Chapter V: Development of improved lipid based delivery systems for
2. Manufacturing of extrudates by twin screw extrusion
2.5. Influence of the manufacturing procedure on the in-vitro release kinetics of IFN-α.175
high PEG concentration within the pores might restrict the release of IFN-α over prolonged periods. This would explain why the burst release (of surface adjacent protein) is followed by a non-release period of IFN-α.
However, in contrast to the release of IFN-α from compressed matrices, a lag-phase was observed with extrudates prepared by twin screw extrusion (diameter 1.9 mm).
Therefore, in the following section the inherent differences of the manufacturing methods, which might contribute to the observed differences in the release profiles, were discussed.
2.5. INFLUENCE OF THE MANUFACTURING PROCEDURE ON THE IN-VITRO RELEASE
compression or by ram extrusion. Interestingly, despite of the reduction of the implant diameter associated with the change of the manufacturing procedure, twin screw extrusion resulted in a more sustained protein delivery. This suggested that twin screw extrusion per se causes more delayed protein release. However, it should be noted that concomitantly to the reduction of the implant diameter the implant height was varied. By compression implants with a height of 2.5 mm were produced, whereas extruded rods were cut into pieces of 2.3 cm.
To get a further insight into the impact of the twin-screw extrusion procedure on the release of IFN-α, extrudates prepared by twin screw extrusion were ground and then compressed to cylindrical implants. This manufacturing procedure should eliminate the influence of the implant geometry on the in-vitro release. If the blending and compaction during twin screw extrusion has an influence on the protein liberation the release profiles should markedly differ from those obtained from implants prepared by compression of the physical protein/lipid/PEG mixture.
Indeed, release profiles from implants prepared from the extruded lipid/protein blend revealed a triphasic release curve (Figure 101). After the burst release a lag-period over 9 days was followed. Finally, the delivery rates of IFN-α increased.
0 20 40 60 80 100
0 5 10 15 20 25
time, d cumulative IFN-α release, %
Figure 101: Influence of the extrusion procedure on the protein release.
For the preparation of lipidic implants a physical powder blend of 10 % IFN-α/HP-β-CD co-lyophilisate, 20 % PEG, and 70 % H12/tristearin ¼ was compressed (). Alternatively the powder was extruded with the twin screw extruder, ground and compressed (S). For comparison only the protein release kinetics from extrudates with a diameter of 1.9 mm (prepared by twin-screw extrusion) are included (z) (average +/- SD; n = 3).
Three possible process inherent features might explain the more sustained release from extrudates prepared by twin screw extrusion: (1) the intense compaction, (2) the fine compounding during extrusion, and (3) the melting of the low melting lipid during extrusion.
As shown in Chapter V.1.6.1 the acceleration of IFN-α release by using different triglyceride matrices was associated with a decrease in the compactness of the matrix. The change of the manufacturing procedure from ram to twin screw extrusion also significantly improved the mechanical stabilities of the extrudates (Table 9).
Therefore, the more compact matrix structure might be one reason for the more sustained protein release from twin screw extrudates.
Table 9: Mechanical properties of implants prepared by various manufacturing methods.
The implants were based on a lipidic powder blend of H12 and tristearin in a ratio of ¼ (average +/- SD; n = 5).
manufacturing method tensile strenght, N
compression 15.2 +/- 2.3
ram extrusion 10.7 +/- 1.5
twin screw extrusion 37.1 +/- 2.6
In addition, homogeneity studies by the admixture of methylene blue to the extrudate formulation showed a uniform staining of extruded rods prepared by twin screw extrusion. In contrast, implants of the same formulation processed by compression or ram extrusion revealed darker and brighter zones (Figure 102).
Figure 102: Optical appearance of the different implant systems.
A lipidic powder blend of H12 and tristearin in a ratio of ¼ was admixed with 1 % methylene blue in mortar. The powder blend was (A) compressed at 19.8 kN for 30 seconds, (B) extruded with a ram extruder, or (C) extruded with a twin-screw extruder.
Based on these observations it can be assumed that the compounding during twin screw extrusion accounts for a more finely distribution of PEG and IFN-α within the lipidic matrix.
It has been suggested recently, that the homogeneity of the protein distribution within a lipidic matrix correlates with the resulting release profiles [126]. Considering this information, the high homogeneity after twin screw extrusion may contribute to the more delayed protein recovery from extrudates prepared by twin screw extrusion.
In addition, in can be assumed that the melting of the low melting lipid during extrusion resulted in a welding of the lipid matrix, which in turn would account for a very dense structure with a low amount of pores and void spaces. Such effects of a melting step during implant manufacturing were reported, for instance, by Pongjanyakul [174]. The implants were prepared by casting the molten lipid into polyethylene tubes (see Chapter I.3.2.3), which entailed a significantly lower water uptake and a lower porosity of the implants compared to compressed implants. This was considered as reason for the reduced overall protein release from molten lipidic implants [174].
Taken together – the compaction, the blending and the melting – during twin screw extrusion can be considered as complementary reasons for the more sustained release of IFN-α from implants prepared by this manufacturing technique.
Furthermore, assuming that these features of twin screw extrusion might also affect the release kinetics of the incorporated excipients, in comparison to ram extrudates and to compressed implants, a more sustained release of HP-β-CD and of PEG can be expected. As explained above, the latter would contribute to the understanding of the observed lag-phase during release studies from twin screw extruded implants.
The verification of these hypothesises was beyond the scope of the present thesis.
However, further studies should investigate the release of the excipients from the implants prepared by twin screw extrusion. In addition, it would be interesting to explore the matrix morphology of twin screw extruded implants before and after in-vitro release and to compare those observations with the morphology of compressed (Chapter IV) and of ram extruded (Chapter V.1) implants. Finally, in order to evaluate whether the uniform distribution of the model compound methylene blue is conferrable to the distribution of IFN-α within the implant, a staining of the protein embedded within the lipidic matrix should be performed. For instance, van de Weert et al. revealed that the use of the red dye Ponceau S allowed a visualisation of
lysozyme embedded within PLGA microspheres [228]. Alternatively, FTIR-microscopy or the incorporation of a model protein labelled with fluorescent dyes could provide further information on the drug distribution [126].
2.6. PROTEIN STABILITY DURING RELEASE
In Figure 103 the monomer content of released IFN-α versus incubation time is illustrated. Over the entire liberation period the IFN-α monomer content remained at a high level (>95 %). Beside monomeric IFN-α only dimer specimen were detected by SE-HPLC. Mostly, the released protein resembled between 0.5 % and 2 % dimer.
Extrudates with a diameter of 1.0 mm delivered an increased amount of dimer of up to 5 % after 16 days. In comparison to that, implants prepared by ram extrusion or by compression revealed a higher integrity of released protein. The delivered dimer fraction from these kinds of implant was smaller than 1.5 %.
As protein destabilisation during the manufacturing procedure is unlikely, one reason for this more pronounced deterioration of the protein might be a destabilisation during release. Such a destabilisation during release is mostly associated with a time-depended decrease in the monomer content. However, such a dependency of the protein stability on the incubation time was not observed. Considering that the protein raw material used for twin screw extrusion already comprised a higher content of dimer specimen this might further explain the reduced protein integrity. SE-HPLC of the reconstituted lyophilisates applied for twin-screw extrusion revealed a dimer content of 0.75 %. In comparison, reconstituted lyophilisates processed by ram extrusion or by compression revealed in average less than 0.1 % dimeric IFN-α. The higher amount of dimer specimen presented within the used raw material used for twin screw extrusion potentially triggered further aggregation during in-vitro release studies from these implants.
As shown in Figure 104 lysozyme was delivered almost entirely in its monomeric form (>98 %) from extrudates prepared by twin screw extrusion. Size exclusion chromatograms of lysozyme revealed a main protein peak with a retention time of 24.5 minutes. In addition, a small peak with a retention time of 21 minutes was detected, which presumable corresponded to dimeric lysozyme.
80 85 90 95 100
0 1 4 7 10 13 16
incubation time, d IFN-α monomer, %
0 10 20 30 40 50 60 70 80 90 100
cumulative IFN-α release, % monomer content, % IFN-α release,%
A
80 85 90 95 100
0 1 4 7 10 13 16 19 23 30 37
incubation time, d IFN-α monomer, %
0 10 20 30 40 50 60 70 80 90 100
cumulative IFN-α release, % monomer content, % IFN-α release,%
B
80 85 90 95 100
0 1 4 7 10 13 16 19 23 30 37 45 53 60 incubation time, d
IFN-α monomer, %
0 10 20 30 40 50 60 70 80 90 100
cumulative IFN-α release, % monomer content, % IFN-α release,%
C
Figure 103: IFN-α integrity during in-vitro release.
Symbols indicate the total amount of delivered protein from extrudates comprising 10 % (open symbols) and 20 % (closed symbols) PEG. The bars illustrate the monomer content of delivered IFN-α (brighter bars: extrudates loaded with 10 % PEG, darker bars: extrudates loaded with 20 % PEG) Extrudates with a diameter of 0.5 mm (A), 1.0 mm (B) and 1.9 mm (C) were investigated (average +/-SD; n = 3).
80 85 90 95 100
0 1 4 7 9 13 16 20 25
incubation time, d
lysozymemonomer, %
0 10 20 30 40 50 60 70 80 90 100
cumulative lysozyme release, %
monomer content monomer content monomer content diameter 1.9 mm:
diamter 1.4 mm:
diameter 1.0 mm:
lysozyme release,%
lysozyme release, % lysozyme release, %
Figure 104: Lysozyme integrity during in-vitro release.
Symbols indicate the total amount of delivered lysozyme from extrudates with different diameters. The bars illustrate the monomer content of delivered lysozyme (average +/- SD; n = 3).