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

到目前为止，药物相关蛋白质的配制和给药仅限于溶液或液体剂型的非肠道给药。这通常是由于蛋白质缺乏稳定性或缺乏组织特异性。此外，翻译后修饰必须保留在配方中，因为它们通常对蛋白质活性至关重要。除了生物利用度外，在评估替代制剂和给药方式时，也需要考虑这些方面。特别是，口服、鼻腔或肠外给药的固体药物将显著扩大或改善蛋白质类药物的治疗用途和依从性。除了稳定性外，在微尺度上评估配方和工艺参数及其在实验室和中试规模上的可扩展性至关重要，因为由于蛋白质的成本，常规开发一克或千克规模在经济上是不可行的。该项目提案的目的是建立用于生产固体剂型的可扩展配方技术(熔融挤压、流态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压和喷雾冷冻过程将转移到蛋白质上。蛋白质的稳定应该通过嵌入到侵蚀或溶解的聚合物基质中进行。此外，还将通过制定高孔性冻干颗粒的物质，来考察喷雾冷冻干燥的快速冷冻步骤是否能为蛋白质的稳定性带来决定性的优势。各种多肽/蛋白质(例如三氢甘氨酸、胰岛素、康普肽等)可作为模型使用，因此，从低剂量到高剂量以及从口服靶标到植入剂，都可以代表配方发现的整个范围。关于稳定性、活性和体外释放动力学的声明必须在每个标尺上做出。这些物质是多个二硫键桥联的分子，其正确的折叠对它们的活性至关重要。获得正确的二硫键对于蛋白质的形成是至关重要的，也是为它们的分析制定适当的标准所必需的。在过去的几十年里，人们通常使用层析-偶联质谱法来分析这类序列，而忽略了Edman的N-末端蛋白质测序方法。鉴于目前已知的质谱学蛋白质分析的局限性，两种方法的组合对于全面的蛋白质分析似乎是有意义的，并且将在本申请的上下文中提供有关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.