植物源重组人血清白蛋白的残留宿主杂质研究

发布时间：2018-01-18 02:33

本文关键词：植物源重组人血清白蛋白的残留宿主杂质研究　出处：《武汉大学》2014年博士论文　论文类型：学位论文

【摘要】：人血清白蛋白是血浆中含量最大的一种蛋白,含量达到40-50g/L,对维持体内渗透压具有重要的作用,此外它还有运输脂类、激素、药物等重要功能。白蛋白已被广泛应用于烧伤、肝腹水和低白蛋白症等的临床治疗,另外白蛋白还被用于药物的赋形剂,稳定剂,以及细胞培养的添加剂量。由于极其广泛的应用,人源血清白蛋白已不能满足现在的市场需求。实验室利用水稻种子这一新型的表达体系表达重组人血清白蛋白,并实现经济有效的大规模生产。临床应用中,通常人血清白蛋白的使用剂量为每人每次10-20g,因此,在利用水稻种子生产重组人血清白蛋白时,急需对其宿主杂质的安全性进行相关评估,并对其进行有效的质量控制。宿主杂质中,宿主蛋白和宿主DNA是最主要的两个宿主杂质,也是重组蛋白药质量控制中最为关注的要素。欧洲药品审评署(EMEA)明确指出,所有生物制品都需要对这两种宿主杂质进行详细的说明以确保最终产品的临床应用安全性。本论文的主要结果如下： 1.残留宿主DNA定量方法的建立：试验通过定量PCR的方法,以水稻5S核糖体基因为内参扩增序列,建立水稻宿主残留DNA定量方法。实验比较了两个常用的定量PCR体系,SYBRGreen Ⅰ荧光染料和Taqman探针法,在没有明显差异的情况下,基于成本考虑,选择SYBRGreen Ⅰ荧光染料为最终的定量体系。通过引物的选择,引物浓度的优化,最终建立的定量方法具有高度的线性相关性,相关系数R2达到0.9991,扩增效率接近100%,检测区间为2×104pg到0.2pg每个反应体系,最低定量限度为0.2pg每个反应体系。qPCR具有很好的重复性与特异性,回收实验的回收率在80%到122.2%之间,且标准样品的选择不会对结果有明显的影响。对一个重组人血清白蛋白典型样品的定量分析,最终样品的宿主残留DNA为每剂量(10g)含3.5ng,少于法规每剂量10ng的要求。 2.残留宿主蛋白定量方法的建立：利用水稻种子抗原制备抗体建立夹心ELISA定量检测方法,制备的抗体具有很好的覆盖性且不对目的蛋白白蛋白有交叉反应,实验比较了分别以HRP标记抗体为检测抗体和Biotin标记抗体为检测抗体的灵敏度,最后选择灵敏度较高的Biotin标记抗体为检测抗体的反应体系。实验主要通过包被抗体和检测抗体的浓度优化,建立的ELISA定量方法具有良好的非线性相关性,相关系数R2达到0.99,定量区间从25ng/ml到1.25ng/ml之间,最低定量限为1.25ng/ml。ELISA具有很好的重复性及精确度,定量结果的cv值小于11.5%,添加回收试验中宿主蛋白的回收率在86%到110.6%之间,样品的最终定量纯度达到99.9998%。 3.残留宿主蛋白的鉴定研究：为了避免目的产物人血清白蛋白对残留宿主蛋白鉴定的干扰,实验设计抗体免疫层析的方法去除目的蛋白-重组人血清白蛋白,并达到浓缩残留宿主蛋白的效果。实验通过人源血清白蛋白免疫兔子制备抗人血清白蛋白多克隆抗体,用制备的人血清白蛋白抗体与CNBr-activatived SepharoseTM4Fast Flow进行偶联,获得能够吸附约100mg人血清白蛋白的抗体免疫层析柱。利用该层析柱分离目的蛋白(重组人血清白蛋白)和残留宿主蛋白。实验建立的方法对99.8%样品的宿主蛋白回收率高于90%。收集的残留宿主蛋白经过label free NanoLC-ESI-MS/MS鉴定分析,结果显示,几乎所有的残留宿主蛋白都能被有效鉴定。 4.残留宿主蛋白反复给药的毒性研究：实验收集99.9%纯度产品中的白蛋白宿主蛋白,以1倍(0.2mg/kg)、5倍(1mg/kg)、25倍(5mg/kg)的剂量进行28天的反复静脉注射大鼠的毒性试验。整个试验过程中,大鼠没有出现样品相关的致命性毒性引起的死亡现象,主要的毒性表现为贫血症状以及脾功能亢进,初步推测与低渗性溶血或免疫性溶血有关。
[Abstract]:Human serum albumin is a protein content in the plasma of the largest content reached 40-50g/L, to maintain the osmotic pressure plays an important role, in addition it has important function in transport of lipids, hormones, drugs and so on. Albumin has been widely used in clinical treatment of burns, liver ascites and low serum albumin in addition, albumin is also used to pharmaceutical excipients, stabilizer, dosage and cell culture. Because of widespread application, human serum albumin has been unable to meet the current market demand. This new type of rice seed expression system of recombinant human serum albumin expression in the laboratory, and realize large-scale production effectively. The clinical application of human serum albumin, usually dose for each time 10-20g, therefore, in the use of rice seed production of recombinant human serum albumin, the safety need of its host of impurities The relevant assessment, and effectively control the quality of the host. Impurities, host proteins and host host DNA is the two main impurity, is the most concerned factor recombinant protein drug quality control. The European Medicines Evaluation Agency (EMEA) clearly pointed out that all biological products are required for the detailed explanation of this two host impurity to ensure the clinical safety of the final product. The main results of this thesis are as follows:
The establishment of 1. residual host DNA quantitative method: Test methods used by PCR, a rice 5S gene as internal ribosome sequences, establish DNA quantitative method of residue in rice host. The experimental comparison of two commonly used quantitative PCR system, SYBRGreen 1 and Taqman fluorescent probe method, no obvious differences in the situation, based on the cost consider, choose SYBRGreen I dye for quantitative system finally. Through the selection of the primers, optimized primer concentration, quantitative method established has a high linear correlation, the correlation coefficient reached 0.9991 R2, expanding the efficiency rate close to 100%, the detection range of 2 * 104pg to 0.2pg each reaction system, the minimum limit for each quantitative 0.2pg.QPCR reaction system has good repeatability and specificity, recovery experiments at the rate of 80% to 122.2%, and the standard sample selection does not have obvious influence on the results The quantitative analysis of a typical sample of recombinant human serum albumin shows that the final residual DNA of the sample is 10g with 3.5ng, which is less than the requirement of 10NG per regulation.
The establishment of 2. quantitative methods: residual host protein antibody sandwich ELISA quantitative detection method established by antigen preparation of rice seed, antibody preparation has good coverage and not objective protein albumin cross reaction, were compared respectively with HRP labeled antibody as detection antibody and Biotin labeled antibody for the sensitivity of antibody detection and response the final choice of Biotin system with high sensitivity for the detection of antibody labeled antibody. The experiment of coating concentration optimization of antibody and detection antibody, ELISA method has good linear correlation, the correlation coefficient R2 is 0.99, the quantitative range from 25ng/ml to 1.25ng/ml, the limit of quantitation was 1.25ng/ml.ELISA has good repeatability and the accuracy of quantitative results of CV value is less than 11.5%, the recovery recovery test of host proteins at the rate of 86% to 110.6% between the samples. The final quantitative purity is 99.9998%.
The 3. residue identification of host proteins: to avoid interference to the product of human serum albumin on residual host protein identification methods, experimental design of antibody immune chromatography removal protein recombinant human serum albumin, and achieve the concentrated residual host protein effect. Through the experiment of human serum albumin preparation of rabbit polyclonal anti human serum albumin antibody preparation of human serum albumin antibody and CNBr-activatived SepharoseTM4Fast Flow were coupled to obtain antibody immune chromatography column to 100mg adsorption of human serum albumin. The target protein is separated using the chromatography column (recombinant human serum albumin) and residual host protein. The developed method in 99.8% samples of host protein recovery rate is higher than the residual host after the label free NanoLC-ESI-MS/MS protein identification and analysis, 90%. collected results show that almost all of the residual The host protein can be effectively identified.
4. residual host protein repeatedly to drug toxicity studies: collected 99.9% albumin host protein purity in the product, with 1 times (0.2mg/kg), 5 times (1mg/kg), 25 times (5mg/kg) toxicity tests were repeated dose intravenous injection of rats for 28 days. During the experiment, the rats did not appear the phenomenon of death caused fatal toxicity related to the sample, the main toxicity showed symptoms of anemia and hypersplenism, speculated and hypotonic hemolysis or immune hemolytic.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R977.6;Q946.1

【共引文献】