层析工艺设计以及优化1例析.ppt

* * * * * * * * * * * * * * * * * 确定蛋白质的变性温度 差热扫描分析（DSC）是常用的确定变性温度的一种方法。在DSC中蛋白质的变性是一个吸热的峰，而蛋白质的聚集是一个放热的峰。一些因素可以影响测定。首先就是所采用的方法,不同的测定方法对于相同的蛋白可以给出不同的值。最近红外光谱（IR）和毛细管电泳（CE）技术也有在确定变性温度中的应用。 吸附状态下蛋白质结构的检测 纳米颗粒 圆二色光谱 需要大量的重复实验取平均，文献中报道为32-128次 吸附状态下蛋白质结构的检测 平面 表面等离子体共振——测量反射光 石英微天平——晶体压电效应测量表面质量变化 双偏振极化干涉——测量干涉条纹，信息全 原子力显微镜——隧道效应 吸附状态下蛋白质结构的检测 介质颗粒 荧光 H-D交换结合核磁共振 H-D交换结合质谱 共聚焦拉曼光谱 仪器检测部分小结 注意事项 越灵敏的仪器影响因素越多——如原子力显微镜 多种手段互相验证，不仅检测不同级别的结构，二级结构也通常用红外和圆二色相互验证 对仪器不恰当的使用以及对原理的忽视往往带来很多错误的结果 目前的科研原来越依赖于先进的仪器，但人和思想是最重要的 * * * * * * * * * * * * * * * * * In any chromatographic step there are four main performance properties to adjust to reach a fully optimized procedure (see slide), resolution, speed, capacity and recovery. In general, optimization of any of these can only be realized at the expense of the others. The relative priority of these parameters will vary depending on whether a particular chromatographic step is for Capture, Intermediate Purification or Polishing. This will steer the optimization of the critical processing parameters in any particular step, as well as the selection of the most suitable chromatography matrix to be used in the step. * This application provides a good illustration of the 3-stage strategy for Downstream processing described in the previous slides, and a strong positioning statement for our media. Note that STREAMLINE is used for Capture, Sepharose Fast Flow and SOURCE 30 for Intermediate Purification and SOURCE 15 for Polishing. Gram quantities of inactivated Pseudomonas aeruginosa exotoxin A were needed for the preparation of several polysaccharide conjugated vaccines. The inactivated exotoxin A serves as the carrier protein moeity which is covalently connected to the polysaccharide. By forming a conjugate between a polysaccharide and the modified exotoxin A it is possible to increase the immunogenicity of the polysaccharide and to confer T-cell dependent properties (booster response) in infants and young children. It is thus possib