(T1130-06-32) Solid-State Stabilization of Proteins in Highly-Loaded Melt Extrudates – Characterization of Protein Integrity

Purpose: Biopharmaceuticals play an increasingly important role in the treatment of various diseases. The administration of biopharmaceuticals has hitherto been limited to parenteral administration. One of the challenges is that many biologics are not stable in liquid formulations, or require a time-, and resource-wasting formulation development to achieve stable formulations. Therefore, solid-state is preferred for a wide range of protein drugs [1]. Besides the mainly used techniques (e.g., lyophilization, and spray-drying) to convert liquid protein formulations into solid-state, one of the most promising fields is processing of proteins by extrusion. However, during extrusion, proteins are exposed to thermal and mechanical stress, leading to changes of their native structure. The main concern for solid-state protein formulation is to ensure protein integrity and stability during the manufacturing process. Embedment of protein drugs in a polymeric or lipid-based matrix by extrusion has been previously published [1,2,3]. However, an intensive understanding of extrusion process parameters and correlation of experimental data on protein integrity was incompletely considered. The aim of this work was the establishment of small-scale extrusion for production of highly-loaded protein extrudates at batch sizes below 3 g and evaluation of protein integrity after extrusion.

Methods: Physical mixtures (3g) composed of PEG 20,000 and protein powder (lysozyme or BSA: 20, 40, or 60%, or insulin human: 40% (w/w)) were blended for 10 min at 50 rpm using a turbula mixer (Willy A. Bachofen AG). The properly mixed powder blends were either manually fed into a preheated barrel (60-63°C) of a ram extruder or fed via a conveying belt in a 5-mm TSE (ZE5, Three-Tec). For DSC studies, ∼10 mg of protein powder or grounded extrudates (DSC2, Mettler Toledo) were accurately weighed in pinned aluminum crucibles. DSC scans were recorded using a ramp from 25 to 230 °C (heating rate: 20 °C/min). Protein content was characterized by RP-HPLC (Agilent 1100 series, RP-C18 Nucleosil, 125×4 mm, MN). Solvent system consisted of water/acetonitrile/trifluoroacetic acid (A: 100/0/0.1, B: 0/100/0.1, V/V). Linear gradient method was applied (0-10 min, 5-95%B) at 1 mL/min flow and a column temperature of 30°C. Extrudates were dissolved in Milli‑Q^® water and 10 µL were injected. For SEC analysis a Superdex 75 Increase 10/300 GL column (Cytiva) was used with 50 mM sodium phosphate pH 7.0 and 400 mM sodium chloride buffer at 0.6 mL/min flow. Chromatograms obtained with a UV-detector were quantified at 214 and 280 nm. Peak area, relative areas of monomer, fragments, and aggregates were determined. Protein particle distribution was examined by elemental mapping of cross sections of extrudates for the characteristic X-ray peak of nitrogen (Hitachi S-2700, EDX spectroscopy). The extrudates were coated with platinum. Activity of lysozyme was measured using EnzChek^® Lysozyme Assay Kit (ThermoFisher) and microplate reader (EnSpire^®, PerkinElmer).

Results: Temperature in combination with shear stress during extrusion, can potentially affect protein integrity and consequently lead to a loss in activity. Stability of proteins after ram (lower shear stress) and TS (higher shear stress) extrusion was evaluated and correlated with extrusion process parameters. Protein particles were homogeneously embedded into PEG 20,000 (Fig. 1). Lysozyme was recovered from extrudates with full biological activity (ram: 98.5±6.3%, TSE conveying: 94.8±8.0%, kneading: 92.8±7.7%). SEC results showed no higher order protein aggregates or fragments after ram or TS extrusion for lysozyme-loaded extrudates. BSA-loaded ram and TS extrudates showed formation of < 2% protein aggregates or fragments, whereas insulin showed up to 3% fragment formation after TS extrusion, but without forming aggregates. Insulin monomer content after ram extrusion was over 98.5%, indicating that ram extrusion introduced less shear stress compared to TSE. DSC measurements showed melting temperature (T_m) of 204.2, 218.2, and 212.7°C for native lysozyme, BSA and insulin, respectively. T_m of protein-loaded extrudates were compared to unprocessed protein. Due to higher shear stress during TSE, decrease in T_m for all proteins was higher compared to ram extrusion and native protein (Fig. 2). T_m data were also correlated with specific mechanical energy (SME). The higher the SME the material is exposed to during TSE processing, the higher the decrease in T_m. However, T_m of proteins were still high, there was no indication for thermal protein denaturation as a consequence of exposure to temperature and shear stress during extrusion.

Conclusion: The feasibility of extrusion for processing of proteins was evaluated with regard to protein instability due to thermo-mechanical stress. Nearly complete recovery of (active) and stable protein from extrudates showed that extrusion did not disrupt the protein integrity. Correlation of experimental and simulation data helps to facilitate extrusion process development. Both ram and TSE could be applied as formulation technology embedding and stabilizing proteins with a high drug load (up to 60%) in a hydrophilic matrix. However, in the case of shear-sensitive proteins, ram extrusion should be preferred. Thus, HME offers a potential stabilization technology of proteins in solid state at high drug load.

References: [1] Cossé, A. et al., Hot Melt Extrusion for Sustained Protein Release: Matrix Erosion and In Vitro Release of PLGA-Based Implants. AAPS PharmSciTech, 1;18(1):15-26 (2017)
[2] Even, M.-P. et al., Twin-screw extruded lipid implants containing TRP2 peptide for tumour therapy. Eur. J. Pharm. Biopharm. 114, 79-87 (2017).
[3] Stanković, M., Frijlink, H. W. & Hinrichs, W. L. J. Polymeric formulations for drug release prepared by hot melt extrusion: application and characterization. Drug Discov. Today 20, 812-823 (2015).

Acknowledgements: We would like to thank the German Research Foundation (DFG) for funding within priority program SPP 1934.


Figure 1. Protein distribution illustrated with EDX-SEM elemental mapping of nitrogen (green spots) on cross section of 40% lysozyme-, BSA-, or insulin human-containing PEG-extrudates prepared by ram or TS extrusion at 63°C.


Figure 2. Melting temperatures (left y-axis) of 20, 40, and 60% protein-loaded extrudates prepared by ram or twin-screw extrusion at 63°C. Spheres represent the calculated specific mechanical energy (right y-axis). Dotted lines show the melting temperature of unprocessed proteins (reference). Error bars represent the standard deviation of three measurements.