因子类血液制品中蛋白质含量对效价检测结果影响的研究
发布时间：2019-05-30 03:38
【摘要】：人凝血因子Ⅷ、人凝血酶原复合物、人凝血因子Ⅸ等因子类血液制品,是以健康人血浆为原料,经分离纯化和病毒灭活制成,在临床上主要用于各种凝血因子缺乏的治疗,这些因子类血液制品的凝血因子效价是药品有效性的指标,凝血因子效价的测定也是药品质量检测中的关键检测项目。血液制品生产企业及各级检测机构一般采用全自动血凝仪并按照仪器使用说明来进行因子效价的检测,然而在不同检测方法结果比对中,发现全自动血凝仪检测结果与中国药典中一期法的检测结果在部分因子效价检测中存在检测差异,差异可能是样品稀释中使用的稀释液的蛋白含量不同引起的,故本课题主要研究因子类血液制品中蛋白质含量对效价检测结果影响。因子类血液制品是血液中凝血因子的高纯度浓缩物,在进行效价测定时需要对样品进行稀释,而稀释液中蛋白含量的不同对其检测结果有着影响,国内因子类血液制品的生产企业极少,对因子产品效价检测方法的研究也很少,并主要对因子效价检测的操作方式、实验条件、辅料影响等进行了研究,本课题以寻找实际工作中不同检测方法间结果差异的原因为出发点,确立了不同因子类产品的自身性质对效价检测结果的影响以及不同种类稀释液中蛋白含量对效价检测结果的影响,最后通过方法学验证确立了适用于因子效价检测的稀释方法,从而完善了因子类血液制品效价检测的方法。全自动血凝仪主要设计用于临床血浆样本的检测,故在建立因子类血液制品的全自动血凝仪法效价检测时,需要消除产品本身性质对检测结果的影响,并对建立的检测方法进行方法学验证。本课题通过蛋白含量对因子效价检测影响的研究建立起的全自动血凝仪因子效价检测方法,消除了产品本身性质的影响,可以准确进行因子效价的测定,方法的建立不但可以准确控制因子类血液制品的质量,保证生产企业持续稳定生产出质量均一的药品,而且可以减少因子类产品在临床使用中的风险。本课题从工作过程中检测结果的差异入手,对蛋白质含量在因子效价检测中的影响进行了研究,并以国外同类产品进行检测比对,最后确立了全自动血凝仪因子效价检测的方法并对方法进行了验证。具体研究内容如下：(1)对同一批人凝血因子Ⅷ、人凝血酶原复合物样品选用不同的稀释液进行全自动血凝仪法的效价检测,以确定不同稀释液稀释检测结果的差别,并对连续生产的人凝血因子Ⅷ、人凝血酶原复合物进行不同稀释液的效价检测,确定不同稀释液稀释检测结果的差别。(2)对同一批人凝血因子Ⅷ、人凝血酶原复合物用含不同蛋白质浓度的稀释液或凝血因子缺乏血浆进行稀释,确定蛋白质浓度对因子效价检测的影响,并对2批国外生产的同类产品进行同样的试验,确定出适用于因子效价检测的稀释方法。(3)建立起人凝血因子Ⅷ、人凝血酶原复合物的全自动血凝仪因子效价检测方法并进行方法验证,选择适用于因子效价检测的稀释方法和稀释液,对方法的线性关系、准确度、精密度、日间精密度、专属性等进行了方法学验证,确认所建立方法适用于因子类血液制品效价的检测。
[Abstract]:human blood coagulation factor VIII, human prothrombin complex, human blood coagulation factor IX and other factor-like blood product are prepared from human plasma as raw material, separated and purified and virus-inactivated, and is mainly used for treating various blood coagulation factor deficiency in clinic, The coagulation factor titer of these factor-based blood products is an index of the effectiveness of the drug, and the determination of the potency of the blood coagulation factor is also a key detection item in the quality detection of the drug. The blood product production enterprise and the detection mechanism at all levels generally adopt a full-automatic blood coagulation instrument and carry out the detection of the factor titer according to the instructions of the instrument, It is found that the detection result of the full-automatic blood-coagulation instrument and the detection result of the first-phase method in the Chinese pharmacopoeia are different from the detection result of the first-phase method in the Chinese pharmacopoeia, and the difference can be caused by the difference in the protein content of the diluent used in the sample dilution, Therefore, the content of protein in the main research factor blood product of the subject is affected by the result of the potency test. The factor-based blood product is a high-purity concentrate of the blood coagulation factor in the blood, The research on the method for detecting the potency of the factor product is very small, and the operation mode, the experimental conditions and the influence of the auxiliary materials of the factor potency test are also studied. The main cause of the difference between the different detection methods in the actual work is the starting point. The effect of the self-nature of the different factor products on the potency test result and the effect of the protein content on the titer detection result in different kinds of diluent are established, and finally, the dilution method applicable to the detection of the factor titer is established through the method verification, So as to improve the method for detecting the potency of the factor-like blood product. The full-automatic blood-coagulation instrument is mainly designed for the detection of clinical plasma samples, so that the effect of the nature of the product on the detection result needs to be eliminated when the full-automatic blood-coagulation instrument of the factor-based blood product is established, and the established detection method is validated. The method for detecting the potency of the full-automatic blood-coagulation instrument established by the research on the influence of the protein content on the titer detection of the factor eliminates the influence of the properties of the product, and can accurately carry out the measurement of the factor titer, The establishment of the method not only can accurately control the quality of the factor-based blood products, ensure the continuous and stable production of the drugs with uniform quality, but also can reduce the risk of the factor products in clinical use. In this paper, the influence of the protein content in the detection of the factor titer was studied by the difference of the detection results in the working process, and the method for detecting the potency of the full-automatic blood-coagulation instrument was established and the method was validated. The specific research contents are as follows: (1) the same batch of human blood coagulation factor VIII and human prothrombin complex samples are selected to carry out the full-automatic blood-coagulation instrument titer detection to determine the difference of the dilution detection result of different dilutions, and the human blood coagulation factor VIII is continuously produced, The value of different dilutions of human prothrombin complex was tested to determine the difference in the dilution test results for different dilutions. (2) the same batch of human blood coagulation factor VIII and human prothrombin complex are diluted with a diluent or a blood coagulation factor containing different protein concentration to determine the influence of the protein concentration on the detection of the factor titer, and the same test is carried out on the similar products produced in the two batches, A dilution method suitable for the detection of a factor titer is determined. and (3) establishing a full-automatic blood coagulation instrument factor titer detection method for the human blood coagulation factor VIII and the human prothrombin complex and carrying out a method verification, selecting a dilution method and a diluent suitable for the detection of the factor titer, the linear relationship, the accuracy, the precision and the day precision of the method, The specificity and the like are verified by the methodology, and the established method is applicable to the detection of the potency of a factor-based blood product.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R977.8
[Abstract]:human blood coagulation factor VIII, human prothrombin complex, human blood coagulation factor IX and other factor-like blood product are prepared from human plasma as raw material, separated and purified and virus-inactivated, and is mainly used for treating various blood coagulation factor deficiency in clinic, The coagulation factor titer of these factor-based blood products is an index of the effectiveness of the drug, and the determination of the potency of the blood coagulation factor is also a key detection item in the quality detection of the drug. The blood product production enterprise and the detection mechanism at all levels generally adopt a full-automatic blood coagulation instrument and carry out the detection of the factor titer according to the instructions of the instrument, It is found that the detection result of the full-automatic blood-coagulation instrument and the detection result of the first-phase method in the Chinese pharmacopoeia are different from the detection result of the first-phase method in the Chinese pharmacopoeia, and the difference can be caused by the difference in the protein content of the diluent used in the sample dilution, Therefore, the content of protein in the main research factor blood product of the subject is affected by the result of the potency test. The factor-based blood product is a high-purity concentrate of the blood coagulation factor in the blood, The research on the method for detecting the potency of the factor product is very small, and the operation mode, the experimental conditions and the influence of the auxiliary materials of the factor potency test are also studied. The main cause of the difference between the different detection methods in the actual work is the starting point. The effect of the self-nature of the different factor products on the potency test result and the effect of the protein content on the titer detection result in different kinds of diluent are established, and finally, the dilution method applicable to the detection of the factor titer is established through the method verification, So as to improve the method for detecting the potency of the factor-like blood product. The full-automatic blood-coagulation instrument is mainly designed for the detection of clinical plasma samples, so that the effect of the nature of the product on the detection result needs to be eliminated when the full-automatic blood-coagulation instrument of the factor-based blood product is established, and the established detection method is validated. The method for detecting the potency of the full-automatic blood-coagulation instrument established by the research on the influence of the protein content on the titer detection of the factor eliminates the influence of the properties of the product, and can accurately carry out the measurement of the factor titer, The establishment of the method not only can accurately control the quality of the factor-based blood products, ensure the continuous and stable production of the drugs with uniform quality, but also can reduce the risk of the factor products in clinical use. In this paper, the influence of the protein content in the detection of the factor titer was studied by the difference of the detection results in the working process, and the method for detecting the potency of the full-automatic blood-coagulation instrument was established and the method was validated. The specific research contents are as follows: (1) the same batch of human blood coagulation factor VIII and human prothrombin complex samples are selected to carry out the full-automatic blood-coagulation instrument titer detection to determine the difference of the dilution detection result of different dilutions, and the human blood coagulation factor VIII is continuously produced, The value of different dilutions of human prothrombin complex was tested to determine the difference in the dilution test results for different dilutions. (2) the same batch of human blood coagulation factor VIII and human prothrombin complex are diluted with a diluent or a blood coagulation factor containing different protein concentration to determine the influence of the protein concentration on the detection of the factor titer, and the same test is carried out on the similar products produced in the two batches, A dilution method suitable for the detection of a factor titer is determined. and (3) establishing a full-automatic blood coagulation instrument factor titer detection method for the human blood coagulation factor VIII and the human prothrombin complex and carrying out a method verification, selecting a dilution method and a diluent suitable for the detection of the factor titer, the linear relationship, the accuracy, the precision and the day precision of the method, The specificity and the like are verified by the methodology, and the established method is applicable to the detection of the potency of a factor-based blood product.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R977.8
【参考文献】
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1 李青;刘晓;张t，
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