Nucleation Kinetics during Suspension Crystallization of Proteins

蛋白质结晶是蛋白质生产过程中的重要环节。它用于蛋白质溶液的纯化和生产稳定性高、保质期长的蛋白质制剂。尽管悬浮结晶在生物技术、制药和食品等行业具有很高的潜力和相关性，但除了经验方法外，几乎没有其他方法可用于此类过程的设计。在悬浮结晶过程中，产生的颗粒尺寸分布由晶体的成核和生长决定。在工业结晶器中，成核要么是由于初级机制，特别是由于二级机制。一个典型的次要机制是已经存在的晶体与搅拌器叶片碰撞而形成原子核。对于低分子物质，文献中讨论了三种二次成核机制：剪切成核、接触成核和磨损。本提案的第一部分的目的是证明或否定这些蛋白质结晶机制的存在，并在适用的情况下确定相关的影响参数。本提案的第二部分的目的是从机理上和定量地描述蛋白质结晶过程中的磨损。蛋白质晶体的机械稳定性不如非蛋白质晶体，因为它们的晶内相互作用相当弱。因此，蛋白质晶体应该比那些更容易磨损。在该项目中，应寻求一种描述磨损的定量模型，该模型考虑到晶体上的机械载荷以及它们的机械强度。对已建立的低分子晶体模型在蛋白质晶体磨蚀成核过程中的适用性进行了研究，目的在于定性描述成核机理和定量了解成核动力学，从而使蛋白质结晶过程的质量和效率有较大的提高。

Protein crystallization is an important step along the process chain of protein production. It is applied for purification of protein solutions and for producing protein formulations with high stability and long shelf life. Despite of its high potential and relevance for industries in the biotechnological, pharmaceutical and food branch, there are only few other than empirical approaches available for the design of such processes.In suspension crystallization processes the produced particle size distribution is determined by nucleation and growth of the crystals. In technical crystallizers nucleation takes place either due to primary and, in particular, due to secondary mechanisms. A typical secondary mechanism is the creation of nuclei by collision of already existing crystals with stirrer blades. For low molecular substances 3 secondary nucleation mechanisms are discussed in the literature: shear nucleation, contact nucleation and attrition. It is the aim of the first part of this proposal to prove or disapprove the existence of these mechanisms for protein crystallization, and if applicable to identify the relevant influencing parameters.It is the aim of the second part of this proposal to come to a mechanistic and quantitative description of attrition during protein crystallization. Protein crystals are mechanically less stable than non-protein crystals because of their rather weak intracrystalline interactions. Therefore, protein crystals should be more prone to attrition than those. In the project it shall be searched for a quantitative model like description of attrition which takes into account the mechanical load on the crystals as well as their mechanical strength. A model, which has already been developed for low-molecular crystals, shall be scrutinized with regard to its applicability for attrition nucleation of protein crystals.The aimed at qualitative mechanistic description of nucleation mechanisms and the quantitative knowledge about nucleation kinetics in technical protein crystallizers shall make possible a considerable improvement of quality and efficiency of protein crystallization processes.