Effects of Formulation and Manufacturing Conditions on Protein Structure and Physical Stability

Nathan Wilson, Purdue University

Abstract

With expanding interest in the use of novel processing methods for biologics, it remains critical to develop formulations capable of stabilizing the conformational state of proteins and ensuring long-term physical stability. Under manufacturing conditions, proteins are exposed to a variety of stresses that can be detrimental to the physical stability of their native structure. In Chapter 1, a review of the effects of physical stresses induced by manufacturing methods will be discussed, with emphasis on their effect on initiating denaturation and aggregation. The common physical stresses discussed will include temperature, surface-induced stresses, pH effects, freezing, dehydration, and pressure. Specific examples of degradation under these stresses will be mentioned, with formulation approaches that can be used to protect against these factors. Studies in Chapter 2 examined the effects of formulation and manufacturing methods (lyophilization and spray drying) on protein structure and physical stability. Powders containing one of four model proteins (myoglobin, bovine serum albumin, lysozyme, β-lactoglobulin) were formulated with either sucrose, trehalose, or mannitol and dried using lyophilization or spray drying. The powders were characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), solid-state fluorescence spectroscopy, differential scanning calorimetry (DSC) and solid-state hydrogen/ deuterium exchange mass spectrometry (ssHDX-MS). SsFTIR and fluorescence spectroscopy identified minor structural differences among powders with different excipients and drying methods for some proteins. Using ssHDX-MS, differences were observed among protein formulations containing sucrose or trehalose and mannitol, and/or with varying processing conditions, including proteins like β-lactoglobulin, for which standard characterization techniques showed no differences. Proteins processed by spray drying typically showed greater heterogeneity by ssHDX-MS than those lyophilized; these differences were not detected by ssFTIR or solid-state fluorescence spectroscopy. The ssHDX-MS metrics were better correlated with protein physical instability measured by size-exclusion chromatography in 90- day stability studies (40°C, 33% RH) than with the results of DSC, ssFTIR, or fluorescence spectroscopy. Thus, ssHDX-MS detected subtle changes in conformation and/or matrix interactions for these proteins that were correlated with storage stability, suggesting that the method can be used to design robust solid-state protein drug products and processing methods more rapidly. From this work, it was established that population heterogeneity in spray-dried formulations was higher than those that were lyophilized, potentially due to the impact of the air-liquid interface. In order to investigate how excipients can influence the composition of protein at the surface and population heterogeneity, Chapter 3 and 4 focus on spray drying of proteins formulated with sugar-containing excipients and on the effects of surfactant inclusion, respectively. For examining the impact of saccharide-containing formulations, spray-dried formulations of myoglobin or BSA were prepared without excipient or with sucrose, trehlaose, or dextrans. Samples were characterized by ssFTIR, DSC, size exclusion chromatography (SEC), and scanning electron microscopy (SEM). Protein surface coverage was determined by X-ray photoelectron spectroscopy (XPS), while population differences were determined by ssHDX-MS. From these techniques, structural differences were exhibited with the inclusion of different excipients, with dextran formulations indicating perturbation of secondary structure. XPS indicated sucrose and trehalose reduced protein surface concentration better than dextrancontaining formulations.

Degree

Ph.D.

Advisors

Topp, Purdue University.

Subject Area

Energy|Analytical chemistry|Chemistry|High Temperature Physics|Industrial engineering|Pharmaceutical sciences|Physics|Thermodynamics

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