本文選題：重組人生長激素 + 宿主菌蛋白殘留量　； 參考：《長春理工大學》2017年碩士論文

【摘要】：生長激素(Human Growth Hormone,hGH)是一種肽類激素,其主要生理作用是促進機體蛋白質(zhì)的合成。但這種天然生長激素來源于垂體提取,價格昂貴,也不易獲得,限制了其臨床應用。重組人生長激素(Recombinant Human Growth Hormone,rhGH)是以大腸桿菌表達的細胞因子類生物重組制品。在純化過程中,大腸桿菌的宿主蛋白(Host Cell Protein,HCP)殘留是衡量重組制品純度的關(guān)鍵問題。精細純化過程能夠有效去除HCP殘留量,但在純化過程中能夠精準定量HCP殘留量的檢測方法成為了目前企業(yè)和學者們的瓶頸問題。本文旨在建立rhGH原液中HCP殘留量及蛋白含量檢測方法并進行方法學驗證。并將HCP殘留量與蛋白含量兩種檢測方法相結(jié)合,共同應用于rhGH原液純化工藝生產(chǎn)中,并對每一步純化工藝進行HCP殘留量質(zhì)控。檢測HCP殘留量的方法學建立主要通過比較直線回歸擬合與三次樣條曲線擬合分析方法,對比是否采用封閉液封閉,對比四種稀釋液檢測效果,利用正交試驗設計對實驗條件進行多因素篩選,對重建的HCP殘留量檢測方法進行方法學驗證;采用藥典方法對rhGH原液蛋白含量進行檢測,并對檢測方法進行方法學驗證;按照建立的兩種方法檢測rhGH原液原有純化工藝每一步純化產(chǎn)物的HCP殘留量及蛋白含量,根據(jù)檢測結(jié)果進行指導并優(yōu)化純化工藝,以每毫克rhGH原液中含有HCP殘留量的濃度作為標準,在新舊純化工藝生產(chǎn)中,比較HCP殘留量改善情況。比較結(jié)果顯示,選取三次樣條曲線擬合對數(shù)據(jù)進行分析,不采用封閉液封閉,選用pH 6.0檸檬酸緩沖液作為樣品稀釋液,正交試驗設計篩選結(jié)果顯示,最佳實驗條件為24℃、600rpm/min、孵育170min,經(jīng)過方法學驗證后,各驗證項目均符合相關(guān)規(guī)定;rhGH原液蛋白含量方法學驗證結(jié)果顯示,各檢驗項目均符合相關(guān)規(guī)定;優(yōu)化后的純化工藝能夠使HCP殘留量由優(yōu)化前的13-16ng/mg減低到2ng/mg,使純化產(chǎn)物符合藥典要求(10ng/mg)。本文成功建立了重組人生長激素原液中HCP殘留量及蛋白含量的檢測方法,并對兩種檢測方法進行了方法學驗證。對每一步純化環(huán)節(jié),控制重組人生長激素原液含有HCP殘留量具有重要的指導意義,為重組人生長激素的生產(chǎn)和研究奠定了基礎。
[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.

【學位授予單位】：長春理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R392-33

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