Lyophilization of proteins - an in-situ study on structural changes and molecular interactions

随着生物技术的飞速发展，越来越多的治疗蛋白进入市场。这些蛋白质大分子中的大多数都是天生不稳定的，因此稳定它们以保持足够的储存和运输保质期是很重要的。这些宏观功能分子有几个层次的结构，分别是一级、二级、三级和四级结构，它们的结构与它们的功能直接相关。建立它们的结构往往能保留它们的功能。这通常是通过添加各种稳定剂（赋形剂）并将其加工成稳定状态来完成的-这一过程称为配方。几乎所有的结构变化都涉及与水的相互作用，因此延长保质期的主要方法是通过完全去除水并将含水配方转化为干粉来降低水的可用性和活性，而干粉可以很容易地在室温下储存和运输。由于许多蛋白质对高温很敏感，冷冻干燥一直是蛋白质配方实现可接受的保质期的选择。冷冻干燥或“冻干”是一种干燥过程，首先将溶剂（通常是水）冷冻，然后在低温和低压（通常为6.6Pa至66Pa）下通过升华除去。然而，冷冻干燥会破坏稳定力，使分子展开、变性和聚集。因此，需要稳定的配方和工艺设计，以避免结构和/或化学改变的蛋白质分子的生物活性损失和可能的免疫原性效应。通常使用添加具有稳定潜力的赋形剂（例如糖）和/或改变冷冻干燥条件的“试错”方法来解决稳定性问题。然而，无论是稳定的机制，还是在冷冻干燥过程中每个个体应力的相对贡献，都没有被完全理解。了解蛋白质在冻干过程中何时发生降解，将有助于从产品质量的角度设计更稳定的配方和优化冻干工艺。技术上的挑战是如何在冷冻干燥的每个阶段识别蛋白质分子结构并表征它们与辅料的相互作用。在这个项目中，我们将采用两种先进的成像技术，(i)焦平面阵列(FPA)-傅立叶变换红外光谱（FTIR），它提供蛋白质结构信息，（ii）多光子显微镜（MPM），它将研究扩展到三维，以便在由冷冻台提供的明确协议下对冷冻干燥进行现场实时研究。在冷却、冷冻和干燥过程中，蛋白质与稳定赋形剂之间的相互作用以及蛋白质在微米和纳米尺度上的二级结构的数据将得到，这将导致更好的配方。结果将通过工业冻干工艺进行验证，我们将开发有关蛋白质配方的实用指导。

More and more therapeutic proteins are getting into market due to the rapid development of biotechnology. Most of these protein macromolecules are inherantly unstable and it is important to stabilise them to preserve sufficient shelf life for storage and transport. These macro functional molecules have several levels of structure, referred to as primary, secondary, tertiary and quaternary structure, and their structure is directly linked to their functions. Stablising their structure often preserves their functions. This is usually done by adding various stablisers (excipients) and processing them into a stable status - a process referred as formulation. Almost all structural changes involve interactions with water, and hence the primary approach to elongate shelf life is to reduce water availability and activity by preferably removing water completely and transferring the aqueous formulation into a dry powder, and the latter can be easily stored and transported at ambient temperature. As many proteins are sensitive to elevated temperature, freeze-drying has been the process of choice to accomplish acceptable shelf life for protein formulations. Freeze-drying or 'lyophilization' is a drying process where the solvent, usually water, is first frozen and then removed by sublimation at low temperature and low pressure (typically 6.6Pa to 66Pa). However, freeze drying can disrupt the stabilizing forces and allowing the molecule to unfold, denature and aggregate. Hence stable formulation and process design is required to avoid biological activity loss and possible immunogenic effect of the structurally and/or chemically altered protein molecules. Time and material consuming 'trial-and-error' approaches adding excipients that have the potential to stabilize, e.g. saccharides, and/or varying the freeze-drying conditions are usually used to address stability issues. However, neither the mechanisms of stabilisation nor the relative contributions of each individual stress during freeze-drying are completely understood. The knowledge and understanding of when the degradation of proteins occur during lyophilization would be beneficial in designing more stable formulations and optimizing the freezing-drying process from a product quality perspective. A technical challenge has been how to identify the protein molecular structures and characterise their interactions with excipients during each stage of freeze drying. In this project we will adopt two advanced imaging techniques, (i) focal plane array (FPA)- Fourier Transform Infrared Spectroscopy (FTIR), which provides protein structural information, and (ii) multiphton microscopy (MPM), which extends the study into three dimensional for the in situ real-time study of freeze drying at well defined protocols provided by a cryostage. The data on interactions between proteins and stabilizing excipients as well as on the protein secondary structure, at micron and nanometre scales, during cooling, freezing and drying will be obtained, which will lead to better formulation. The results will be validated with industrial lyophilisation process and we will develop practical guidance on protein formulation.