types of transport phenomena were of importance when the release modifier PEG was present. In contrast, the release of PEG as well as of HP-β-CD remained purely diffusion controlled, irrespective of the presence of PEG.
In literature the role of PEG within controlled release systems has so far mostly been explained by its function as a pore forming agent [40, 108, 138, 174]. Due to an increase in the matrix porosity elevated levels of drug were recovered with an accelerated release rate.
As PEG of various molecular weights was reported to precipitate IFN-α [201], it was assumed that a reduced protein solubility or even an in-situ protein precipitation may explain the sustained release of IFN-α from PEG-containing tristearin implants and the observed deviations from pure diffusion control. The assumption was backed in Chapter IV.2 where it was shown that IFN-α spontaneously precipitates in the presence of more than 3 % (wt/vol) PEG. Importantly, protein precipitation was completely reversible. IFN-α was recovered in its monomeric form without chemical degradation according to SE- and RP-HPLC measurements after re-dissolving the precipitates. Furthermore, FTIR- and fluorescence spectroscopy indicated a preservation of the native secondary and tertiary structure after precipitation and re-dissolution.
The in-situ precipitation of IFN-α during the delivery from PEG-containing matrices was proven in Chapter IV.3. A “macropore model” was developed, which revealed that the dissolution of IFN-α in the presence of PEG was the rate-limiting factor for protein release. Furthermore, the pH-dependence of IFN-α solubility in the presence of PEG was reflected by the protein release kinetics from lipidic implants. Finally, evidence for an in-situ precipitation of IFN-α was provided by both the replacement of PEG by an alternative porogen as well as by the substitution of IFN-α by lysozyme.
In summary, the in-situ precipitation mechanism had two main benefits. The reversible precipitation of IFN-α in PEG-containing lipidic implants facilitated a low burst effect and a sustained protein release with nearly constant release rates.
Moreover, the precipitation also ensured low concentrations of dissolved protein within the implant pores. Therefore, the tendency towards protein aggregation was reduced.
Chapter IV.4 deals with the effects of the second hydrophilic excipient of the matrix formulation – HP-β-CD. In brief, the obtained findings indicated that HP-β-CD influenced the release of IFN-α rather by increasing the matrix porosity than by
protein complexation. In addition, a stabilisation of IFN-α during release can be supposed, as thermal protein denaturation studies, monitored with attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), figured out that HP-β-CD reduced the tendency of IFN-α to undergo protein denaturation and aggregation at elevated temperatures (50-60 °C).
In order to overcome the restrictions of administration associated with the large size of the above described implant system, various extrusion techniques were evaluated regarding their potential to produce small-sized lipidic protein delivery devices (Chapter V).
Initially, the possibilities of ram extrusion were evaluated and an extrusion procedure was developed which did neither induced a polymorphic transformation nor compromised protein integrity. Moreover, the achieved geometry enabled filling of the extrudates in an injection device approved for subcutaneous application of polymeric implants.
However, protein release was only sustained over 16 days and various attempts, such as variations in the particle sizes of the used raw materials or the change of the lipidic matrix material, did not substantially extend the release period. The less sustained protein liberation, compared to compressed implants, can be ascribed to the transfer of the manufacturing procedure from compression to ram extrusion, which provoked a reduction of the diffusion pathways as well as a decrease in matrix density. Since the changes of implant geometry and compactness also affect the in-vitro release kinetics of the excipients, a more accelerated release of HP-β-CD and of PEG occurred from extruded implants. The enhanced leaching out of PEG was, furthermore, shown to be associated with distinctions in the underlying drug release mechanism between the implant systems. As the actual concentrations of PEG within the water-filled pores of extruded implants seemed to be lower compared to that generated within compressed implants, IFN-α release was purely governed by diffusion irrespective of the initial PEG loading.
In Chapter V.2 twin screw extrusion was evaluated as alternative manufacturing technique. Apart from the initial problems associated with the handling of tristearin material, different formulation strategies enabling extrusion were identified. Among them the processing of proteins with a combination of low-melting point and high-melting point lipids was deemed as most promising. Implants with diameters between
0.5 mm to 1.9 mm could be prepared, allowing subcutaneous injection via a large gauge needle.
The produced implants based on a blend of the mixed-acid triglyceride H12 and of tristearin were shown to contain IFN-α in the quality of the raw material. In order to render investigations on the secondary structure of IFN-α embedded within lipidic implants, FTIR spectroscopy was utilised. As the obtained absorption spectra in the amid I region revealed no significant alterations compared to the lyophilised IFN-α prior extrusion, perturbations of the secondary protein conformation due to the extrusion procedure could be regarded as unlikely. Moreover, the produced extrudates comprised the lipids with their stable modification. Based on these positive outcomes regarding protein stability and lipid polymorphism, the main criteria for a new lipidic delivery device were met and release studies could be performed on a meaningful basis.
During in-vitro release the developed implant system delivered IFN-α mainly in its monomeric form over time periods of up to 60 days. Moreover, the employment of small implant diameters or high initial PEG loadings facilitated a complete protein recovery.
Importantly, the in-vitro release profiles could be easily controlled. One option to adapt the in-vitro release kinetics of IFN-α was the variation of the implant diameter.
Protein liberation could be controlled in a prolonged manner over 15, 40, or 60 days by producing extrudates with a diameter of 0.5 mm, 1.0 mm or 1.9 mm, respectively.
Furthermore, protein delivery could be tailored by the admixing of various amounts of PEG.
The comparison of the release of IFN-α and lysozyme pointed out that in-situ protein precipitation is again important for the delayed protein liberation from extrudates prepared by twin screw extrusion. Although the lysozyme liberation occurred less delayed compared to the IFN-α delivery, a sustained lysozyme release over 25 days could be achieved. Furthermore, release studies carried out with implants prepared either by compression, by ram, or by twin screw extrusion indicated that twin screw extrusion per se resulted in more sustained protein delivery. Presumably, the blending, melting, and compaction during extrusion may explain this observation.
Summing up the attainments, twin screw extrusion can be considered as promising manufacturing technique for lipidic implants. The developed extrudate formulation can be easily manufactured. As twin screw extrusion is commonly applied to produce
commercial polymeric implants, up-scaling of the manufacturing appears feasible. It was shown that the designed manufacturing procedure did not compromise protein integrity. Furthermore, IFN-α as well as lysozyme could be delivered in their monomeric form over prolonged periods of time.
In conclusion two major achievements were reached with respect to the development of lipid-based sustained release devices for pharmaceutical proteins. First, a novel release mechanism based on an in-situ precipitation within inert matrices has been identified. Second, a new extruded lipidic implant system has been developed. As former concerns regarding implant administration and manufacture were overcome, the developed lipidic implant system can be deemed as promising platform for the controlled delivery of pharmaceutical proteins. The system was shown to be particularly suitable for the delivery of IFN-α, however, the knowledge obtained on the mechanisms controlling IFN-α release, should enable to transfer the developed system to a variety of other pharmaceutical proteins.