【作者】 许峰；

【导师】 章良；

【作者基本信息】 苏州大学 ， 制药工程（专业学位）， 2014， 硕士

【摘要】 目的：通过一系列的实验室小试研究，确定治疗用单克隆抗体上游制备工艺。根据大规模工业设备所能实现的控制参数，以及各控制参数所能达到的范围及精度，核对、校正和补充实验室获得的数据，修订为商业化生产用工艺用参数。方法：实验室小试阶段通过对比，筛选不同的基础培养基及补料培养基，得到最优化的培养工艺。放大生产阶段，通过查阅设备文献以及通过测定工业级主要生产设备的关键参数，如细胞发酵罐的KLa，搅拌桨线速度等参数，确定细胞培养及蛋白表达的参数范围，然后与实验室小规模条件下得到的工艺进行比较，修正。结果：1.不同的培养基条件下，培养同样长的时间，7#培养基的整体活细胞密度有85E6个/ml，而11#培养基仅有18E6个/ml，两种培养基的总体活细胞密度相差5倍左右。其余培养基中，活细胞密度≤40E6个/ml的，有4种；在40E6个/ml到50E6个/ml的，有5种；在50E6个/ml到70E6个/ml的，有7种；在70E6个/ml到85E6个/ml的，有5种。2.不同的基础培养基，表达目的蛋白的差别也较大。其中5#和16#培养基，分别有1.7g/L和1.6g/L。但8#培养基中，目的蛋白仅有0.2g/L。11#和9#由于在第12天活率就降低到60%以下，且细胞密度最低，无检测表达量意义。3.从各组总细胞密度来看，不管是化学成分限定的多肽混合物和传统的蛋白水解物，加入到两种培养基中，都不会对细胞生长产生大的影响，其波动范围与未添加水解物的对照组没有明显的区别。4.从蛋白表达量看来，media1-cd4和media2-cd3的实验组结果最高。而且，加入化学成分限定水解物的实验组，其蛋白表达量在同一组实验的不同摇瓶中，表现更为稳定。5.流加培养对目的蛋白的表达都具有积极的作用，在不同的基础培养基及水解物的组合中添加任意一种流加培养基都能有效地提高蛋白表达量。在主要质量指标合格的实验组中，以media1-cd4-F1plus实验组的目的蛋白表达量最高，达到2.45g/L。6.在2L的生物反应器内搅拌桨桨尖速度1.6m/s时，细胞活率已经开始下降，略微降低搅拌桨速至1.4m/s，此时未发现细胞有明显的活率下降。7.根据发酵罐的功率计算及放大公式：P Ne n3d5V l2按单位体积输出功率一致的原则放大，从细胞活率角度出发，在50LV规模时，搅拌桨搅拌转速应控制在≤194RPM，在200L规模时，搅拌转速应控制在≤145RPM。8.在2L的小试规模生物反应器上，在搅拌转速100rpm，温度37℃，通气速率20ml/min，反应器内加入为75%基础培养基+25%补料培养基，测得KLa=0.002s-1=7.2h-1。9.分别检测了2L生物反应器、50L生物反应器、200L生物反应器在桨尖速度0.3m/s，0.6m/s，1.2m/s的条件下的KLa。10.采用检测氯化钠浓溶液的稀释时间，验证了混匀时间可以满足细胞培养需求。11.根据KLa的检测结果可知，通入空气已经可以满足细胞对氧气的需求，且通气量应＜0.1vvm以避免产生泡沫。结论：在治疗用单克隆抗体的生产过程中，由于生产规模的变更，会导致蛋白生产条件的变化，从而引起蛋白特性的改变，进而影响药品质量。而由于生物制品的生产过程成本高昂，无法在工业级规模下进行工艺条件摸索，因此，通过关键参数的有针对性的工艺放大，可以在有限的成本及时间资源条件下，测试、锁定工艺，使生产的产品特性与小试阶段一致，从而保证药品的安全性、有效性及质量的稳定可控。更多还原

【Abstract】 Objective: According to a series of laboratory studies, set the upstream preparationparameters of the therapeutic monoclonal antibody. Then set the process for commercialproduction by checking, correcting and supplementing laboratory data and large-scaleindustrial equipment capacity and accuracy which can be achieved,Methods: By screening different basal medium and feed medium, obtain theoptimized process in laboratory phase. In scale up phase, measure key parameters, such asbioreactor’s KLa, stirring paddle line speed and other parameters, based on the industrialequipment’s instruction and user manual, to establish the operating range. And then checkand correcting the process obtained in laboratoryResults:1. Using different basal media, same culture method, the viable medium#7is85E6/ml, and11#medium is only18E6/ml, the difference of cell density is about5times.Other medium, the viable cell density≤40E6cells/ml, there are four kinds;from40E6cells/ml to50E6cells/ml, there are five kinds; from50E6cells/ml to70E6cells/ml,there are seven kinds; from70E6cells/ml to85E6cells/ml, there are five kinds.2. In different basal medium, the titer of the protein of the differs.5#and16#medium, respectively,1.7g/L and1.6g/L. But8#medium protein only0.2g/L.#11and#9in the first12days as a result of living rate is reduced to60%or less, worthless totest the titer.3. Whether chemically defined peptide mixtures or traditional protein hydrolyzate,will not impact cell growth, and there is no significant difference to the control group.`4. Media1-cd4and media in the experimental group2-cd3has the highest titer.Furthermore, addition of a chemical composition defined in the experimental grouphydrolyzate, its titer in the different shake flask experiments of the same, more stable performance.5. fed-batch culture has a positive effect to express the target protein, add any kindof fed-batch culture in different combinations on the basis of media and hydrolyzate caneffectively increase the titer of protein. The media1-cd4-F1plus group is2.45g/L.6. within2L bioreactor impeller tip speed1.6m/s, cell viability has begun todecline, slightly lower impeller speed to1.4m/s, this time did not find the cell viabilitydecreased significantly.P7. Scale up the formula: Ne nd5V l32Voutput per unit volume consistentwith the principles of power amplification, consider cell viability, in the50L scale, paddlestirring speed should be controlled at≤194RPM, while200L scale, stirring speed shouldbe controlled at≤145RPM.8. In the bench scale2L bioreactors, stirring speed100rpm, temperature37℃,aeration rate of20ml/min, the reactor was added75%basal medium+25%feed medium,measured KLa=0.002s-1=7.2h-1.9. Test KLa values in2L bioreactor,50L bioreactor,200L bioreactor at a tip speedof0.3m/s,0.6m/s, at1.2m/s condition.10. Test the mixing time of concentrated solution of sodium chloride, to verify themixing time to meet the needs of the cell culture.11. Accroding to the KLa value, the air will meet the demand for oxygen in the cells,and the ventilation should be <0.1vvm to avoid bubbles.Conclusion: in monoclonal antibody production process, the change of productionscale, will lead to changes in protein production conditions, which will cause changes inprotein characteristics. The changes in protein characteristics will affect the quality ofmedicine. Considering the high cost of biological, it is impossible to develop process inindustrial-scale. Usually, the process is scaled up by amplifying the key parameters toprepare proteins has the same characteristics as in the pilot scale. Therefore, theeffectiveness, safety and quality controllability of the medicine can be guaranteed.更多还原