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Title: Effect of processing techniques and excipients on integrity and stability of protein formulations
Author: Ahmad, Rita Haj
Awarding Body: University of Sunderland
Current Institution: University of Sunderland
Date of Award: 2013
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Production of protein pharmaceutical formulations has been challenging due to the complexity of protein synthesis, purification and physical and chemical instabilities of the proteins. Accordingly, the aim of this research is to assess the effects of spray drying, freeze drying and crystallisation using different concentrations of four excipients (pluronic®F127, cremophor®EL, β-cyclodextrin and inulin) on protein integrity, thermal stability and biological activity. Lysozyme and Bovine serum albumin were chosen as model proteins. The oral administration of protein pharmaceuticals to the systemic circulation has numerous barriers, including, sharp pH gradients, photolytic enzymes and low epithelial permeability. Trying to overcome these barriers, proteins can be encapsulated in niosomes that are known to protect proteins against the surrounding environment. In this study, Pluronic® F-127 and cremophor® EL were used as co-surfactants for niosome preparations. Niosomes of Span™ 65: cholesterol: co-surfactant were prepared for encapsulation of proteins by film hydration method. Lysozyme and insulin were chosen as model proteins for noisome formulations. Unprocessed, spray-dried, freeze-dried and crystallised proteins were characterised by the following methods: thermal analysis using Differential Scanning Calorimetry (DSC), microscopic examination of the protein particles using Scanning Electron Microscopy and spectroscopic analysis employing Fourier Transform Infra-Red (FT-IR). Moisture contents were determined by Karl Fisher titration. Enzymatic assay were used to measure biological activity. The percentage yield of proteins and protein content were also determnined. Morphology and vesicular sizes of the prepared niosomes were investigated by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (ZetaPlus), respectively. The entrapment efficiency of protein in niosomes was determined by complete vesicle disruption using 50:50 isopropanol:buffer, followed by analysis of the resulting solutions by HPLC. Thermal behaviour of the niosomes was investigated using Differential Scanning Calometry (DSC). Protection of proteins against stimulated gastric fluid (SGF) stimulated intestinal fluid (SIF) were also assessed. Spray-dried and freeze-dried forms of proteins with pluronic® F127 better maintained the native conformation, as indicated by enzymatic assay, of lysozyme. The storage stability of unprocessed, spray-dried and freeze-dried lysozyme was studied over a period of 20 weeks at different temperatures. The results suggested that protein samples prepared with pluronic® F127 were more stable after storage. Crystallising lysozyme with (0.05%) of: pluronic®F-127, cremophor®EL and inulin significantly (P<0.05) maintained higher biological activity compared to a high concentration (0.2%) of the same excipients. However, using (0.2%) of ~cyclodextrin in crystallisation medium led to higher (P<0.05) protein activity compared to (0 .05%) β-cyclodextrin. The storage stability of unprocessed and crystallised protein samples, with and without excipients, showed that crystallisation of lysozyme with 0.05% (w/v) of pluronic• F-127 and 0.2% (w/v) of β-cyclodextrin better maintained the protein activity when compared to unprocessed and crystallised protein with and without excipients. The protein niosomes that prepared with different molar ratios of Span n.1 65, cholesterol and co-surfactants showed different encapsulation efficiency. For Span™ 65, cholesterol and cremophor® EL niosomes, they were not able to encapsulate any protein. Whilst, Span™ 65, cholesterol and pluron ic® F-127 formulations successfully produced insulin and lysozyme niosomes. For insulin containing niosomes, the ratio of 64.7 (Span™ 65): 32.3 (cholesterol): 3.0 (pluronic@ F-127) produced the highest protein encapsulation efficiency and the smallest vesicle size of O.74j..1m . For lysozyme containing niosomes, the maximum protein encapsulation was found in 72.75/24.25/3 .00% molar ratio of Span™ 651 cholesterol/pluronic®F-127 niosomes with vesicle size of 648.0IJm. The release study of proteins from the niosomal preparations in simulated gastric fluid (SGF) and simulated intestinal fiuid (SIF) revealed that insulin and lysozyme efflux from the niosomes is a biphasic process. The release of both proteins (insulin and lysozyme) from the niosomal formulations was found to be dependent on vesicle size, vesicle lamilarity, protein encapsulation, the position of the protein within the niosomal lamilarity and niosomal formulation component ratio . In conclusion , protein formulations using pluronic@F-127 increased protein stabilisation. Pluronic® F-127 is a good candidate to be used for protein formulations employing different processing techniques. The overall results show promise for protein drug delivery systems.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available