【作者】 刘晶晶；

【导师】 胡仰栋； 王学重；

【作者基本信息】 中国海洋大学 ， 海洋化学工程与技术， 2010， 博士

【摘要】 大分子生物药以晶体形式作为给药的方式具有药物稳定性高,药物有效成分浓度高以及容易实现药物的可控性释放等许多优点。然而,、截至2004年的150多种生物药中,以晶体为其主要的生产和销售形态的药物只有胰岛素。造成这种情况的主要技术原因是因为蛋白质结晶和小分子结晶相比具有更为复杂的结晶环境和影响因素,大规模生产蛋白质晶体在方法和技术上还很不成熟。相对于传统的蛋白质结晶中实验研究以及模拟研究所针对的单晶体尺度,本论文主要在过程尺度上对蛋白质结晶过程中晶体群体的生长行为进行了系统的模型化和实验研究。蛋白质晶体的形状以及产品的粒度分布是衡量结晶产品质量的主要指标,但是目前对蛋白质结晶的群体粒数衡算模型的研究还非常少,而且只能模拟粒度分布,不能模拟形状分布,因为模型中的晶体尺寸被简单的定义为该晶体的体积当量直径。本论文提出了一个适用于蛋白质结晶过程的基于晶体形状结构的群体粒数衡算模型。在该模型中,蛋白质晶体的形状被定量描述为每个晶面到晶体几何中心的距离,由此提出了基于蛋白质晶形的多维粒度分布,即形状分布的概念。论文以溶菌酶结晶过程为例,对溶菌酶晶体结晶的热力学和动力学行为进行了分析和研究,应用非线性回归的方法,确定了四方系溶菌酶的结晶动力学方程参数。结合本论文提出的基于晶体形状结构的群体粒数衡算模型,质量衡算方程以及能量方程建立了溶菌酶冷却结晶过程模型。利用该模型可以描述不同操作条件下晶体群体的形状分布和粒度分布随时间的动态变化,即在过程系统尺度上蛋白质结晶过程的晶体生长行为。利用提出的溶菌酶冷却结晶过程模型,论文模拟研究了鸡蛋清溶菌酶冷却结晶过程中的冷却速率,晶种负荷以及晶种的尺寸对结晶过程的影响,,尤其是对产品晶体的形状和粒度分布以及过饱和度的影响。模拟研究发现,结果表明：低晶种负荷(0.001)生产的溶菌酶晶体颗粒较大而且长宽比较小；较低的冷却速率(0.7℃/day)下溶菌酶晶体生长很慢；较高的冷却速率(高于2.0℃/day)会产生较大的晶体颗粒和较小的长宽比。论文还提出了适于二维晶种负荷预估的模型,用于估算蛋白质结晶过程所需添加的晶种负荷量。应用基于晶型结构的群体粒数衡算模型可以研究和控制不同晶面的生长,以达到所期望的晶体外形和尺寸,因此本论文进一步对溶菌酶结晶过程中的产品晶体形状分布和粒度分布进行了优化研究。对于期望的产品晶体的形状和尺寸分布,通过调节操作参数,可以优化得到最优的冷却温度曲线和过饱和度曲线。模拟晶体群体的生长行为是一个极为复杂,非线性很强的过程,而且模拟计算非常耗时,本文利用遗传算法对群体粒数模型进行优化,证明是有效的。通过调节结晶器夹套中冷却剂的流量实现了冷却温度曲线和过饱和度曲线的跟踪控制,从而使控制晶体产品形状分布和尺寸分布成为可能。由于温度条件易于操作控制,重复性好,因此被广泛应用于小分子工业结晶。然而,目前还缺乏对连续降温冷却法进行蛋白质结晶的系统研究。本论文通过多槽微孔板并行结晶实验(24槽坐滴式微结晶器)找到了适合研究溶菌酶冷却结晶的适宜的初始条件,包括适宜的溶菌酶溶液初始浓度,沉淀剂浓度以及溶液pH值。利用实时照相技术观测记录了在溶菌酶降温结晶过程的晶体生长变形轨迹。通过对大区域冷却台反应器冷却速率的调节,研究了冷却速率对晶体形变的影响。这些结果对本论文的模拟研究提供了实验依据,也验证了连续降温对蛋白质结晶过程是可行和有效的。更多还原

【Abstract】 Large molecule protein crystals have shown significant benefits in the delivery of biopharmaceuticals to achieve high stability, high concentration of active pharmaceutical ingredients (API), and controlled release of API. However, among the about 150 biopharmaceuticals on the market by 2004, only insulin has been marketed in crystalline form. The main technical reason led to this situation is due to the fact that protein crystallisation has a more complicated crystallisation environment and is affected by many factors. There is currently a lack of knowledge on commercial scale production of protein crystals. Therefore, in contrast to the majority of previous work on experimental and computational studies of protein crystallisation that has been centered on single crystal scale, the focus of the research reported in this thesis is placed on the computational and experimental study of protein crystallisation at process scale, investigating the growth behavior of the population of crystals grown from a crystallizer.The morphology and size distribution of protein crystals are key measures in quality control. However, research on population balance modelling of protein crystallisation processes is still very limited and restricted to simulation of crystal size distribution, ignoring crystal shape information because the size of a crystal is simply defined as the diameter of a sphere having the same volume as the crystal. A new morphological population balance model is proposed in this work for modelling protein crystallisation processes. In the model, the shape of a crystal is quantitatively defined as the distances of all crystal faces to the geometric centre of the crystal. For all the crystals in the crystalliser, a concept of multi-dimensional size distributions, which we call shape distribution for the crystal population, is proposed.The case study protein is Hen-Egg-White (HEW) lysozyme for which the thermodynamics and kinetics of crystallisation were examined. Faced crystal growth kinetic models were developed using non-linear regression. Process models were built for the crystallisation process of HEW lysozyme by combining the newly proposed morphological population balance model with mass and energy balance equations. The models were applied to simulate the evolving behaviour of shape distribution as well as size distribution of crystals of tetragonal HEW lysozyme.The morphological population balance model was applied to crystallization of HEW lysozyme for study of the effect of cooling rate, seed loading and seed size on size and shape distributions of product crystals as well as supersaturation. It was found that for growth only crystallization, low seed loading (0.001) and high cooling rate (2.0℃/day) lead to large crystals of low aspect ratio, but care has to be taken to avoid nucleation and major shape change such as width becoming larger than the length. Low cooling rate (0.7℃/day) results in slow growth. In addition, a two dimensional model for estimating seed loading was developed.In principle, the proposed morphological population balance model for protein crystallisation can be applied to investigate and control the growth of individual faces with the aim of obtaining desired crystal shape and size. Therefore process optimisation techniques were introduced to the model for optimising the product crystal shape distribution and size distribution. Optimal temperature and supersaturation profiles leading to the desired crystal shape and size distributions were derived. Genetic algorithm was investigated and found to be an effective optimisation technique for the current application. Since tracking an optimum temperature or supersaturation trajectory can be easily implemented by manipulating the coolant flowrate in the reactor jacket, the proposed methodology provides a feasible closed-loop mechanism for protein crystal shape tailoring and control.The modelling and optimisation study in this work is based on cooling protein crystallisation. Cooling rate control can be easily achieved and has good repeatability, and as a result has been widely used in industrial crystallisation of small molecules. However, there is a noticeable lack of systematic study on cooling crystallisation of proteins. In this study Linbro parallel crystallization experiments (24-well sitting-drop plate) was conducted to find the suitable conditions for carrying out cooling crystallisation investigation, including the initial concentration of HE W lysozyme solutions, precipitate concentration and pH value of solution. Real-time in-process imaging was used to record the morphological evolution of HEW lysozyme crystals during cooling crystallization using a hot-stage. Using a hot-stage, the effect of cooling rates on HEW lysozyme crystallization was investigated. The results provide experimental proof of the feasibility and effectiveness of cooling as a means for protein crystallisation.更多还原