2015 Development of lipid based depot formulation s using interferon-beta-1b as a model protein

Authors:Christian Thomas Karl-Heinz Neuhofer

Journal: Dissertation


Faculty of Chemistry and Pharmacy, Ludwig-Maximilians-University Munich


Due to their specificity and potency, biologicals allow entering new therapeutic application areas. Therefore, therapeutic proteins are a very important part of the global pharmaceutical market. A common characteristic of proteins is their short half-life which makes frequent parenteral application necessary and consequently might decrease patient compliance. Drug loaded depot systems are able to address this challenge.

The goal of this project was to investigate the suitability of triglyceride based extrudates (SLIs) and vesicular phospholipid gels (VPGs) as a depot system for protein delivery for the very hydrophobic and labile model protein interferon-β-1b(IFN-β-1b). To improve the physicochemical properties of the protein, a side specific PEGylation technique was used. This gives also the opportunity to investigate the influence of PEGylation on the release. Furthermore, it gives the option to combine two principles of half-life extension: PEGylation and a depot system. Because of the combination of a hydrophobic depot system and a hydrophobic protein, protein-lipid interactions are likely to occur. The investigation of the influence of protein-lipid-interactions on protein stability and the influence of these interactions on the protein release was also part of this project.

IFN-β-1b was successfully PEGylated using a 40 kDa branched PEGaldehyde. PEGylation of IFN-β-1b does significantly improve the solubility and also the stability of the protein. However, the changed physicochemical properties result in a new molecule with completely different characteristics.

SLIs were successfully prepared by mixing the protein lyophilisate and the lipids using hot-melt extrusion. Therefore, a human serum albumin free lyophilisate was developed for IFN-β-1b and PEG-IFN-β-1b. For both, the native and the PEGylated protein species, a sustained release over seven days was achieved. While the addition of a surfactant was necessary to release native IFN-β-1b at all, a release under physiological conditions was possible for PEG-IFN-β-1b. Also the effect of protein-lipid interactions was reduced. Even though the preparation and short-term storage did not harm the protein stability, a drop of biological activity was observed during the later stage of the release. This loss of activity was the result of instabilities of both protein species in the release medium in the presence of lipids.

Due to the occurrence of strong protein-lipid interactions, a release of the native IFN-β-1b from VPGs was not possible. PEGylation of IFN-β-1b made the investigation of VPGs as a depot system possible. However, the release rate was very slow and incomplete. This was also due to protein-lipid interactions, which were reduced by PE Gylation but not completely avoided. Those interactions did not only affect the release, but did also change the mechanical properties of the VPGs by increasing their viscosity. During the release, a significant drop of the biological activity was observed even at the first day which indicates a degeneration of the protein during VPG preparation.

In order to get a better understanding regarding the observed protein-lipid interactions and to find a quick and material saving tool to get information of possible effects that might influence the release from VPGs, different tools and methods were investigated. Both IFN-β-1b species were compared to other more hydrophilic proteins.

Depending on the model, the effect of interaction between some proteins and phospholipids could even be seen macroscopically. However, other proteins needed more sensitive methods. Therefore, a quartz crystal microbalance or a Langmuir film balance and different models like liposomes or monolayers were used. Due to the complexity of the interactions no consistent correlation between the methods and no prediction regarding the influence on the release behavior was possible.