The effect of a water/organic solvent interface on the structural stability of lysozyme

Abstract

The effect of emulsification of lysozyme solutions with methylene chloride on protein recovery and structural integrity was investigated. Total lysozyme recovery in the aqueous phase was found to be concentration dependent, and ranged between 65 and 80%. The unrecovered lysozyme was observed at the interface as a white precipitate. No structural changes of the soluble lysozyme were observed by enzymatic activity assay, size-exclusion chromatography (SEC), gel electrophoresis (SDS–PAGE), and circular dichroism (CD). The lyophilized precipitated protein was analyzed by FTIR, and evidence of intermolecular β-sheet formation was found. In addition, the precipitate was analyzed after redissolution in 1 M guanidine hydrochloride by enzymatic activity assay, CD, SDS–PAGE, and SEC. No differences with control lysozyme samples or samples in aqueous buffer solutions were observed. This indicates that lysozyme precipitates as non-covalent aggregates upon emulsification, and these precipitates can refold into their native state in 1 M guanidine hydrochloride. Protein recovery could not be improved by the addition of sucrose, Tween 20, or Tween 80. Excipients competing for the water/organic solvent interface, such as BSA and partially hydrolyzed polyvinylalcohol (PVA) significantly improved lysozyme recovery to >95%. Emulsions which contained poly(lactic-co-glycolic acid) (PLGA) in the organic phase gave irreproducible protein recovery. Here also, partially hydrolyzed PVA significantly increased lysozyme recovery. Thus, we found that emulsification of lysozyme-containing aqueous solutions with methylene chloride causes incomplete protein recovery and non-covalent aggregation of lysozyme. These aggregates are also encapsulated in controlled drug delivery systems which are prepared using a water-in-oil emulsification procedure. The use of surface-active additives, such as partially hydrolyzed PVA significantly reduces lysozyme aggregation, and can be used to prevent encapsulation of inactive and potentially immunogenic protein species.