治疗用抗人T淋巴细胞CD25单克隆抗体（WuTac）的工艺研究及其杂质鉴定

发布时间：2018-01-18 08:17

本文关键词：治疗用抗人T淋巴细胞CD25单克隆抗体（WuTac）的工艺研究及其杂质鉴定　出处：《武汉生物制品研究所》2010年硕士论文　论文类型：学位论文

【摘要】： 治疗用鼠抗人T淋巴细胞CD25单克隆抗体(WuTac),以下简称WuTac,是武汉生物制品研究所自主研发的用以预防临床肾移植排斥反应的单抗制品,目前已完成Ⅰ期临床研究,正在进行Ⅱ期临床研究。为了进一步提高制品的安全性和有效性,本实验论文对该单抗制品的中试生产工艺进行了优化和验证,并对纯化后的产品进行了杂质分析和鉴定。针对WuTac纯化工艺中的疏水层析步骤,以流动相为研究对象,对其盐种类、盐浓度和梯度层析条件进行摸索从而初步确定中试放大的疏水层析条件:吸附条件为1.0M氯化钠+20 mM PB(pH 7.4),流速为10 ml/min;洗脱条件为20 mM PB(pH7.4),20 ml/min。WuTac腹水经辛酸硫酸铵沉淀法联合疏水层析纯化后,采用SDS-PAGE法和HPLC法对纯化后的产品进行纯度鉴定:SDS-PAGE扫描纯度达到100%,HPLC单体含量达到100%。采用流式细胞术(FCM)对其效价进行鉴定,效价不小于1:10000。采用SDS-PAGE联合质谱法对疏水层析去除的杂蛋白(分子量介于130-170kD)进行分析,杂蛋白经质谱鉴定为小鼠血浆铜蓝蛋白。采用双向电泳法联合质谱法对疏水层析纯化后的产品进行杂质分析,质谱分析结果表明,双向电泳结果中除抗体重、轻链外的杂蛋白为小鼠的抗体片段。利用等电聚焦(IEF)测定其等电点(pI)为5.65～5.94。利用生物传感器(BIAcore 3000)测定WuTac疏水层析(HIC)前、后的亲和常数, KA分别为5.85×1010 L/mol和3.49×1011 L/mol。本论文初步确定了WuTac中试生产工艺中疏水层析步骤的条件。疏水层析法能够去除WuTac制品中的二聚体、多聚体以及小鼠血浆铜蓝蛋白。经疏水层析精纯后的WuTac制品纯度高,除小鼠抗体片段外无其它鼠源性杂蛋白。为制定和完善该产品的质量控制体系提供了实验依据。
[Abstract]:CD25 monoclonal antibody against human T lymphocytes (WuTac) was used for treatment. Wuhan Institute of Biological products (Wuhan Institute of Biological products) has developed a monoclonal antibody to prevent renal allograft rejection. At present, we have completed phase I clinical research. In order to further improve the safety and effectiveness of the product, the pilot-scale production process of the McAb was optimized and verified in this paper. The impurity of the purified product was analyzed and identified. According to the hydrophobic chromatography in the purification process of WuTac, the mobile phase was used as the research object, and the salt species were analyzed. Salt concentration and gradient chromatography conditions were explored to determine the hydrophobic chromatography conditions of the pilot scale up: the adsorption condition was 1.0m NaCl 20mm PB(pH 7.4). The flow rate was 10 ml / min; The elution conditions were as follows: (1) the pH value of 20 mm PBL was 7. 4% and 20 ml/min.WuTac. The ascites were purified by ammonium octanoate sulfate precipitation and hydrophobic chromatography. The purity of purified product was identified by SDS-PAGE and HPLC. The scanning purity of the purified product was 100%. The content of HPLC monomer was 100%. Flow cytometry was used to identify its titer. The titer was not less than 1: 10000.The hetero proteins (molecular weight ranged from 130 to 170 KD) removed by hydrophobic chromatography were analyzed by SDS-PAGE combined with mass spectrometry. The hybrid protein was identified as mouse ceruloplasmin by mass spectrometry. The impurity of the purified product was analyzed by two-dimensional electrophoresis and mass spectrometry. The result of mass spectrometry showed that anti-body weight was removed from the result of two-dimensional electrophoresis. The heterologous protein outside the light chain is the antibody fragment of mice. The isoelectric point (Pi) determined by isoelectric focusing (IEF) is 5.65 ~ 5.94. BiAcore 3000 is used as biosensor. Before the determination of WuTac hydrophobic chromatography. The subsequent affinity constants were 5.85 脳 10 10 L / mol and 3.49 脳 10 11 L / mol, respectively. In this paper, the conditions of hydrophobic chromatography in the pilot-scale production of WuTac were determined. Hydrophobic chromatography can remove dimer from WuTac products. Polymers and mouse plasma ceruloplasmin. Purified by hydrophobic chromatography, WuTac products were of high purity. There were no other murine hybrids except mouse antibody fragments, which provided experimental basis for establishing and perfecting the quality control system of this product.
【学位授予单位】：武汉生物制品研究所
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【相似文献】