Micro-Scale Vacuum Compression Molding as a Predictive Screening Tool of Protein Integrity for Potential Hot-Melt Extrusion Processes

Hot-melt extrusion (HME) is used for the production of solid protein formulations mainly for two reasons: increased protein stability in solid state and/or long-term release systems (e.g., protein-loaded implants). However, HME requires considerable amounts of material even at small-scale (>2 g batch size). In this study, we introduced vacuum compression molding (VCM) as a predictive screening tool of protein stability for potential HME processing.

The focus was to identify appropriate polymeric matrices prior to extrusion and evaluation of protein stability after thermal stress using only a few milligrams of protein. The protein stability of lysozyme, BSA, and human insulin embedded in PEG 20,000, PLGA, or EVA by VCM was investigated by DSC, FT-IR, and SEC. The results from the protein-loaded discs provided important insights into the solid-state stabilizing mechanisms of protein candidates.

We demonstrated the successful application of VCM for a set of proteins and polymers, showing, in particular, a high potential for EVA as a polymeric matrix for solid-state stabilization of proteins and the production of extended-release dosage forms. Stable protein-polymer mixtures with sufficient protein stability after VCM could be then introduced to a combination of thermal and shear stress by HME and further investigated with regard to their process-related protein stability.

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Materials

Human insulin was kindly donated by Sanofi Deutschland GmbH (Frankfurt am Main, Germany). Lysozyme from chicken egg-white, lyophilized powder (Cat. No. L6876) was obtained from AppliChem (AppliChem GmbH, Darmstadt, Germany). Bovine serum albumin (BSA), sodium chloride, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, acetonitrile, and dichloromethane were purchased from Merck (Merck KGaA, Darmstadt, Germany). PLGA (Resomer^® RG502 H) was purchased from Evonik (Evonik Nutrition & Care GmbH, Darmstadt, Germany). Polyethylene glycol (PEG) 20,000 was obtained from Carl Roth (Karlsruhe, Germany). Poly(ethylene/vinyl acetate) (EVA; 60:40 (wt)) granules were purchased from Polysciences (Polysciences Inc., Warrington, PA, USA). All chemicals were of analytical grade or equivalent purity.

Preparation

PEG 20,000 flakes were milled utilizing a high-shear mixer (Krups Mixette type 210, Krups, Frankfurt am Main, Germany). EVA granules were milled using a cryogenic mill (6775 Freezer/Mill^® Cryogenic Grinder, SPEX SamplePrep LLC, Metuchen, NJ, USA). The EVA granules were loaded into the sample holder and placed in the grinding chamber, which maintains cryogenic temperatures due to continuous immersion in liquid nitrogen. EVA granules were pre-cooled for 10 min and then milled through 3 grinding cycles (10 cycles per second) at 2 min each with 2 min intercool time. For the preparation of protein-loaded discs, a physical mixture composed of polymer and 20% protein powder was manually blended with a spatula. The physical mixtures of polymer and protein powder were then vacuum compression molded. VCM was conducted using a VCM tool (MeltPrep GmbH, Graz, Austria) with a 5 mm diameter disc geometry. Approx. 15 mg of each blend was loaded into the VCM device and heated under vacuum [17] for 6 or 12 min at a temperature of 65 °C for PEG 20,000, or 70 °C for PLGA, and EVA (Table 1).

Dauer, K.; Wagner, K.G. Micro-Scale Vacuum Compression Molding as a Predictive Screening Tool of Protein Integrity for Potential Hot-Melt Extrusion Processes. Pharmaceutics 2023, 15, 723. https://doi.org/10.3390/pharmaceutics15030723