As protein-based therapeutics often exhibit a limited stability in liquid formulations, there is a growing interest in the development of solid protein formulations due to improved protein stability in the solid state. We used small-scale (<3 g) ram and twin-screw extrusion for the solid stabilization of proteins (Lysozyme, BSA, and human insulin) in PEG-matrices. Protein stability after extrusion was systematically investigated using ss-DSC, ss-FTIR, CD spectroscopy, SEM-EDX, SEC, RP-HPLC, and in case of Lysozyme an activity assay. The applied analytical methods offered an accurate assessment of protein stability in extrudates, enabling the comparison of different melt extrusion formulations and process parameters (e.g., shear stress levels, screw configurations, residence times). Lysozyme was implemented as a model protein and was completely recovered in its active form after extrusion. Differences seen between Lysozyme- and BSA- or human insulin-loaded extrudates indicated that melt extrusion could have an impact on the conformational stability. In particular, BSA and human insulin were more susceptible to heat exposure and shear stress compared to Lysozyme, where shear stress was the dominant parameter. Consequently, ram extrusion led to less conformational changes compared to TSE. Ram extrusion showed good protein particle distribution resulting in the preferred method to prepare highly-loaded solid protein formulations.
Keywords: BSA, bovine serum albumin; BSE, backscattered electron; CD, circular dichroism; DSC, Differential Scanning Calorimetry; EDX, energy-dispersive X-ray detector; EVA, Ethylene-vinyl acetate; FTIR, Fourier transformation infrared spectroscopy; HME, hot-melt extrusion; HMWS, high molecular weight species; Hot-melt extrusion; PEG, polyethylene glycol; PEO, polyethylene oxide; PLGA, Poly Lactic-co-Glycolic Acid; Protein stability; SEM, scanning electron microscopy; Small-scale; Solid-state characterization; TSE, twin-screw extrusion; ss, solid-state.
© 2022 The Authors.