【作者】 刘兴茂；

【导师】 陈昭烈；

【作者基本信息】 中国人民解放军军事医学科学院 ， 细胞生物学， 2009， 博士

【摘要】 中国仓鼠细胞(Chinese hamster ovary cell)及其无血清流加培养是目前包括工程抗体和重组蛋白在内的生物技术药物生产的最为重要的表达系统和工艺技术。CHO工程细胞无血清流加培养工艺技术的提升,在很大程度上有赖于对无血清流加培养条件下CHO工程细胞代谢动力学的深入了解。据此,本研究的主要目的在于应用MATLAB软件,采用全局收敛的Levenberg-Marquardt法,对CHO工程细胞无血清流加培养的细胞生长、底物消耗及流加速率的模型参数进行非线性规划,获得全局性收敛的最优参数估计值,建立CHO工程细胞无血清流加培养的生长及代谢动力学模型,为CHO工程细胞无血清流加培养的优化控制提供可参考的理论依据。同时,结合基因组学研究对细胞培养工程渗透和推动的技术发展趋势,本研究拟通过运用基因芯片技术考察CHO工程细胞在无血清流加培养模式下的转录谱特征,借于了解CHO工程细胞不同生长及代谢状态的内在原因,为探寻促使细胞处于更为有效的生长和代谢状态的可能途径提供依据。建立能够在一定程度上体现流加培养工艺特色和技术指标的CHO工程细胞无血清流加培养技术是开展课题研究的基础和前提。课题研究选用表达重组人尿激酶原的CHO工程细胞为研究对象,在细胞悬浮体系中引入Pluronic F-68和硫酸葡聚糖,有效地消除了CHO工程细胞从静止贴附培养转入悬浮培养所引发的流体剪切力损伤和细胞聚结成团现象。与初转入悬浮培养的CHO工程细胞相比,悬浮培养适应的CHO工程细胞的平均比生长速率由0.27 d-1提高到0.48 d-1,最大细胞密度由2.5×106 cells/ml提高到6.3×106 cells/ml,悬浮适应前后CHO工程细胞的代谢特征基本相同。应用Plackett-Burman方法及响应面方法,设计了一种适合CHO工程细胞悬浮培养的无血清培养基。CHO工程细胞在无血清培养基中批次培养的最大细胞密度为4.2×106 cells/ml,较CHO工程细胞在含血清培养基中批次培养的最大细胞密度约提高1.7倍;相应地CHO工程细胞表达的目标蛋白的最大生产浓度较其在含血清培养基中约提高1.6倍。与国外商品化的CHO细胞无血清培养基相比,所设计的CHO工程细胞无血清培养基不逊于国外同类培养基的培养效果。在此基础上通过对流加起始时间、初始接种密度、流加培养基中的不同营养成分及底物控制策略对CHO工程细胞流加培养效果的考察,初步建立了CHO工程细胞无血清流加培养的工艺技术。在12 d流加培养过程中CHO工程细胞的最大生长密度达到7.8×106 cells/ml、目标蛋白最大累积活性为8875 IU/ml,分别比批次培养提高85.7%和50.9%。基于完整的流加培养过程可大致分为批次培养及流加培养两个阶段,为了更翔实的反映细胞在两个阶段的生长及代谢特征,分别研究了细胞无血清批次培养及流加培养的生长及代谢动力学。在细胞无血清批次培养的动力学研究中,依据细胞生长、底物消耗及产物形成的相关经验模型,并根据实际检测的实验数据,应用MATLAB软件,采用全局收敛的Levenberg-Marquardt法,对CHO工程细胞无血清批次培养对数生长期的细胞生长、葡萄糖消耗及乳酸生成的模型参数进行非线性规划,获得全局性收敛的最优参数估计值,建立了无血清批次培养细胞对数生长期的生长及代谢动力学模型。所建模型与实测数据吻合度较好,基本反映了CHO工程细胞无血清批次培养对数生长期的细胞生长、葡萄糖消耗及乳酸产出的内在规律。在此基础上,依据流加培养相关的细胞生长、底物消耗及流加速率的经验模型及实际检测数据,应用MATLAB软件,采用全局收敛的Levenberg-Marquardt法,对CHO工程细胞无血清流加培养的细胞生长、底物消耗及流加频率的模型参数进行非线性规划,获得全局性收敛的最优参数估计值,建立了CHO工程细胞无血清流加培养的生长及代谢动力学模型和依此为指导近似指数的间歇式流加策略,并在2 L搅拌罐式生物反应器中验证了代谢动力学模型用于指导CHO工程细胞无血清流加培养的优化控制的可行性。运用基因芯片技术比较了CHO工程细胞在批次培养及流加培养不同培养阶段基因表达水平的差异,同时采用Genmapp软件,结合已知的细胞代谢途径或信号通路图,着重分析了两种培养模式下有关细胞糖代谢、凋亡及细胞周期的基因差异。结果表明基因芯片大致涉及的19191个目标基因中,下调表达的基因数量多于上调表达基因数目,且两种培养模式下的基因差异表达有着明显的不同,尤其是在培养的衰退期,流加培养下调表达的基因数量明显多于批次培养。根据有关糖酵解及三羧酸循环关键调控基因的差异表达情况,提示在批次培养方式下,CHO细胞对葡萄糖的利用更多的是进行糖酵解途径;在流加培养阶段由于酵解途径关键调控基因表达的下调,有可能减少细胞了葡萄糖不完全氧化的比例,进而提高了细胞葡萄糖完全氧化的比例,解释了细胞在两种培养方式下代谢的差异性。CHO细胞在两种培养模式下涉及细胞凋亡的两条主要途径中的大多数促使细胞凋亡的关键基因,在培养的整个过程中的表达均没有明显差异,仅有少数基因表达发生了变化,印证了流式细胞仪检测细胞凋亡的比例结果。依据有关调控细胞周期的关键基因的表达,表明CHO细胞主要是通过下调表达CDKs、cyclin及CKI家族中的Cdk6、Cdk2、Cdc2a、Ccne1、Ccne2基因及上调表达Smad4基因,来达到调控细胞增殖及维持自身活力的目的,较好的解释了细胞在两种培养模式下细胞周期分布的差异性。综合上述结果表明,基于MATLAB软件及采用全局收敛的Levenberg-Marquardt法的CHO工程细胞无血清流加培养代谢动力学模型,能较为真实的反映实际培养过程中细胞生长及流加速率的变化,并为实现CHO工程细胞无血清流加培养的优化控制提供预见性指导。此外,针对CHO工程细胞无血清流加培养的基因转录谱特征的研究,不仅有助于阐明CHO工程细胞批次培养及流加培养的细胞生长及代谢差异的内在原因,也为探寻促使细胞保持更为有效的生长和代谢状态的可能途径提供线索。更多还原

【Abstract】 CHO cells (Chinese hamster ovary cell) is presently the most important expression systems for engineering antibodies and recombinant protein and serum-free fed-batch culture is also the most important process for biopharmaceuticals production. Deeply Understanding CHO cells metabolic dynamics would be favor of improving serum-free fed-batch CHO cells culture technology. Accordingly, the main purpose of this study is to establish the dynamic model of cell growth and metabolism and build the approximate index intermittent fed-batch strategy in order to provide the reference theory for achieving optimal control of serum-free fed-batch culture. The global convergence optimal parameters of cell growth, substrate consumption and feed frequency were estimated by non-linear programming through using the global convergence Levenberg-Marquardt method based on MATLAB software. At the same time, according to the infiltration and promotion impact of genomics research on cell culture technology development, the gene transcription characteristics of the cells cultured in serum-free fed-batch mode was investigated by using gene chip technology in order to understand the underlying reasons about the growth and metabolic characteristics of the cells cultured in serum-free fed-batch mode and provide clues for exploring the possible way to promote cells in more effective growth and metabolism state.Establishment of a serum-free fed-batch CHO cells culture paltform which can reflect a certain extent about the characteristics and technical indexes of fed-batch culture process was the basis and premise for this research. The recombinant CHO cells expressing recombinant human pro-urokinase (Pro-UK) as the object of study. And both the cells cluster and injury in suspension culture due to the shear stress were effectively eliminated by adding Pluronic F-68 and dextran sulfate. Compared with the original cells, the average growth rate of the cells adapted to suspension culture in logarithmic phase increases from 0.27-1 to 0.48-1, and the highest viable cell density, increased from 2.5×106 cells/ml to 6.3×106 cells/ml. It was shown that the cells adapted to suspension culture showed similar metabolism characteristics to the original cells.An appropriate serum-free medium for CHO cell suspension culture was formulated by application of Plackett-Burman and response surface method. The maximum cells density reached 4.2×106 cells/ml in serum-free suspension batch culture mode and was about 1.7 fold of that in 1% serum-containing suspension batch culture, correspondingly the maximum activity of interest increased about 1.6 fold.A serum-free fed-batch CHO cells culture platform was established through investigating the impact of the start time of feeding, the initial cell inoculation density, the different culture components and substrate limits strategy on fed-batch culture. The results showed that the maximum cells density reached 7.8×106 cells/ml and the largest protein activity reached 8875 IU/ml in fed-batch culture during 12 d culture process, which were about 85.7% and 50.9% increase as compared with those of the batch culture, respectively.In consideration of the fact that a whole fed-batch culture process is actually compose of both a batch culture stage and a feeding culture stage, The growth and metabolic characteristics of the cells in both serum-free batch culture stage and serum-free fed-batch culture stage was investigated individually. According to the established mathematical model of cell growth, substrate consumption and product formation, the optimal mathematical model parameters of logarithmic cell growth phase was estimated by non-linear programming using the global convergence of the Levenberg-Marquardt method and the MATLAB software based on the actual experimental data. The dynamic models of cell growth and metabolism in serum-free batch culture, which could match the experimental results and reflected essentially inherent orderliness about cell growth, glucose consumption and lactic acid production of the cells in logarithmic growth phase stage were established Further, according to the established mathematical model of cell growth, substrate consumption and feed frequency, the optimal mathematical model parameters of the cells in feeding culture stage were estimated by non-linear programming using the global convergence of the estimated Levenberg-Marquardt method and the MATLAB software again, based on the actual experimental data. Thus, the dynamic models of growth and metabolism of the cells in fed-batch serum-free culture and the approximate index for intermittent feeding strategy were established, which were approved to be feasibility in guiding serum-free fed-batch culture control in a 2 L bioreactor.The differences of gene expression levels of the cells in different culture phase in both batch and fed-batch modes were revealed by using gene chip technology. And based on the known metabolic pathway or the cell signaling pathway, the expression level differences of genes related to glucose metabolism, apoptosis and cell cycle of the cells cultured in batch and fed-batch modes were analysed by using Genmapp software. Results from this study showed that among approximate 19,191 the target gene in gene chip, the number of down-regulated genes was higher than that of up-regulated genes of the cells in both batch and fed-batch culture. Further, the number of down-regulated genes of the cells in fed-batch culture was much higher than that of the cells in the recession phase of batch culture. The results suggested that more glucose was utilized through glycolysis pathway by the cells in batch culture according to the key regulatory genes expression of glycolysis and TCA cycle in batch culture and fed-batch culture process. And, due to down-regulated of the key genes regulated glycolysis in the fed-batch culture stage, its would potentially bring about reducing the ratio of glucose incomplete oxidation and increasing the ratio of glucose of complete oxidation of glycolytic. There was no significant difference in the gene expression, which were related to the key apoptosis pathways of the cells cultured in batch and fed-batch mode, only a few gene expression changed, which illuminated the results of cell apoptosis proportion by using flow cytometry. According to the key regulation gene expression of cell cycle in both culture mode, the results indicated that the cell proliferation and cells viability were mainly regulated through down-regulating Cdk6, Cdk2, Cdc2a, Ccne1, Ccne2 genes of CDKs, cyclin and CKI family and up-regulating Smad4 genes, which also explained clearly the differences of cell cycle distribution in culture mode. Overall, in two culture modes, the cells achieved controlling cell metabolism, proliferation and the cycle and responded to changed culture conditions through down-regulating gene expression with reference to cell metabolic pathway or signaling pathway.The above results show that metabolism dynamics model of serum-free fed-batch cells culture based on the MATLAB software and the global convergence Levenberg-Marquardt method reflected the actual cell growth and culture volume change of fed-batch cell culture process and provided a theoretical reference basis for realizing optimal control in serum-free fed-batch CHO cells culture. At the same time, the study about gene transcription characteristics of the CHO cells cultured in serum-free fed-batch culture will not only help to clarify the underlying causes of cell growth and metabolism differences between the CHO cells cultured in batch mode and fed-batch mode as well as to provide clues for exploring possible ways to promote cells in a more effective growth and metabolism state.更多还原