血清抗体提取工艺及抗体冻干工艺的研究

发布时间：2018-10-19 13:14

【摘要】：抗体（Antibody）又称免疫球蛋白，存在于人和动物血液（血清）、组织液及其他外分泌液中。抗体是机体对抗原物质产生免疫应答的重要物质，具有各种免疫功能，在疾病爆发时可通过被动免疫方式用于紧急治疗。但是目前为止提取抗体的生产工艺依然是传统的硫酸铵盐析法，此方法由于所提抗体得率低、提取过程繁琐致使抗体价格昂贵而得不到广泛应用。因此寻找新的提取工艺代替传统盐析法来大量提取抗体、降低生产成本是解决抗体价格昂贵的根本方法。为寻找适合提取血清中抗体的新工艺，，本研究以双水相系统为对象，筛选并优化出适合提取血清中抗体的双水相系统。对双水相系统提取血清中抗体的得率、纯度及扩大后的稳定行进行检测。通过对比传统工艺分析双水相系统的优越性，探讨双水相系统的应用价值。最后对提取的抗体进行冻干保存。根据抗体的特性筛选出适合抗体液冻干的最佳保护剂，将提取的抗体进行冻干保存。方便了抗体的运输，保证了抗体的长期存放。本研究主要包括以下几个方面： 1.萃取血清抗体的双水相系统建立与优化。首先对双水相系统的组成成分进行筛选，得出最佳聚合物和亲液盐种类。然后优化双水相系统萃取条件，主要包括盐水相浓度及中性盐的浓度、pH大小等条件的优化。得出双水相系统的组成为50%（w/w）PEG6000、20%（w/w）硫酸铵、pH=7.0、加入5%（w/w）氯化钠后效果更佳。对双水相系统与血清添加比例进行筛选。得出双水相系统与血清的最佳比例为聚合物水相体积：盐水相体积：血清体积=3：3：1。 2.利用双水相系统工业化生产血清中抗体。通过扩大500倍(500mL)、10000(10000mL)倍，所提抗体得率及纯度均不变，工艺稳定性好。本研究还从生产成本、人力、物力、生产周期、所提抗体纯度及得率等方面与目前用于工业生产的硫酸铵盐析法进行对比分析。此工艺可提高得率近1倍，且工艺简单、成本低、节省时间、人力和物力。 3.抗体冻干条件的筛选。本研究对不同冻干保护剂以及保护剂的浓度进行筛选，确定适合保护抗体液冻干的保护剂为蔗糖。为确保冻干后抗体的物理性状良好且效价无降低，在反复筛选后得出抗体最佳冻干保护剂为5%（w/w）的蔗糖。根据筛选的冻干曲线加入适当浓度保护剂冻干后抗体常温放置一年后效价无影响。双水相系统的研究为血清中抗体的提取提供新工艺，为替换传统工艺提供依据。抗体冻干工艺的研究为抗体的保存与运输提供方法。也为今后抗体的广泛应用提供条件。
[Abstract]:Antibody (Antibody), also called immunoglobulin, is present in human and animal blood (serum), tissue fluid and other exocrine fluids. Antibody is an important substance that produces immune response to antigens. It has various immune functions and can be used in emergency treatment by passive immunization when the disease breaks out. However, the traditional ammonium sulfate salting-out method is still used in the production of the antibody, which is not widely used because of the low yield of the antibody and the complicated extraction process, which results in the high price of the antibody. Therefore, to find a new extraction process instead of the traditional salt-out method to extract a large number of antibodies and reduce the production cost is the fundamental method to solve the high price of antibodies. In order to find a new technology for extracting antibody from serum, the two-phase system was selected and optimized. The extraction rate, purity and expanded stability of antibody from serum were determined by aqueous two-phase system. By comparing the advantages of two-phase water system with traditional technology, the application value of two-phase water system is discussed. Finally, the extracted antibody was freeze-dried and preserved. According to the characteristics of antibody, the best protective agent for freeze-drying of antibody solution was selected, and the extracted antibody was freeze-dried and preserved. It facilitates the transport of antibodies and ensures the long-term storage of antibodies. This research mainly includes the following aspects: 1. Establishment and Optimization of Aqueous Two-phase system for extraction of Serum Antibodies. Firstly, the composition of aqueous two-phase system was screened, and the best polymer and hydrophilic salt were obtained. Then the extraction conditions of aqueous two-phase system were optimized, including the concentration of brine phase and neutral salt, the size of pH and so on. The results show that the composition of aqueous two-phase system is 50% (w / w) PEG6000,20% (w / w) ammonium sulfate, and the effect of adding 5% (w / w) sodium chloride to pH=7.0, is better. The ratio of two-phase water system to serum was screened. The results show that the optimum ratio of aqueous phase system to serum is polymer aqueous phase volume: brine phase volume: serum volume = 3: 3: 1.2. The antibody in serum was produced by using two-phase aqueous system. By expanding 500-fold (500mL) and 10000 (10000mL) times, the yield and purity of the antibody were unchanged, and the process stability was good. In this study, the production cost, manpower, material resources, production cycle, purity and yield of antibody were compared with ammonium sulfate salting-out method used in industrial production. This process can increase the yield by nearly 1 times, and the process is simple, low cost, save time, manpower and material resources. 3. Screening of freeze-drying conditions for antibodies. In this study, sucrose was chosen as the protective agent suitable for freeze-drying of protective antibody by screening different concentration of freeze-drying protectant and protective agent. In order to ensure that the physical properties of freeze-dried antibodies were good and their titers were not decreased, the optimum freeze-dried sucrose with 5% (w / w) sucrose was obtained after repeated screening. According to the selected freeze-drying curve, the titer of antibody was not affected after a year's storage at room temperature. The study of aqueous two-phase system provides a new process for the extraction of antibodies in serum and a basis for replacing the traditional process. The study of antibody freeze-drying technology provides a method for the preservation and transportation of antibody. It also provides conditions for the wide application of antibodies in the future.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.5

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