本文選題：質(zhì)量標(biāo)準(zhǔn) + dTMP-GH　； 參考：《第三軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：血小板減少癥在臨床治療中較為常見(jiàn)，嚴(yán)重的血小板水平低下直接威脅著人體生命安全。血小板減少性紫癜、腫瘤放/化療、再生障礙性貧血及骨髓移植等許多因素都能引起血小板減少。目前，臨床上應(yīng)對(duì)血小板減少的主要治療方法仍然是直接輸注血小板；然而，血小板輸注存在著輸血反應(yīng)、病原體感染等多種隱患。已知血小板由骨髓巨核細(xì)胞產(chǎn)生，因而通過(guò)對(duì)巨核細(xì)胞增殖分化進(jìn)行調(diào)控可以達(dá)到促進(jìn)血小板生成的目的。所以，能夠促進(jìn)巨核細(xì)胞增殖分化的造血生長(zhǎng)因子類基因工程藥物越來(lái)越受到人們的廣泛關(guān)注。 血小板生成素(thrombopoietin, TPO)，也稱為巨核細(xì)胞生長(zhǎng)發(fā)育因子(megakaryocyte growth and development factor, MGDF)，是調(diào)節(jié)巨核細(xì)胞增殖分化和血小板生成的重要細(xì)胞因子，可通過(guò)與受體(c-Mpl)的結(jié)合，激活巨核細(xì)胞中相應(yīng)的信號(hào)通路，發(fā)揮其促巨核細(xì)胞增殖分化的作用。人們利用基因重組方法，研制出聚乙二醇化的重組人巨核細(xì)胞生長(zhǎng)發(fā)育因子(pegylated recombinant human megakaryocyte growthand development factor, PEG-rhMGDF)和重組人血小板生成素(recombinant humanthrombopoietin, rhTPO)兩種第一代促進(jìn)血小板生成基因工程藥物。但是，PEG-rhMGDF在美國(guó)進(jìn)行二期臨床試驗(yàn)時(shí)，，發(fā)現(xiàn)其能誘導(dǎo)產(chǎn)生與內(nèi)源性TPO起交叉反應(yīng)的中和性抗體，反而使患者血小板水平進(jìn)一步降低。因此，美國(guó)FDA隨即全面禁止了重組人TPO基因工程藥物的臨床實(shí)驗(yàn)。之后，人們又用噬菌體表面展示技術(shù)篩選出一種由14個(gè)氨基酸組成的TPO模擬肽(thrombopoietin mimetic peptide, TMP)，它與TPO無(wú)同源序列，但與c-Mpl具有較高親和力。研究表明，TMP尤其是其二聯(lián)體（dTMP）不僅能促進(jìn)巨核細(xì)胞增殖，并且不會(huì)誘導(dǎo)產(chǎn)生TPO中和性抗體。然而，由于TMP分子量只有1.5KD，通過(guò)基因工程制備難度較大，并且在體內(nèi)循環(huán)中極易被降解。 已有研究表明，人生長(zhǎng)激素(human growth hormone, hGH)對(duì)骨髓造血干細(xì)胞的增殖和/或分化具有重要調(diào)控作用，并且發(fā)現(xiàn)在體內(nèi)應(yīng)用后可加速血小板水平恢復(fù)。hGH可通過(guò)與其受體(growth hormone receptor, GHR)結(jié)合發(fā)揮生物學(xué)活性，該受體與c-Mpl為同類細(xì)胞因子受體，可激活相似的信號(hào)通路。因此，本室將GH與dTMP進(jìn)行融合，構(gòu)建出重組融合蛋白分子dTMP-GH，以期發(fā)揮協(xié)同促進(jìn)巨核細(xì)胞增殖及血小板生成作用。另一方面，融合蛋白dTMP-GH的分子量為26KD，較單純TMP更容易進(jìn)行基因工程制備，且可望延長(zhǎng)其體內(nèi)半衰期。 前期工作中，我們已構(gòu)建并篩選出特異性表達(dá)dTMP-GH的工程菌株，并進(jìn)行了實(shí)驗(yàn)室水平的小規(guī)模制備。在此基礎(chǔ)上，為放大生產(chǎn)，滿足新藥申報(bào)要求，本研究進(jìn)一步對(duì)融合蛋白dTMP-GH在大腸桿菌中的高效表達(dá)和純化制備工藝進(jìn)行了優(yōu)化，最終實(shí)現(xiàn)了該重組融合蛋白的中試放大生產(chǎn)，獲得純度大于98%的目標(biāo)產(chǎn)品，并對(duì)該融合蛋白的質(zhì)量標(biāo)準(zhǔn)及體內(nèi)分布進(jìn)行了初步研究。主要研究結(jié)果與結(jié)論如下： 1.基于已有的dTMP-GH表達(dá)菌株，通過(guò)對(duì)培養(yǎng)基成分、培養(yǎng)溫度和培養(yǎng)時(shí)間的優(yōu)化，成功將從一個(gè)單克隆到獲得目的蛋白菌體整個(gè)酵表達(dá)過(guò)程控制在30小時(shí)以內(nèi)，含目的蛋白的濕菌產(chǎn)量達(dá)50g/L，目的蛋白表達(dá)量占總蛋白的25%，高壓勻漿破菌后dTMP-GH包涵體含量達(dá)到50%以上。 2.為提高效率和處理量，采用超濾法代替透析法對(duì)包涵體進(jìn)行復(fù)性。通過(guò)對(duì)純化條件的優(yōu)化，目前單次純化所得融合蛋白dTMP-GH的量達(dá)到克級(jí)，產(chǎn)品純度大于98%。 3.對(duì)融合蛋白dTMP-GH的分子量、等電點(diǎn)、氨基酸序列、純度等理化特性及其凍干制劑成品的水分、pH值、滲透壓摩爾濃度、蛋白含量、純度、質(zhì)譜分子量、質(zhì)量肽譜分析、宿主菌蛋白殘留、外源性DNA殘留、細(xì)菌內(nèi)毒素含量、熱原、生物學(xué)活性等進(jìn)行了檢測(cè)，各項(xiàng)指標(biāo)均達(dá)到治療用生物制品的申報(bào)標(biāo)準(zhǔn)要求。在此基礎(chǔ)上，與中國(guó)食品藥品檢定研究院共同草擬了融合蛋白dTMP-GH的質(zhì)量標(biāo)準(zhǔn)草案。 4.用放射性碘標(biāo)記融合蛋白dTMP-GH，制備得到標(biāo)記率為71.5%、放射純度為96.5%、比活度為0.22MBq/μl的125I-dTMP-GH。BALB/c小鼠經(jīng)尾靜脈注射放射性125I標(biāo)記的dTMP-GH后，于30分鐘時(shí)股骨放射性計(jì)數(shù)達(dá)到最高值，占到注射總量的10%，隨時(shí)間推移經(jīng)肝、腎代謝排出體外。證實(shí)融合蛋白dTMP-GH具有明顯骨髓偏向性分布特點(diǎn)。 通過(guò)本研究，成功建立了融合蛋白dTMP-GH快速高效的中試規(guī)模制備工藝及質(zhì)量標(biāo)準(zhǔn)草案。單次發(fā)酵純化可獲得克級(jí)且純度98%的dTMP-GH，可制備出3000支凍干產(chǎn)品。抽樣檢測(cè)結(jié)果均符合dTMP-GH質(zhì)量標(biāo)準(zhǔn)要求，為融合蛋白dTMP-GH的進(jìn)一步開發(fā)和申報(bào)新藥奠定了基礎(chǔ)。
[Abstract]:Thrombocytopenia is more common in clinical treatment. Severe thrombocytopenia is a direct threat to human life safety. Thrombocytopenic purpura, tumor radiotherapy / chemotherapy, aplastic anemia and bone marrow transplantation can cause thrombocytopenia. At present, the main treatment methods for thrombocytopenia are still in clinical practice. It is a direct infusion of platelets. However, there are many hidden dangers such as blood transfusion reaction and pathogen infection. The known platelets are produced by bone marrow megakaryocytes. Therefore, the regulation of proliferation and differentiation of megakaryocytes can promote the production of platelets. Therefore, it can promote the proliferation and differentiation of megakaryocytes. Genetic engineering drugs of factor type have attracted more and more attention.
Thrombopoietin (TPO), also known as megakaryocyte growth and development factor (megakaryocyte growth and development factor, MGDF), is an important cytokine that regulates the proliferation, differentiation and platelet formation of megakaryocyte. It can activate the corresponding signaling pathway in megakaryocyte by combining with the receptor (c-Mpl) and play its role in stimulating megakaryocyte. Two first generations of recombinant human megakaryocyte growth factor (pegylated recombinant human megakaryocyte growthand development factor, PEG-rhMGDF) and recombinant human thrombopoietin (recombinant humanthrombopoietin, rhTPO) were developed by gene recombination method. However, when PEG-rhMGDF was conducted in two clinical trials in the United States, it was found that it could induce neutralizing antibodies that produce a cross reaction with endogenous TPO, instead making the patient's platelet level further reduced. Therefore, FDA in the United States immediately banned the clinical trials of recombinant human TPO engineering drugs. Later, people use phage surface display technology to screen out a TPO thrombopoietin mimetic peptide (TMP), which is composed of 14 amino acids. It has no homologous sequence with TPO, but has a high affinity with c-Mpl. The study shows that TMP especially its binary (dTMP) not only promotes megakaryocyte proliferation, but does not induce the production of TPO. However, because the molecular weight of TMP is only 1.5KD, it is difficult to prepare by genetic engineering, and it is easily degraded in the circulation of the body.
It has been shown that human growth hormone (hGH) plays an important role in regulating the proliferation and / or differentiation of bone marrow hematopoietic stem cells, and it is found that after application in vivo, it can accelerate the recovery of platelets,.HGH can be combined with its receptor (growth hormone receptor, GHR) to play biological activity, and the receptor is the same as c-Mpl. The cell factor receptor activates a similar signaling pathway. Therefore, GH and dTMP are fused to construct a recombinant fusion protein molecule dTMP-GH, in order to play a synergistic role in promoting megakaryocyte proliferation and platelet formation. On the other hand, the molecular weight of the fusion protein dTMP-GH is 26KD, which is easier to be prepared by genetic engineering than that of pure TMP. It is expected to prolong the half-life of the body.
In the previous work, we have constructed and screened the engineering strains specifically expressing dTMP-GH, and carried out a small scale preparation in the laboratory level. On this basis, in order to enlarge the production and meet the requirements of the new drug application, this study further optimized the process of efficient expression and purification of fusion protein dTMP-GH in Escherichia coli. The pilot scale production of the recombinant fusion protein was achieved, the target products with purity greater than 98% were obtained, and the quality standard and the distribution of the fusion protein were preliminarily studied. The main results and conclusions were as follows:
1. based on the existing dTMP-GH expression strain, through the optimization of culture medium composition, culture temperature and culture time, the whole process of fermentation from a monoclonal to the target protein body is controlled within 30 hours. The yield of the wet bacteria containing the target protein is up to 50g/L, the expression of the target protein is 25% of the total protein, and the high pressure homogenate is d after breaking the bacteria. The content of inclusion body of TMP-GH is above 50%.
2. in order to improve the efficiency and the amount of treatment, the inclusion body was refolded by ultrafiltration instead of dialysis. Through the optimization of the purification conditions, the amount of the fusion protein dTMP-GH obtained by single purification reached the level of gram, and the purity of the product was more than 98%..
3. the molecular weight, isoelectric point, amino acid sequence and purity of fusion protein dTMP-GH and the moisture of the finished product, pH value, osmotic pressure mol concentration, protein content, purity, mass spectrometry, mass peptide spectrum analysis, host protein residue, exogenous DNA residue, bacterial endotoxin content, pyrogen, biological activity, etc. On the basis of this, the quality standard of the fusion protein dTMP-GH was drafted together with the China Institute of food and drug verification.
4. the radioiodine labeled fusion protein dTMP-GH was used to prepare a 125I-dTMP-GH.BALB/c mouse with a labeling rate of 71.5%, a radioactive purity of 96.5%, and a 125I labeled dTMP-GH injected into the tail vein of a 125I-dTMP-GH.BALB/c mouse with a specific activity of 0.22MBq/ L. The femoral radioactivity count reached the highest value at 30 minutes, accounting for 10% of the total injection, passing through the liver and kidney over time. The excretion of metabolism in vitro showed that fusion protein dTMP-GH showed a significant distribution of bone marrow bias.
Through this study, a rapid and efficient pilot scale preparation process and a draft quality standard for fusion protein dTMP-GH were successfully established. A single fermentation and purity of 98% purity of dTMP-GH could be obtained, and 3000 freeze-dried products were prepared. The results of the samples were all in accordance with the requirements of the dTMP-GH quality standard and the further development and extension of the fusion protein dTMP-GH. The new medicine laid the foundation.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 申明強(qiáng);陳默;王崧;粟永萍;王軍平;;~(125)I標(biāo)記重組融合蛋白dTMP-GH在小鼠體內(nèi)分布的實(shí)驗(yàn)研究[J];局解手術(shù)學(xué)雜志;2014年03期

2 王崧;王軍平;陳默;許楊;陳芳;申明強(qiáng);王艾平;粟永萍;開麗;;重組融合蛋白rTMP-GH在巴斯德畢赤酵母中表達(dá)的研究[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2008年21期

3 許楊;王軍平;趙景宏;粟永萍;陳芳;申明強(qiáng);陳默;王崧;開麗;;重組融合蛋白rTMP-GH對(duì)體外培養(yǎng)巨核細(xì)胞增殖的影響[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2008年01期

本文編號(hào)：1868117