Stabilization of proteins against methylene chloride/water interface-induced denaturation and aggregation.
The purpose of this study was to stabilize proteins during the commonly used microencapsulation process of methylene chloride/water emulsification. The model proteins used in this study included bovine serum albumin (BSA), S-carboxymethylated BSA (CM-BSA), reduced-S-carboxymethylated BSA (RCM-BSA), ovalbumin, and lysozyme. Emulsification of a 0.5-mg/ml protein solution in methylene chloride brought about changes in the composition of water-soluble species and interfacial coagulation. As a result, 37.8, 71.8 and 98.7% of ovalbumin, lysozyme and BSA were recovered from the aqueous phase after emulsification, respectively. Experiments with BSA, CM-BSA, and RCM-BSA demonstrated that a free thiol group and/or disulfide bond participated in interfacially induced dimerization and polymerization of proteins. Interfacial reactions that led to the aggregation of ovalbumin and lysozyme were inhibited by adding hydroxypropyl-beta-cyclodextrin or BSA into their aqueous solutions. Moreover, such beneficial effect of the excipients was observed to be concentration dependent. Under our experimental conditions, the recovery of ovalbumin and lysozyme was improved up to 97.7 and 95.6%, respectively. This study substantiated that an adequate formulation could overcome denaturing effects of the methylene chloride/water interface upon a protein of interest to be encapsulated into microspheres.
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Room 214, 26 S. Dunlap Street, Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee, Memphis, TN 38163, USA. hsah@utmem1.utmem.edu
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004655:Emulsions; D008752:Methylene Chloride / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry; D010047:Ovalbumin / Q000737:chemistry; D011489:Protein Denaturation / Q000187:drug effects*; D011506:Proteins / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012995:Solubility; D014867:Water / Q000737:chemistry*
D004655:Emulsions; D011506:Proteins; D014867:Water; D012710:Serum Albumin, Bovine; D008752:Methylene Chloride; D010047:Ovalbumin; D009113:Muramidase