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抗体作为临床应用最广泛的治疗性蛋白药物，主要是通过CHO（Chinese Hamster Ovary）细胞培养来表达生产。与前一代的生物技术产品如细胞因子类药物相比，抗体类生物药的主要特点是临床用药剂量大，因此需要大规模工业化生产来满足市场需求，而CHO细胞培养的工艺优化和规模放大具有较大挑战性。提高细胞培养工艺表达量，扩大细胞培养生产规模，保证表达抗体质量稳定成为目前国内抗体产业界在抗体类蛋白药物规模生产过程中亟待解决的问题。本文围绕着以上问题，系统研究了CHO细胞的流加培养小试工艺优化，中试和产业化规模工艺放大，并探索性开发了基于切向流换液（ATF）技术的浓缩灌流工艺，以进一步提升我国抗体类药物规模产能。首先考察了培养基中金属离子的浓度对抗体质量的影响，培养基中将铜离子浓度从1000 nM降低到500 nM时，抗体产物MAb A的生产细胞可以维持1.2′107 vc/mL密度的生长，抗体产量保持不变，电荷异构体中碱性异构体的比例明显降低。说明铜离子作为多种酶的辅因子，除了维持细胞生长代谢，还会对C-端脯氨酸酰胺化产生影响。在搅拌式反应器培养抗体MAb A生产细胞时，将溶氧从60%降低至20%，细胞生长及抗体产量没有明显变化，抗体N-糖基化中G0F比例明显升高，G1F比例明显降低，说明溶氧水平对半乳糖糖基化有明显影响。另一方面延长培养时间观察到抗体酸性异构体比例明显上升，主要原因是培养液环境中自由基和氧化还原作用对抗体理化性质的稳定性存在一定影响。随后基于机械搅拌鼓泡式反应器内气-液传质理论，开发了一套动物细胞反应器传质模型。此模型结合了反应器传质属性和细胞培养代谢参数对细胞在大规模反应器培养过程中的氧气需求和二氧化碳累积水平做出了预测。在此模型的指导下，MAb A细胞培养工艺从2L实验室规模一次性放大到了1500L产业化规模，最终细胞生长、抗体产量和抗体质量在小试规模和商业化生产规模之间保持了一致。针对传统CHO细胞流加培养条件下由于细胞代谢废物积累造成抗体产量难以进一步大幅提高的问题，我们开发了基于切向流过滤换液ATF系统的CHO细胞浓缩灌流培养工艺。在浓缩灌流培养工艺的优化过程中，培养液中游离氨基酸浓度的检测起了很重要的作用，为灌流培养基配方的优化及灌流速率的调整提供了依据。最终通过浓缩灌流培养工艺，MAb A细胞生长密度提高了7.5倍，抗体产量提高了7.7倍。;As the most widely used therapeutic protein drugs in clinical, antibodies are mainly expressed and produced by CHO (Chinese Hamster Ovary) cell culture. Compared with the previous generation of biotechnology products such as cell factor drugs, the clinical dosage of antibody is rather high. Therefore, antibodies need to be produced in industrial large scale to meet the market demands. And the process development, optimization and scale-up of CHO cell culture are rather challenging. Productivity improvement, cell culture scale enlargement and stable quality of antibodies become the main problems to be solved in the large-scale production of antibody protein drugs in the domestic antibody industry. Focusing on the above problems, this paper systematically studied the optimization of the fed-batch cell culture process of CHO cells in small scale, the scale up of the pilot and commercial scales and explored the enhanced concentrated fed-batch based on alternative tangential flow (ATF) technology, so as to further improve the scale productivity of antibody drugs in China.In this study, the effect of the concentration of metal ions in the medium on the quality of antibodies was investigated. When the concentration of copper ions in the medium was reduced from 1000 nM to 500 nM, the viable cell density of MAb A cell can be maintained at 1.2′107 vc/mL. Productivity remained unchanged, and the proportion of basic variants in charge variants decreased significantly. These results suggest that copper ions, as the cofactors of various enzymes, not only can maintain cell growth and metabolism, but also affect C-terminal proline amidation. When antibody MAb A cell was cultured in stirred reactor, dissolved oxygen was reduced from 60% to 20%. As a result, cell growth and antibody production were not significantly changed. The ratio of G0F in antibody N-glycosylation was significantly increased, while the ratio of G1F was decreased obviously, which indicating that dissolved oxygen level had a significant effect on galactose glycosylation. On the other hand, the increase of the proportion of acidic variants of antibody was observed when the culture time was prolonged. The main reason was that the free radicals and redox effects in the culture medium had certain effects on the physicochemical properties of antibody.Based on the gas-liquid mass transfer theory in mechanical stirred bubbling reactor, a set of animal cell reactor mass transfer model was developed. The model combines the bioreactor mass transfer properties and mammalian cell metabolic parameters to predict the cell culture process oxygen demands and carbon dioxide accumulation in bioreactors. This mass-balance model has been successfully applied during MAb A process one-step scaling up from 2-L to 1500-L bioreactor. The cell growth, antibody production and quality were consistent between the bench scale and commercial production scale.To solve the problem that it is difficult to further improve the antibody productivity significantly due to the accumulation of cell metabolic waste in the traditional CHO cell culture process, we developed a concentration fed-batch culture of CHO cells based on the alternative tangential flow (ATF) system. During the optimization of concentration perfusion culture process, the detection of free amino acid concentration in the medium played an important role, which provided a basis for the optimization of the perfusion medium component and the adjustment of perfusion rate. Finally, by concentrated fed-batch culture, the cell growth density of MAb A cells was increased by 7.5 times, and the antibody production was increased by 7.7 times.
|何川. CHO细胞培养生产抗体药物的工艺优化与放大研究工程[D]. 中国科学院大学,2019.|
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