Breakage of unstable protein crystals in mechanical solid-liquid separation processes

作为DFG优先项目“生物技术过程中蛋白质和生物凝聚体的分散、结构和相变化(DIP Biotech)”的一部分，申请人正在研究机械不稳定的蛋白质晶体的过滤和断裂行为。从微观到宏观对生物技术过程从培育到配方的全面分析旨在更好地理解和优化整个过程链。Cornehl等人。结果表明，晶体的大小、形状和破碎度对溶菌酶晶体的过滤性能有很大影响。蛋白质的表达和结晶都影响晶体的形态和断裂行为。蛋白质或晶体的修饰和优化对后续工艺步骤有影响。最终，必须确保目标产品顺利通过完整的工艺链，在工程第一阶段通过小规模离心式过滤确定的工艺函数(通量密度函数和压缩屈服应力)没有考虑滤饼的任何不均匀性。在离心过滤的情况下，最大压力施加在滤饼的底部，这导致了不均匀的破碎行为。压力作用下的滤饼显示出晶体尺寸分布向更小的晶体移动。除压应力外，工艺过滤过程中还会产生剪切应力，导致晶体破碎，使晶体尺寸变小。剪切应力对蛋白质晶体的滤饼结构和加工功能的影响必须在本研究项目的范围内得到澄清。μCT分析可以研究滤饼中的结构差异，并确定晶体的尺寸分布。对于机械不稳定的蛋白质晶体，不存在将过程功能从小型过滤单元修正和转移到较大过滤单元的模型。然而，这是应用过程函数来计算较大分离设备上的过滤行为的先决条件。因此，在这个项目中必须开发这样的模型。在CFD模拟的支持下，将对考虑破碎机理的分离装置进行优化。通过3D打印和快速成型，可以实现过滤装置的建设性改进，并在过滤行为和晶体破碎方面进行比较。

As part of the DFG priority program “Disperse, structural and phase changes of proteins and biological agglomerates in biotechnological processes (DiSP Biotech)”, the applicant is investigating the filtration and fracture behavior of mechanically labile protein crystals. The comprehensive analysis of biotechnological processes from cultivation to formulation on the micro to macro scale aims to better understand and optimize the entire process chain. Cornehl et al. showed that crystal size, shape and breakage have a strong influence on the filtration performance of lysozyme crystals. Both, protein expression and crystallization, affect crystal morphology and breakage behavior. Modifications and optimizations of the proteins or crystals have an effect on the subsequent process steps. Ultimately, it must be ensured that the target product passes the complete process chain successfully.The process functions (flux density function and compressive yield stress), which were determined by centrifugal filtration on a small scale in the first project phase, do not take any inhomogeneity of the filter cake into account. In the case of centrifugal filtration the maximum pressure is applied to the bottom of the filter cake, which leads to an inhomogeneous breakage behavior. Pressure stressed filter cakes show a shift in crystal size distribution towards smaller crystals. In addition to the compressive stress, shear stress also occurs in technical filtration processes, which results in crystal breakage and reduces the crystal size. The influence of shear stress on the cake structure and the process functions for protein crystals has to be clarified within the scope of this research project.The μCT analysis allows to investigate structural differences in the filter cake and to determine the crystal size distribution. Models for correcting and transferring process functions from a small-scale to larger filtration units do not exist for mechanically labile protein crystals. However, this is a prerequisite for the application of the process functions to calculate the filtration behavior on larger separation apparatuses. Hence, such a model has to be developed in this project. Supported by CFD simulations, the optimization of separation apparatuses under consideration of breakage mechanisms will be carried out. With 3D printing and rapid prototyping constructive improvements of the filtration apparatus can be realized and compared with regard to the filtration behavior and crystal breakage.