由于基于蛋白质的治疗剂通常在液体制剂中表现出有限的稳定性,由于在固体状态下改善的蛋白质稳定性,人们对固体蛋白质制剂的开发越来越感兴趣。我们使用小规模(<3g)活塞和双螺杆挤出来稳定蛋白质(溶菌酶,BSA,和人胰岛素)在PEG基质中。使用ss-DSC系统研究了挤出后的蛋白质稳定性,ss-FTIR,CD光谱学,SEM-EDX,SEC,RP-HPLC,在溶菌酶的情况下,进行活性测定。应用的分析方法提供了对挤出物中蛋白质稳定性的准确评估,能够比较不同的熔融挤出配方和工艺参数(例如,剪切应力水平,螺钉配置,停留时间)。溶菌酶被用作模型蛋白质,并在挤出后以其活性形式完全恢复。溶菌酶和BSA或人胰岛素负载挤出物之间的差异表明熔融挤出可能对构象稳定性有影响。特别是,与溶菌酶相比,BSA和人胰岛素更容易受到热暴露和剪切应力的影响,其中剪切应力是主要参数。因此,与TSE相比,RAM挤出导致更少的构象变化。RAM挤出显示出良好的蛋白质颗粒分布,导致制备高负载固体蛋白质制剂的优选方法。
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.