Microencapsulation of mPEG-Modified Lysozyme in PLGA by Spray Drying

Abstract

Protein-based drugs hold great promise as new therapeutic agents because of their high specificity. Unfortunately, they are limited by their short half-life in the organism to be treated. This drawback is caused by premature proteolytic degradation, rapid renal clearance, and instabilities of the protein itself. In this thesis, methods to improve the applicability of protein based drugs were investigated. PEGylation and microencapsulation are two commonly used methods which already proved great potential in drug development processes. The combination of these methods is applied in this thesis using lysozyme as a model protein and mPEG-pNp and PLGA as polymers. First, the PEGylation process was closely analyzed. The reaction showed a great dependence on the reaction time, the polymer-to-protein mass ratio, and the pH. PEGylated lysozyme acts like a molecule many times larger than its actual size. Hence, the rapid clearance from an organism will be prolonged. Mixtures containing PEGylated conjugates of more and higher degrees of modification show a decrease in their enzymatic activity. The purification of the modified protein from the unreacted lysozyme and the remaining polymer was performed by a SEC. A complete separation of the single modification degrees was not possible, but mixtures containing predominantly higher or lower degrees of PEGylation were produced. These mixtures show an improved stability and an improved resistance to proteolysis, which reduces instability and premature degradation. The microencapsulation in spherical particles with a rough surface was conducted using solvent evaporation and spray drying. Suitable particle sizes for injectable microspheres were produced by spray drying with an average yield of 40 %, but solvent evaporation achieved 20 % higher yields. The BCA assay does not appear to be an appropriate method for the determination of the protein release from PLGA microspheres. The PEGylation and the microencapsulation of lysozyme in PLGA were successfully conducted. Positive effects of these formulation methods on proteins could be determined during this thesis, but further studies, especially on the release kinetics of the encapsulated proteins, are necessary.