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

随着生物技术的迅速发展，越来越多的治疗性蛋白质进入市场。这些蛋白质大分子中的大多数在遗传上是不稳定的，并且重要的是稳定它们以保持足够的储存和运输保质期。这些大功能分子具有几个层次的结构，称为一级、二级、三级和四级结构，并且它们的结构与它们的功能直接相关。稳定它们的结构往往会保留它们的功能。这通常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-这一过程称为配制。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优选完全除去水并将水性制剂转移到干粉中来降低水的可用性和活性，并且后者可以在环境温度下容易地储存和运输。由于许多蛋白质对升高的温度敏感，因此冷冻干燥一直是实现蛋白质制剂可接受的保质期的选择方法。冷冻干燥或“冻干”是一种干燥过程，其中溶剂，通常是水，首先被冷冻，然后通过在低温和低压（通常为6.6Pa至66 Pa）下升华除去。然而，冷冻干燥可以破坏稳定力，并允许分子展开，变性和聚集。因此，需要稳定的制剂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。通常使用耗时耗料的“试错法”来解决稳定性问题，即添加具有稳定性潜力的赋形剂，例如冻干和/或改变冷冻干燥条件。然而，无论是稳定的机制，也不是每个单独的应力在冷冻干燥过程中的相对贡献是完全理解。了解和理解冻干过程中蛋白质何时发生降解，将有助于设计更稳定的制剂，并从产品质量的角度优化冻干工艺。一个技术挑战是如何识别蛋白质分子结构，并在冷冻干燥的每个阶段验证它们与赋形剂的相互作用。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）-傅里叶变换红外光谱（FTIR），它提供蛋白质的结构信息，和（ii）多光子显微镜（MPM），它扩展到三维的研究，在冷冻干燥的原位实时研究在由冷冻台提供的明确定义的协议。将获得在冷却、冷冻和干燥过程中蛋白质与稳定辅料之间的相互作用以及蛋白质二级结构（微米和纳米级）的数据，这将导致更好的配方。结果将通过工业冻干工艺进行验证，我们将制定蛋白质配方的实用指南。

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.