Effects of mixed surfactant system on chemical and physical stability of human growth hormone

Abstract

In protein formulation, addition of surfactants is commonly executed to enhance product stability and shelf-life. The reason is that surfactants can protect proteins against surface-induced aggregation. However, the interaction between surfactant and protein depends on the type of surfactant used. Anionic surfactants can unfold proteins and form complexes in equilibrium manner. The complex structure depends on the ratio of anionic surfactant-to-protein. On the other hand, nonionic surfactants can weaken interactions between anionic surfactants and proteins. Here, human growth hormone (hGH) is used. Sodium dodecyl sulfate (SDS) in the SDS-hGH complex was slowly extracted to form mixed micelles with n-dodecyl-β-D-maltopyranoside (DDM). This gentle process allows the protein to refold back to its native form with less aggregation. From previous studies, the DDM/SDS ratio is pivotal for the protein refolding procedure using a nonionic surfactant, regardless of mixing order. This study is aimed at assessing the stability of refolded SDS-unfolded hGH after incubating at 37°C for 42 days. Regarding hGH properties, deamidation seems to be dominant in chemical degradation. One of the formulations was designed for less deamidation, at pH 5.4, to eliminate other destabilizing effects compared to pH 7. In the physical stability aspect, the SDS-hGH formulation at pH 5.4 had the lowest stability of refolded hGH, as seen in the results from FlowCAM, DLS, and Probe Drum. However, the refolded hGH structure in this formulation was the most chemically stable as seen from HPLC analysis. Nuclear magnetic resonance is needed to assess the complex conformation in this formulation.

Popular Abstract

The protein-based treatment provides an advantage with great specificity. This kind of product has been increasing in the pharmaceutical market recently. In the development of protein formulations, the addition of surfactants is commonly executed to enhance product stability and shelf-life. The reason is that surfactants can protect proteins against surface-induced aggregation. However, the interaction between surfactant and protein depends on the type of surfactant used. Anionic surfactants, like sodium dodecyl sulfate (SDS), can unfold proteins and form as complexes in equilibrium. The complex structure depends on the ratio of anionic surfactant to protein. On the other hand, nonionic surfactants, like n-dodecyl-β-D-maltopyranoside (DDM), can weaken interactions between anionic surfactants and proteins. Here, human growth hormone (hGH) was used. SDS in the SDS-hGH complex was slowly extracted to form mixed micelles with DDM. This gentle process allows the protein to refold back into its native form. From previous studies, the DDM/SDS ratio was important for the protein refolding procedure using a nonionic surfactant, regardless of mixing order. This study was aimed at assessing the stability of refolded hGH after being unfolded at 37°C for 42 days. Regarding hGH properties, deamidation was dominant in chemical degradation. One of the formulations was designed for less deamidation, at pH 5.4, to minimize other destabilizing effects compared to pH 7. After removing SDS, particle size and particle number were determined. The conformation of hGH was tracked by measuring the fluorescence spectra. In the physical stability aspect, the hGH formulation with SDS at pH 5.4 had the lowest stability of refolded hGH as it developed the most particle number and had the largest particle size. Moreover, the fluorescence spectra were different from the initial point. The trend of particle number was contradictory to particle size in pH 7 formulations. The chemical stability was evaluated by separating the native form of hGH from degraded products, which were then detected with an ultraviolet detector. By comparing the proportion of the native form, the formulation at pH 5.4 had the highest percentage. However, this formulation developed white particles after adding DDM during the SDS removal step.
In conclusion, the unfolded hGH can retrieve its native structure by using this refolding procedure at pH 7, but not in pH 5.4. The pH of the formulation had an impact on its physical and chemical stability. The more acidic pH reduces the deamidation rate, as seen in the chemical stability result. In the future, the stability study is recommended to be conducted at 25°C for 7 days to gain the most useful and informative data. Lastly, the complex structure of hGH with SDS in pH 5.4 should be investigated to better understand how pH affects degradation.

• author: Viriyayudhakorn, Karnsinee
• supervisor: Marie Wahlgren; Johanna Hjalte; Ingrid Ramm
• organization: Food Technology and Nutrition (M.Sc.)
• course: KLGM05 20231
• year: 2023
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: human growth hormone, surfactant, refolding, protein stability, physical degradation, chemical degradation, pharmaceutical technology
• language: English
• id: 9124261
• date added to LUP: 2023-06-14 09:44:38
• date last changed: 2023-06-14 09:44:38