一种新型升血小板基因工程产品制备工艺优化及质量标准研究

发布时间：2018-05-10 06:07

本文选题：质量标准 + dTMP-GH　；参考：《第三军医大学》2014年硕士论文

【摘要】：血小板减少症在临床治疗中较为常见，严重的血小板水平低下直接威胁着人体生命安全。血小板减少性紫癜、肿瘤放/化疗、再生障碍性贫血及骨髓移植等许多因素都能引起血小板减少。目前，临床上应对血小板减少的主要治疗方法仍然是直接输注血小板；然而，血小板输注存在着输血反应、病原体感染等多种隐患。已知血小板由骨髓巨核细胞产生，因而通过对巨核细胞增殖分化进行调控可以达到促进血小板生成的目的。所以，能够促进巨核细胞增殖分化的造血生长因子类基因工程药物越来越受到人们的广泛关注。血小板生成素(thrombopoietin, TPO)，也称为巨核细胞生长发育因子(megakaryocyte growth and development factor, MGDF)，是调节巨核细胞增殖分化和血小板生成的重要细胞因子，可通过与受体(c-Mpl)的结合，激活巨核细胞中相应的信号通路，发挥其促巨核细胞增殖分化的作用。人们利用基因重组方法，研制出聚乙二醇化的重组人巨核细胞生长发育因子(pegylated recombinant human megakaryocyte growthand development factor, PEG-rhMGDF)和重组人血小板生成素(recombinant humanthrombopoietin, rhTPO)两种第一代促进血小板生成基因工程药物。但是，PEG-rhMGDF在美国进行二期临床试验时，，发现其能诱导产生与内源性TPO起交叉反应的中和性抗体，反而使患者血小板水平进一步降低。因此，美国FDA随即全面禁止了重组人TPO基因工程药物的临床实验。之后，人们又用噬菌体表面展示技术筛选出一种由14个氨基酸组成的TPO模拟肽(thrombopoietin mimetic peptide, TMP)，它与TPO无同源序列，但与c-Mpl具有较高亲和力。研究表明，TMP尤其是其二联体（dTMP）不仅能促进巨核细胞增殖，并且不会诱导产生TPO中和性抗体。然而，由于TMP分子量只有1.5KD，通过基因工程制备难度较大，并且在体内循环中极易被降解。已有研究表明，人生长激素(human growth hormone, hGH)对骨髓造血干细胞的增殖和/或分化具有重要调控作用，并且发现在体内应用后可加速血小板水平恢复。hGH可通过与其受体(growth hormone receptor, GHR)结合发挥生物学活性，该受体与c-Mpl为同类细胞因子受体，可激活相似的信号通路。因此，本室将GH与dTMP进行融合，构建出重组融合蛋白分子dTMP-GH，以期发挥协同促进巨核细胞增殖及血小板生成作用。另一方面，融合蛋白dTMP-GH的分子量为26KD，较单纯TMP更容易进行基因工程制备，且可望延长其体内半衰期。前期工作中，我们已构建并筛选出特异性表达dTMP-GH的工程菌株，并进行了实验室水平的小规模制备。在此基础上，为放大生产，满足新药申报要求，本研究进一步对融合蛋白dTMP-GH在大肠杆菌中的高效表达和纯化制备工艺进行了优化，最终实现了该重组融合蛋白的中试放大生产，获得纯度大于98%的目标产品，并对该融合蛋白的质量标准及体内分布进行了初步研究。主要研究结果与结论如下： 1.基于已有的dTMP-GH表达菌株，通过对培养基成分、培养温度和培养时间的优化，成功将从一个单克隆到获得目的蛋白菌体整个酵表达过程控制在30小时以内，含目的蛋白的湿菌产量达50g/L，目的蛋白表达量占总蛋白的25%，高压匀浆破菌后dTMP-GH包涵体含量达到50%以上。 2.为提高效率和处理量，采用超滤法代替透析法对包涵体进行复性。通过对纯化条件的优化，目前单次纯化所得融合蛋白dTMP-GH的量达到克级，产品纯度大于98%。 3.对融合蛋白dTMP-GH的分子量、等电点、氨基酸序列、纯度等理化特性及其冻干制剂成品的水分、pH值、渗透压摩尔浓度、蛋白含量、纯度、质谱分子量、质量肽谱分析、宿主菌蛋白残留、外源性DNA残留、细菌内毒素含量、热原、生物学活性等进行了检测，各项指标均达到治疗用生物制品的申报标准要求。在此基础上，与中国食品药品检定研究院共同草拟了融合蛋白dTMP-GH的质量标准草案。 4.用放射性碘标记融合蛋白dTMP-GH，制备得到标记率为71.5%、放射纯度为96.5%、比活度为0.22MBq/μl的125I-dTMP-GH。BALB/c小鼠经尾静脉注射放射性125I标记的dTMP-GH后，于30分钟时股骨放射性计数达到最高值，占到注射总量的10%，随时间推移经肝、肾代谢排出体外。证实融合蛋白dTMP-GH具有明显骨髓偏向性分布特点。通过本研究，成功建立了融合蛋白dTMP-GH快速高效的中试规模制备工艺及质量标准草案。单次发酵纯化可获得克级且纯度98%的dTMP-GH，可制备出3000支冻干产品。抽样检测结果均符合dTMP-GH质量标准要求，为融合蛋白dTMP-GH的进一步开发和申报新药奠定了基础。
[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.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

【参考文献】