Protein delivery from polymeric matrices:From pre-formulation stabilization studies to site-specific delivery

In haar onderzoek heeft Naomi Teekamp de stabiliteit en toediening van therapeutische eiwitten bestudeerd vanuit polymeer-gebaseerde formuleringen voor verlengde afgifte. Eiwitten kunnen potente geneesmiddelen zijn en zijn onmisbaar geworden in de hedendaagse geneeskunde, maar de toediening ervan kan nog drastisch verbeterd worden. Eiwitten kunnen makkelijk denatureren in de waterige injectievloeistoffen en zijn de noodzakelijke frequente injecties niet patiëntvriendelijk. Ofschoon het laatste aspect verbeterd kan worden door toepassing van polymere formuleringen die leiden tot verlengde afgifte, kan stabiliteit nog steeds problematisch zijn. Door eiwitten in te sluiten in suikerglazen worden ze beschermd tijdens de productie van deze formuleringen. In dit proefschrift werd hiervoor een nieuwe combinatie van twee suikers geëvalueerd en waarbij sommige geteste verhoudingen de stabiliteit optimaal was, vooral bij een hoge luchtvochtigheidsgraad. Verdere verbetering van de behandeling kan door modificatie van eiwitten, zodat ze bij specifieke cellen worden afgeleverd, wat bijwerkingen kan verminderen en de therapeutische effectiviteit kan verbeteren. In dit proefschrift werd in verschillende diermodellen aangetoond dat de combinatie van polymere formuleringen en modificatie van eiwitten leidt tot verlengde afgifte van eiwitten die specifiek ophopen in bepaalde cellen in fibrotisch weefsel. Dit proefschrift biedt inzicht in hoe een succesvolle vertaling van veelbelovende, maar vaak instabiele, therapeutische eiwitten naar de kliniek kan worden gemaakt. Het is daarvoor essentieel dat formuleringsstudies uitgevoerd worden met het therapeutische eiwit in plaats van een modeleiwit. Dit eiwit zal uitgebreid gekarakteriseerd moet worden, en bovendien zullen er slimme keuzes voor het (polymere) materiaal voor de matrix en het productieproces van de formulering moeten worden gemaakt

Protein Stability during Hot Melt Extrusion: The Effect of Extrusion Temperature, Hydrophilicity of Polymers and Sugar Glass Pre-stabilization

Purpose Biodegradable polymers have been widely investigated for controlled release formulations for protein delivery. However, the processing stability of proteins remains a major challenge. The aim of this research is to assess the influence of the hot melt extrusion process on the activity of a model protein by varying extrusion temperature, hydrophilicity of polymer and pre-stabilization of proteins with sugar glass technology. Methods The thermolabile model protein alkaline phosphatase (AP) was spray dried with inulin in a 1:10 protein:inulin weight ratio. The spray dried powder and the bare protein were exposed to 55°C, 95°C and 130°C for 10, 30, 60 and 120 minutes to assess the effect of heat stress that can be expected during hot melt extrusion. The spray dried powder and the bare protein were extruded with six different biodegradable polymers: the hydrophobic polymers poly-ε-caprolactone and low and high molecular weight poly (lactic-co-glycolic acid) at 55°C, 85°C and 130°C, respectively. Three hydrophilic polymers, based on the same polymers but with poly (ethylene glycol) incorporated, were extruded at the same temperatures. After extraction of protein from the extrudates by dissolving the polymer in an organic solvent, the activity of AP was determined using an enzymatic activity assay. Results Exposure to heat stress showed a protective effect of inulin against activity loss for AP at 95°C and 130°C, whereas at 55°C almost no activity loss was seen for both bare AP and AP spray dried with inulin. The decrease of activity was in accordance with Arrhenius behavior. After hot melt extrusion at 55°C, the remaining activity of AP was higher than 75% for all formulations, and showed an additional stabilizing effect of inulin on AP in the hydrophilic polymer. The stabilizing effect of inulin was more pronounced with extrusion at intermediate temperature (85°C), as in both the hydrophilic and the hydrophobic polymer the remaining activity of spray dried AP was about twice as high as the remaining activity of bare AP. The hydrophilicity of the polymer affected the protein stability of the formulations that were extruded at 85°C. For both bare AP and AP-inulin, the remaining activity was higher in the hydrophilic polymer than in the hydrophobic polymer. The activity loss of AP after extrusion at 130°C was over 90% for all formulations. Moreover, no stabilizing effects of inulin were seen, most probably due to the extrusion temperature being close to the Tg of the spray dried AP-inulin. Conclusions Heat stress data of proteins can not be used as a predictor for hot melt extrusion formulation development, as the protein activity loss after heat stress does not correlate with the activity loss after extrusion. This discrepancy is most likely due to the shear forces that occur during extrusion. The use of inulin as a protective agent can be beneficial at low and intermediate temperatures. Moreover, the use of hydrophilic polymers can further improve the stability of proteins during hot melt extrusion, especially at intermediate temperatures