注射用rhGH蛋白含量测定和HCP检测方法学建立及应用

发布时间：2018-05-04 19:41

本文选题：重组人生长激素 + 宿主菌蛋白残留量　；参考：《长春理工大学》2017年硕士论文

【摘要】：生长激素(Human Growth Hormone,hGH)是一种肽类激素,其主要生理作用是促进机体蛋白质的合成。但这种天然生长激素来源于垂体提取,价格昂贵,也不易获得,限制了其临床应用。重组人生长激素(Recombinant Human Growth Hormone,rhGH)是以大肠杆菌表达的细胞因子类生物重组制品。在纯化过程中,大肠杆菌的宿主蛋白(Host Cell Protein,HCP)残留是衡量重组制品纯度的关键问题。精细纯化过程能够有效去除HCP残留量,但在纯化过程中能够精准定量HCP残留量的检测方法成为了目前企业和学者们的瓶颈问题。本文旨在建立rhGH原液中HCP残留量及蛋白含量检测方法并进行方法学验证。并将HCP残留量与蛋白含量两种检测方法相结合,共同应用于rhGH原液纯化工艺生产中,并对每一步纯化工艺进行HCP残留量质控。检测HCP残留量的方法学建立主要通过比较直线回归拟合与三次样条曲线拟合分析方法,对比是否采用封闭液封闭,对比四种稀释液检测效果,利用正交试验设计对实验条件进行多因素筛选,对重建的HCP残留量检测方法进行方法学验证;采用药典方法对rhGH原液蛋白含量进行检测,并对检测方法进行方法学验证;按照建立的两种方法检测rhGH原液原有纯化工艺每一步纯化产物的HCP残留量及蛋白含量,根据检测结果进行指导并优化纯化工艺,以每毫克rhGH原液中含有HCP残留量的浓度作为标准,在新旧纯化工艺生产中,比较HCP残留量改善情况。比较结果显示,选取三次样条曲线拟合对数据进行分析,不采用封闭液封闭,选用pH 6.0柠檬酸缓冲液作为样品稀释液,正交试验设计筛选结果显示,最佳实验条件为24℃、600rpm/min、孵育170min,经过方法学验证后,各验证项目均符合相关规定;rhGH原液蛋白含量方法学验证结果显示,各检验项目均符合相关规定;优化后的纯化工艺能够使HCP残留量由优化前的13-16ng/mg减低到2ng/mg,使纯化产物符合药典要求(10ng/mg)。本文成功建立了重组人生长激素原液中HCP残留量及蛋白含量的检测方法,并对两种检测方法进行了方法学验证。对每一步纯化环节,控制重组人生长激素原液含有HCP残留量具有重要的指导意义,为重组人生长激素的生产和研究奠定了基础。
[Abstract]:Human Growth Hormone (hGH) is a peptide hormone and its main physiological role is to promote the synthesis of protein in the body. But this natural growth hormone is extracted from the pituitary, is expensive, and is not easy to obtain. It restricts its clinical application. Recombinant human growth hormone (Recombinant Human Growth Hormone, rhGH) is a form of Escherichia coli. In the purification process, the Host Cell Protein (HCP) residue is the key problem to measure the purity of the recombinant products. The fine purification process can effectively remove the HCP residue, but in the purification process, the detection method of the precision calibration HCP residue has become the enterprise and the current enterprise. The purpose of this paper is to establish the method of detecting the residual and protein content of HCP in the rhGH original solution and to verify the method, and combine the two detection methods of HCP residue and protein content, and jointly apply it to the production of rhGH original liquid purification process, and carry out the quality control of HCP residue in each step of purification process and detect the residual quantity of HCP. The method is mainly through comparing the linear regression fitting and the three spline curve fitting analysis method, comparing whether the sealing liquid is closed, compared with the four kinds of diluent detection effect, the orthogonal experiment design is used to screen the experimental conditions by multiple factors. The method of the rebuilt HCP residue detection method is verified by the method, and the Pharmacopoeia method is adopted. The content of protein in rhGH liquid was detected and the method was verified by methodology. According to the two methods established, the HCP residue and protein content of the purified product of rhGH original purification process were detected, and the purification process was optimized according to the test results. The concentration of HCP residues in the original rhGH liquid per milligram was made. For the standard, in the production of the new and old purification process, the improvement of HCP residue was compared. The results showed that the data were analyzed by three spline curve fitting, no closed liquid was used and pH 6 citric acid buffer solution was selected as the sample diluent. The orthogonal test design screening results showed that the optimum experimental conditions were 24, 600rpm/min, and incubated. 170min, after the validation of the methodology, all the verification items were in accordance with the relevant regulations. The results of the rhGH solution test showed that all the test items were in conformity with the relevant regulations; the optimized purification process could reduce the residual amount of HCP from the optimized 13-16ng/mg to the 2ng/mg, so that the purified product was in conformity with the Pharmacopoeia requirements (10ng/mg). The method of detection of HCP residue and protein content in recombinant human growth hormone original solution was established, and the method of two detection methods was verified. It has important guiding significance for each step of purifying the recombinant human growth hormone original solution containing HCP residue, which lays a foundation for the production and research of recombinant human growth hormone.

【学位授予单位】：长春理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392-33

【参考文献】