Innovative Pharmaceutical Stabilization and Formulation Techniques for Protein Drugs and Their Influence on Sequence and Structure

迄今为止，药物相关蛋白的配制和给药仅限于溶液或液体剂型的肠胃外给药。这通常是由于缺乏稳定性或缺乏蛋白质的组织特异性。此外，翻译后修饰必须保留在制剂中，因为它们通常对蛋白质活性至关重要。除了生物利用度，在评价替代制剂和给药方式时，还需要考虑这些方面。特别地，用于经口、经鼻或肠胃外给药的固体将显著扩展或改善基于蛋白质的药物的治疗用途和顺应性。除了稳定性之外，在微尺度上的配方和工艺参数的评估及其在实验室和中试规模中的可扩展性是至关重要的，因为由于蛋白质的成本，在克或千克规模上的常规开发在经济上是不可行的。本项目提案的目的是为固体剂型的生产建立可扩展的配方技术（熔融挤出、流化床工艺和喷雾冷冻干燥）。在这个过程中，我们已经建立的熔融挤出和喷雾冷冻工艺将转移到蛋白质上。蛋白质的稳定化应该通过包埋在侵蚀或溶解的聚合物基质中来进行。此外，还将通过物质的高度多孔冻干颗粒的配制来研究喷雾冷冻干燥的快速冷冻步骤是否可以为蛋白质稳定性提供决定性优势。各种肽/蛋白质（例如tridegin、胰岛素、锥肽如齐考诺肽）可用作模型，因此可以代表从低剂量到高剂量以及从口服靶标到植入物的整个制剂发现范围。必须对每个规模进行关于稳定性、活性和体外释放动力学的说明。这些物质是多个二硫键桥接的分子，其正确折叠对其活性至关重要。获得正确的二硫键对于蛋白质的配制是必不可少的，而且对于开发用于其分析的适当标准也是必不可少的。色谱-质谱联用方法通常被用来分析这些序列，而根据Edman的N-末端蛋白质测序的方法在过去的几十年中被忽视。鉴于质谱蛋白质分析的现有已知限制，两种方法的组合似乎对于全面的蛋白质分析是有意义的，并且将提供关于本申请上下文中配制物质的稳定性的信息。

The formulation and administration of pharmacologically relevant proteins has hitherto been limited to the parenteral administration of solutions or liquid dosage forms. This is often due to a lack of stability or lack of tissue specificity of the proteins. In addition, post-translational modifications must be retained in a formulation as they are often critical for protein activity. Apart from bioavailability, these aspects also need to be considered when evaluating alternative formulations and modes of administration. In particular, solids for peroral, nasal or parenteral administration would significantly expand or improve the therapeutic use and the compliance of protein-based drugs. In addition to stability, the evaluation of recipe and process parameters on the microscale and their scalability in the laboratory and pilot scale is of crucial importance, as due to the cost of proteins conventional developments on a gram or kilogram scale are not financially feasible. The aim of this project proposal is the establishment of scalable formulation technologies (melt extrusion, fluidized bed processes and spray freeze drying) for the production of solid dosage forms. In the process, the melt extrusion and spray freezing processes we have established will be transferred to proteins. The stabilization of the proteins should take place by embedding in eroding or dissolving polymer matrices. In addition, it will be investigated by the formulation of highly porous lyophilizate particles of the substances, whether the rapid freezing step of the spray freeze drying can give decisive advantages for protein stability. Various peptides/proteins (for example tridegin, insulin, conopeptides such as ziconotide) are available as models, so that from low-dose to high-dose and from oral targets to implants the entire range of formulation finding can be represented. Statements regarding stability, activity and in vitro release kinetics must be made on each scale. The substances are multiple disulfide-bridged molecules whose correct folding is crucial for their activity. Obtaining the correct disulfide bonding is essential for the formulation of proteins, but also for the development of appropriate standards for their analysis. Chromatography-coupled mass spectrometric methods are usually used to analyze such sequences, while the method of N-terminal protein sequencing according to Edman was neglected in the past decades. In view of the now known limits of mass spectrometric protein analysis, a combination of both methods seems to make sense for a comprehensive protein analysis and will provide information on the stability of the formulated substances in the context of the present application.