人血白蛋白制剂中蛋白组分的研究

发布时间：2018-06-02 11:42

本文选题：人血白蛋白 + 分离纯化　；参考：《湖北中医药大学》2017年硕士论文

【摘要】：人血白蛋白(Human serum albumin,HSA)是由健康人的血浆经低温乙醇分离提取并经病毒灭活后制成的生物制品,临床上用于出血性、外伤性体克、烧伤、肝硬化伴腹水和水肿、恶性肿瘤、肾病水肿等。目前国内企业采用低温乙醇法压滤工艺分离血浆蛋白,该工艺具有蛋白收率高,生产成本低,经济效益好,因而在工业化生产中大规模应用。然而低温乙醇法特异性不高,在生产的过程中不能完全除去杂蛋白成分,各企业的工艺参数也不尽相同,因此在终产品中会存在一定量的杂蛋白。据文献表明,制剂中存在的这些杂蛋白是引起的临床不良反应的主要原因之一[1],不良反应类型主要为全身过敏反应和多器官损坏,包括过敏性休克、热原样反应、肾脏损害等。因此探究血液制品中的未知蛋白可为临床不良反应提供参考,本课题旨在建立杂蛋白分离分析的方法并测定人血白蛋白制剂中的杂蛋白的组成,为各企业的生产工艺优化提供了数据支持,同时为产品的一致性评价奠定基础。为了初步了解各企业的杂蛋白含量和种类的差异,对五家企业近两年的批签发数据进行统计分析,共获得323批白蛋白制剂的纯度数据。结果显示,五家企业生产的白蛋白制剂纯度范围在96.2%~98.3%之间,企业间差异较大。其中A企业生产的制剂纯度较为均一,提示生产工艺较为稳定;B企业生产的制剂纯度较其他四所企业低,显示产品中存在一定量的未知蛋白。为了进一步分析各企业制剂的蛋白组成异同,对白蛋白制剂进行聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,初步探究其组成成分的异同,结果显示各企业间生产的白蛋白制剂差异较小,各企业制剂的杂蛋白有8~9条带。鉴于醋酸纤维素薄膜电泳与SDS-PAGE方法的局限性,不能特异性地鉴定出杂蛋白成分,本研究尝试建立未知蛋白谱鉴定方法,进而确定各企业生产的人血白蛋白制剂的未知蛋白组成。在建立质谱分析方法时,首先对供试品浓度进行选择,再对质谱鉴定参数进行优化。结果显示,白蛋白浓度超过5mg/ml时,超过仪器鉴定阈值,导致白蛋白主成分未能被成功鉴定,提示蛋白浓度不能过高。蛋白浓度在1mg/ml-2mg/ml时,主成分白蛋白肽图较为完整,但未知蛋白检出效率低;对白蛋白肽图中的肽段进行b/y离子连续性分析发现,蛋白鉴定得分大于150分,结果具有较高的可信度。以上结果提示主成分蛋白浓度过高,掩盖未知蛋白的的信号,未知蛋白的鉴定需要对未知蛋白进一步分离。结合前期结果,对各企业的白蛋白中未知成分进行分离纯化、酶切方法的优化,确立其分离纯化方法,并分析各企业未知蛋白的种类、数量、分子量、等电点分布及其工艺相关性。采用阴离子交换柱与凝胶过滤层析柱分别对白蛋白进行分离,结果显示,使用阴离子交换层析只能得到主成分单一峰,而使用凝胶过滤层析得三组峰,因此采用凝胶过滤层析对白蛋白中位置组分进行分离。采用胃蛋白酶和胰蛋白酶分别对各分离组分进行酶切,结果显示,胃蛋白酶酶切结果重复性较差且蛋白得分较低,而使用胰蛋白酶重现性良好,蛋白鉴定得分较高。对胰蛋白酶酶切效果进行进一步确证,结果发现,三次平行实验,共有11个蛋白被检出,其中共有8个蛋白每次均检出,说明方法稳定性较好。最终采用胰蛋白酶对人血白蛋白中的未知蛋白成分进行鉴定,结果显示五家生产企业产品,除一家企业杂蛋白较少外,另外四家企业所有批次样品均含有载脂蛋白a-Ⅱ(apolipoproteina-ii)、人结合珠蛋白(haptoglobin)、人血色素结合蛋白(hempoexin)、α-1b-糖蛋白(alpha-1b-glycoprotein)、α-2-hs-糖蛋白(alpha-2hs-glycoprotein),且略有差异。对未知蛋白等电点进行分析发现,未知蛋白等电点与白蛋白较接近,可能在乙醇共沉淀时与白蛋白共同析出,初步可以说明未知蛋白为工艺相关蛋白;对未知蛋白分子量进行分析发现,未知蛋白以多聚体的形式存在于白蛋白中。以上结果表明,在临床应用中,应更加关注上述未知蛋白的过敏效应,在工业生产中,应优化生产工艺,尽可能去除无关蛋白,提高产品质量。本课题建立的白蛋白制剂杂蛋白质谱分析方法为探究临床不良反应提供了数据支持,为白蛋白制剂的一致性评价提供了新的方法,为血液制品的质量控制奠定了基础、提供了新思路。
[Abstract]:Human Albumin (Human serum albumin, HSA) is a biological product made from the plasma of healthy people and extracted by low temperature ethanol and inactivated by virus. It is used clinically for bleeding, traumatic body grams, burns, cirrhosis with ascites and edema, malignant tumor, nephrosis edema and so on. At present, the domestic enterprises adopt the low temperature ethanol filtration process to separate blood Plasma protein, which has high protein yield, low production cost and good economic benefit, is widely used in industrial production. However, the specificity of the low temperature ethanol method is not high. In the process of production, the protein components can not be completely removed, and the technological parameters of each enterprise are not the same. Therefore, there will be a certain amount of protein in the final products. It is shown in the literature that these proteins are one of the main causes of adverse reactions in the preparation of [1], and the types of adverse reactions are mainly systemic anaphylaxis and multiple organ damage, including anaphylactic shock, thermal response, kidney damage, etc. Therefore, the exploration of unknown proteins in the blood can provide a reference for adverse clinical reactions. The purpose of this study is to establish a method of separation and analysis of hetero protein and determine the composition of the heteroprotein in Human Albumin preparation. It provides data support for the optimization of the production process of various enterprises, and lays the foundation for the evaluation of the consistency of the products. In order to understand the differences in the content and types of the egg white in each enterprise, five enterprises have been used for the last two years. A total of 323 batch of albumin preparations were obtained by statistical analysis of the batch data. The results showed that the purity of the albumin preparation produced by the five enterprises was between 96.2%~98.3%, and the difference between enterprises was large. The purity of the preparation produced by the A enterprise was more uniform, indicating that the production process was more stable; the purity of the preparation produced by the B enterprise was more than that of it. In order to further analyze the protein composition and similarities and differences of various enterprise preparations, SDS-PAGE analysis of protein preparation was carried out to explore the similarities and differences of the composition of the components. The results showed that the differences in albumin preparation produced between enterprises were small, and the enterprises were small in different enterprises. The heteroproteins of the preparation have 8~9 bands. In view of the limitations of cellulose acetate membrane electrophoresis and the SDS-PAGE method, the hetero protein components can not be identified specifically. This study attempts to establish an unknown protein spectrum identification method to determine the unknown egg white composition of the Human Albumin preparation produced by the enterprises. The concentration of the sample was selected and the parameters of the mass spectrometry were optimized. The results showed that when the concentration of albumin exceeded 5mg/ml, the concentration of albumin could not be successfully identified and the protein concentration could not be successfully identified. The protein concentration was not too high. When the protein concentration was at 1mg/ml-2mg/ml, the protein peptide map of the principal component was more complete, but the unknown protein was detected. The b/y ion continuity analysis in the peptide map of the protein peptide map showed that the protein identification score was more than 150 points, and the results had high reliability. The above results suggested that the concentration of the principal component protein was too high to cover the signal of the unknown protein. The identification of the unknown protein should be further separated from the unknown protein. The separation and purification of the unknown components in the albumin of the enterprises, the optimization of the enzyme cutting method, the separation and purification method, and the analysis of the species, quantity, molecular weight, the distribution of the electric point and the correlation of the process of the unknown proteins of the enterprises were analyzed. The separation of white protein was carried out by the anion exchange column and the gel filtration column. The results showed that the use of the protein was used to separate the protein. The anion exchange chromatography can only get the single peak of the principal component, and the three groups of peaks are obtained by gel filtration chromatography. Therefore, the gel filtration chromatography is used to separate the position components in the white protein. The protease and trypsin are used to cut the separate components respectively. The results show that the results of the pepsin digestion are poor and the protein score is poor. It was low, and the trypsin reproducibility was good and the protein identification score was higher. The results of trypsin digestion were further confirmed. The results showed that 11 proteins were detected in three parallel experiments, of which 8 proteins were detected each time, indicating that the method was more stable with trypsin in the unknown Human Albumin. The protein components were identified, and the results showed that five production enterprise products, except one enterprise with less clutter, all the other four enterprise batch samples contain apolipoprotein a- II (apolipoproteina-ii), human globin (haptoglobin), human hemoglobin binding protein (hempoexin), alpha -1b- glycoprotein (alpha-1b-glycoprotein), and alpha -2-hs- sugar. Protein (alpha-2hs-glycoprotein), and slightly different. The analysis of the unknown protein and other electrical points found that the unknown protein and other electrical points are closer to the albumin, may precipitate together with the albumin when the ethanol is coprecipitation, and the unknown protein is a process related protein; the unknown protein molecular weight is found to be more than the unknown protein. The form of polymer exists in albumin. The above results show that in clinical application, we should pay more attention to the allergy effect of the above unknown protein. In industrial production, the production process should be optimized and the unrelated proteins should be removed as much as possible to improve the quality of the products. It provides data support, provides a new method for the consistency evaluation of albumin preparation, and lays a foundation for the quality control of blood products, and provides a new idea.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R927.1

【参考文献】