work in this thesis suggests that the intentional design of buffer systems, to have a desired pH and ionic strength at a specific temperature, can be used to enhance the stability of liquid protein formulations towards temperature changes. Furthermore, it suggests that the design of buffers could find application in buffer screening studies and accelerated stability studies to assess the influence of the buffer species on protein stability, independent of the buffers’ temperature-induced pH changes.
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