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.

Popular Abstract

Proteins and peptides are biomolecules that have a wide range of functions in the human body. As such, they are widely used in the treatment and management of various illnesses such as cancer, diabetes, and cardiovascular diseases. This increased the demand for protein-based products such as vaccines, enzymes, and many other drugs. Nowadays, concerted efforts lead to many protein-based drugs in the market and a lot of which are undergoing clinical trials for approval. During the production of the drug, the protein may be affected by various factors such as temperature, humidity, pharmaceutical excipients, and many other stressors. These excipients are the other substances aside from the protein present in a formulation and they typically comprise the majority of the final product. Their function is to ensure that the protein retains its potency and stability until its administration. The loss of stability of a protein is sometimes coupled with the formation of degradation products. This may result in a decrease in the efficacy and shelf-life of the drug and increases the risk to consumers, as they may be toxic.

In this study, various substances were tested for their effects on the stability of two therapeutic proteins, GA-Z, and somatropin. Stability was measured in terms the of chemical degradation of the proteins. Chemical degradation is characterized by a change in the chemical properties of the protein, which leads to changes in its structure and function. For GA-Z, substances such as sucrose, polysorbate 80, and polyethylene glycol 600 in varying concentrations were tested to see if they have stabilizing properties. On the other hand, glycerol was tested for its effect on the chemical stability of somatropin. In the stability studies, both proteins were stored in an incubator at 37°C, with and without excipients, for up to 30 days. After the incubation period, a technique called liquid chromatography was used to determine the amount of the chemically degraded protein. The results showed that sucrose and polysorbate 80 protected GA-Z from chemical breakdown. However, this was not observed in the case of polyethylene glycol.

Unlike GA-Z, a method had to be first created for somatropin prior to testing. In this process, equipment settings were tested out to see how they improved the separation and detection of various components present in the somatropin samples. The use of this developed liquid chromatography method showed that glycerol helped protect somatropin from chemical destruction. The identities of the degradation products from both proteins were then determined using another technique.

Based on the results, the protective abilities of these substances should be further examined. The use of other advanced methods will also provide information on how these substances affect the structure of the protein. The results may then be used as new formulation strategies for the increased stability of protein-based drugs.

• author: Gulangayan Deniola, Emmanuel
• supervisor: Ingrid Ramm; Lars Nilsson
• organization: Food Technology and Nutrition (M.Sc.)
• course: KLGM05 20221
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Chemical stability, protein degradation, GA-Z, somatropin, pharmaceutical excipients, liquid chromatography, pharmaceutical technology
• language: English
• id: 9091794
• date added to LUP: 2022-06-23 09:35:39
• date last changed: 2022-06-23 09:35:39