Effects of pharmaceutical excipients on the chemical stability of therapeutic proteins in a parenteral formulation
Abstract: Proteins and peptides are widely used for the prevention, management, and cure of various illnesses. This vast therapeutic potential represents a significant portion of the pharmaceutical industry in the form of vaccines, antibodies, enzymes, and other protein-based therapeutics. During the drug development process, these proteins/peptides are affected by various environmental factors such as temperature and humidity, as well as the presence of pharmaceutical excipients. These affect the physical and chemical stability of the proteins, which may compromise the safety, efficacy, and shelf-life of the product. With this, stability testing is performed to provide data on how such factors affect the degradation of proteins. In this study, various pharmaceutical excipients were tested for their effect on the chemical stability of two therapeutic proteins, GA-Z, and somatropin over a 30-day incubation period at 37°C. The effects of sucrose (40-120 mg/mL), polysorbate 80 (0.05-1 mg/mL), and polyethylene glycol 600 (20-40%) on the chemical stability of GA-Z were tested. Moreover, glycerol concentrations from 10% to 50% were tested for their effect on somatropin. The results of HPLC-UV analysis of both protein samples provide information on how much native protein remained throughout the incubation period. Sucrose and polysorbate 80 demonstrated an increase in GA-Z stability as their concentrations were increased. Unlike GA-Z, there was no existing LC-UV method for the analysis of somatropin and its degradation products. With this, method development and optimization were performed using a BioResolve reversed-phase column. This process focused on optimizing the flow rate, gradient profile, injection volume, and column oven temperature to improve chromatographic resolution and analyte sensitivity. With the use of this developed method, it was shown that increased glycerol concentrations improved the stability of the somatropin. In conjunction with the LC-UV analysis, further experiments show that the decrease in the degradation of GA-Z is not linked with decreased water activity. However, this decreased chemical degradation in somatropin may be coupled with water activity. Mass spectrometric analyses provided confirmation that the degradation peaks are results of hydrolysis, deamidation, and isomerization. Future work with Asymmetrical Flow Field-Flow Fractionation and Small Angle Neutron Scattering is necessary to provide information on how the excipients affect the tertiary and quaternary structure of the proteins and how these excipients change the protein conformation.
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