动物细胞生物反应器关键技术研究及其结构优化
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摘要
牛顿力学统治了世界几百年，未来生物技术将成为主导21世纪科学技术发展的关键力量。生物反应器是人类进行生物目标产品开发及生产的关键设备。我国在生物反应器的开发研究中存在基础理论研究投入少、细胞的剪切敏感性认知不足、半经验方式设计、反应器效能低下、仿制国外低端产品等问题。针对这些问题，本文从CHO细胞入手，CHO细胞即中国仓鼠卵巢细胞，是动物细胞工程产业应用最为广泛的细胞之一，首先对CHO细胞力学特性进行了研究；以其力学特性为基础分别对生物反应器的搅拌器、罐体结构、温度维持、供料位置进行仿真优化；气泡分布器作为生物反应器中的供氧装置十分关键，但生成的气泡直径不好度量以及破裂过程中高速剪切损伤细胞，通过仿真及实验方法使得这些问题得到很好的解决；本文提出一种新的方法——剪切率参量法，在未制造样机的前提下，利用现有设备可以对新研制反应器剪切环境进行验证评价，剪切率参量法验证方法简单可靠，缩短研发周期，节约成本。
为了给生物反应器设计提供基础性的依据，借助原子力显微镜，对CHO细胞进行成像及力学测量，测量得到生理状态CHO细胞的弹性模量为5.278±0.395kPa，借助数值计算及细胞培养实验分析CHO细胞在生物反应器中的受力环境，得到CHO细胞培养时能够承受的合理的剪切力约为0.392Pa，此时流场剪切率为391.41(1/s)，在此剪切应力作用下对于20μm的细胞来说U方向的最大位移量大约为12.9μm。为反应器的设计中解决流体剪切的问题提供依据。
规模化细胞培养生物反应器研发主要需要解决两个关键要素，首先是保证流场混合的均匀性，同时要解决好流场剪切力对细胞的影响，以CHO细胞承受的合理剪切力为参考，在降低流场剪切作用以及满足流场混合效果的前提下，优化后的反应器结构采用45o叶片夹角的Elephant Ear搅拌器，下吸式安装，罐体高径比为1、罐底半径为罐体直径的一半为、搅拌器安装高度与罐体高度比为、挡板数量为3对，此优化结构流场效果最好。细胞培养过程中温度需要严格控制，建立该反应器的温度场仿真模型，对三种供热保温结构进行对比仿真分析，表明当采用整体壁面加热时，反应器内的流体能较快的达到温度均衡。反应器供料位置对物料混合效果影响明显，利用示踪剂法，找到反应器较优的供料位置，缩短混合时间。
气泡分布器作为反应器的供氧装置用于生成气泡，在细胞培养过程中气泡在分布器孔口形成上升破裂主要与三个参量有关，通过研究得到了一定范围内气泡平均直径大小与孔口通气速度、表面张力系数和孔口直径之间的关系式。定量角度分析了直径在4mm-10mm范围内变化的气泡破裂过程中产生的在0.97-1.91Pa之间变化，根据CHO细胞的承受的最大剪切力为0.392Pa左右，可知在此范围的气泡破裂过程中均会造成细胞的损伤。而气泡越小，气液表面积比越大，这样反应器内溶氧越好，为了满足细胞培养过程中的溶氧需求，因此气泡直径控制在4mm左右为宜。气泡分布器安装时通气孔离生物反应器底部的距离对气含率的影响不大，大分布环的使得气含率更加均匀，而且大分布环通气结构能有效防止气体直接由搅拌器区直接上升至液面溢出，降低气含率，所以实际应用中可以根据需要应尽量选用大分布环气泡分布器。
生物反应器设计制造中的需要解决的关键问题之一就是流场混合过程中产生的剪切力对细胞的损伤，反应器研发初期很难对这一问题进行度量验证，一般通过制造样机进行实验验证，一种新的验证方法剪切率参量法应运而生，它可以在现有实验室设备的基础上通过实验和仿真方法，使得这一问题得到解决。针对本论文提出的多项技术，设计了反应器实验系统，利用该实验系统验证了本文提出的剪切率参量法，当采用上通气的情况下，转速为180r/min时，细胞密度增量最大，其主流场区域平均剪切率为14.5，符合本文提出的剪切率参量法的适宜细胞生长的平均剪切率范围11.6~17.4（1/s）；实验系统包括搅拌器、罐体结构、加热保温装置、气泡分布器均采用本文的优化结果，在此结构参数下采用上通气转速在180r/min时可以获得较好的培养效果，当采用下通气转速为80r/min时气泡直径为在4~8mm范围获得较好的培养效果，其性能与美国NBS商品化生物反应器相近。
本文对CHO细胞生物反应器的关键技术进行了研究，得到了CHO细胞的力学特性、反应器结构及其流场效果、气泡分布器结构及气泡生成破裂过程，反应器剪切率参量验证方法等研究成果，为CHO细胞规模培养生物反应器的实用化奠定了基础。
Newtonian mechanics has ruled the world for hundreds of years;biotechnology will become a pivotal force in leading the development of scienceand technology in the21st century. And bioreactor is the key device for human toproduce and develop the biologic target products. China has many problems in thedevelopment of bioreactor, such as less investment in basic theoretical research,cognitive deficiency on cells’ shear sensitivity semi-empirical method design, lowefficiency reactor, foreign low-end products imitation, etc. CHO cells, also knownas Chinese hamster ovary cells, are one of the most widely used cells in the animalcell engineering. For these problems, the dissertation studied the mechanicalcharacters of CHO cells from the basis; simulation optimized bioreactor’s stirrertank structure, temperature maintenance and feeding mode. The bubble distributoris critical as the supply of oxygen in the bioreactor device, however, the generatedbubble diameter is hard to measure and the high-speed shear can damage cells inthe process of rupture. Thus, these problems can be solved well through simulationand experiments. The dissertation put forward a new method--Shear RateParameter Method (SRPM). It uses existing equipment to verify and evaluate thereactor’s newly developed shear environment in the precondition of non-manufacturing mockup. The SRPM is simple and reliable; it not only shortens thedevelopment cycle, but saves cost.
In order to provide the fundamental basis for bioreactor design, it appliesatomic force microscope to have imaging and mechanical measurement on CHOcells. The measured modulus of elasticity of physiological state CHO cells is5.278±0.395kPa. With the numerical calculation and cell culture experiments, toanalyze the force environment of CHO cells in the bioreactor. It shows that theCHO cells can endure the reasonable shear force is approximately0.392Pa,meanwhile, the shearing rate of flow field is391.41(1/s), the maximumdisplacement of U-direction for20μm cell under such shear force is12.9μm. Thus,it offers the basis for the problems solving of fluid shear in the bioreactor design.
The homogenization of mixed flow fields and the stirring shear force inbioreactor are the two key factors in process of the cell scale culture. Based on the CH cells’ enduring reasonable shear force in the condition of reducing flow filedshear force and satisfying mixed effect, the flow field effect of optimizing structureis the best, when bioreactor adopts the Elephant Ear stirrer with45oblade includedangle by downdraft installation, the tank high-diameter ratio H/T=1, the radium ofthe tank bottom, the installation height of stirrer C/H=1/3and the number of baffleis3pairs. Temperature in the cell culture process requires strict control. It buildsthe bioreactor’s simulation model of temperature field aiming to have comparativesimulation analyzing for the three heating structure. It indicates that when theoverall wall heating, the fluid within the reactor can be faster to reach temperatureequilibrium. The bioreactor feeding position has an obvious influence on materialmixed effect. It can use tracer method to find better feeding position of the reactoras well as to shorten the mixing time.
The bubble distributor as the oxygen supply device of the reactor used togenerate bubble. In process of the cell culture, bubble at reactor orifices from risingto bursting is concerned with three parameters. Study supports, within a certainrange the relation between bubble’s average diameter and orifice ventilation speed,surface coefficient of tension and orifice diameter. It analyzes from the quantitativeangle, the produced in the process of bursting with bubble diameters from4mm-10mm varies from0.97-1.91Pa. It is clear that cell damage happens in the processin terms of the maximum shear force the CHO cell enduring is about0.392Pa.However, the smaller the bubble, the higher the gas-liquid surface area ratio and thebetter the dissolved oxygen in the reactor, in order to meet the dissolved oxygenrequirements in the process of cell culture, the bubble diameter should be controlledat about4mm. During the bubble distributor installation, the distance from orificeto bottom of the bioreactor has little effect on gas holdup. And bubble distributorwith larger distribution ring can balance gas holdup better. Moreover, largedistribution ring ventilation structure can effectively prevent the gas overflowingfrom the area of stirrer raise directly up to the fluid level. It decreases gas holdup;thus, try to choose the large bubble distributor as required in practical application.
One of the key problems needs to be solved in bioreactor design andmanufacture is the damage caused by shear force to cell in the process of flowfields mixed. It is hard to measure and verify the problem at the early stage of thebioreactor’s research and development It normally uses mockup to experiment Anew verification method is developed, namely, shear rate parameter method. It can solve the problem through experiments and simulation based on the existingequipments. The dissertation presented a number of techniques, designed reactorexperiment system and verified the SRPM by it. When adopt upper ventilation androtate speed is180r/min, the increment of cell density is the maximum, meanwhile,the average shear rate in main flow field is14.5. It fits the appropriate cell growthshear rate range from11.6-17.4(1/s) presented in the SRPM. The stirrer, tankstructure, heating insulation device in the experiment system all use theoptimization results. It can get better culture effect with such structural parameterswhen adopts upper ventilation and rotate speed is180r/min. It also can get betterculture effect when the bubble diameter is4-8mm, the rotate speed is80r/min andventilate down. The experiment system and commercialization bioreactor havesimilar performance.
The dissertation studied the key technology of CHO cell bioreactor; the majorfindings including the mechanical characters of CHO cell, bioreactor structure andthe effect of flow field in bioreactor, bubble distributor structure and the process ofits generation to rupture, verification methods for SRPM in bioreactor, etc, whichlaid a foundation for the practicality of CHO cell scale culture bioreactor.
为了给生物反应器设计提供基础性的依据，借助原子力显微镜，对CHO细胞进行成像及力学测量，测量得到生理状态CHO细胞的弹性模量为5.278±0.395kPa，借助数值计算及细胞培养实验分析CHO细胞在生物反应器中的受力环境，得到CHO细胞培养时能够承受的合理的剪切力约为0.392Pa，此时流场剪切率为391.41(1/s)，在此剪切应力作用下对于20μm的细胞来说U方向的最大位移量大约为12.9μm。为反应器的设计中解决流体剪切的问题提供依据。
规模化细胞培养生物反应器研发主要需要解决两个关键要素，首先是保证流场混合的均匀性，同时要解决好流场剪切力对细胞的影响，以CHO细胞承受的合理剪切力为参考，在降低流场剪切作用以及满足流场混合效果的前提下，优化后的反应器结构采用45o叶片夹角的Elephant Ear搅拌器，下吸式安装，罐体高径比为1、罐底半径为罐体直径的一半为、搅拌器安装高度与罐体高度比为、挡板数量为3对，此优化结构流场效果最好。细胞培养过程中温度需要严格控制，建立该反应器的温度场仿真模型，对三种供热保温结构进行对比仿真分析，表明当采用整体壁面加热时，反应器内的流体能较快的达到温度均衡。反应器供料位置对物料混合效果影响明显，利用示踪剂法，找到反应器较优的供料位置，缩短混合时间。
气泡分布器作为反应器的供氧装置用于生成气泡，在细胞培养过程中气泡在分布器孔口形成上升破裂主要与三个参量有关，通过研究得到了一定范围内气泡平均直径大小与孔口通气速度、表面张力系数和孔口直径之间的关系式。定量角度分析了直径在4mm-10mm范围内变化的气泡破裂过程中产生的在0.97-1.91Pa之间变化，根据CHO细胞的承受的最大剪切力为0.392Pa左右，可知在此范围的气泡破裂过程中均会造成细胞的损伤。而气泡越小，气液表面积比越大，这样反应器内溶氧越好，为了满足细胞培养过程中的溶氧需求，因此气泡直径控制在4mm左右为宜。气泡分布器安装时通气孔离生物反应器底部的距离对气含率的影响不大，大分布环的使得气含率更加均匀，而且大分布环通气结构能有效防止气体直接由搅拌器区直接上升至液面溢出，降低气含率，所以实际应用中可以根据需要应尽量选用大分布环气泡分布器。
生物反应器设计制造中的需要解决的关键问题之一就是流场混合过程中产生的剪切力对细胞的损伤，反应器研发初期很难对这一问题进行度量验证，一般通过制造样机进行实验验证，一种新的验证方法剪切率参量法应运而生，它可以在现有实验室设备的基础上通过实验和仿真方法，使得这一问题得到解决。针对本论文提出的多项技术，设计了反应器实验系统，利用该实验系统验证了本文提出的剪切率参量法，当采用上通气的情况下，转速为180r/min时，细胞密度增量最大，其主流场区域平均剪切率为14.5，符合本文提出的剪切率参量法的适宜细胞生长的平均剪切率范围11.6~17.4（1/s）；实验系统包括搅拌器、罐体结构、加热保温装置、气泡分布器均采用本文的优化结果，在此结构参数下采用上通气转速在180r/min时可以获得较好的培养效果，当采用下通气转速为80r/min时气泡直径为在4~8mm范围获得较好的培养效果，其性能与美国NBS商品化生物反应器相近。
本文对CHO细胞生物反应器的关键技术进行了研究，得到了CHO细胞的力学特性、反应器结构及其流场效果、气泡分布器结构及气泡生成破裂过程，反应器剪切率参量验证方法等研究成果，为CHO细胞规模培养生物反应器的实用化奠定了基础。
Newtonian mechanics has ruled the world for hundreds of years;biotechnology will become a pivotal force in leading the development of scienceand technology in the21st century. And bioreactor is the key device for human toproduce and develop the biologic target products. China has many problems in thedevelopment of bioreactor, such as less investment in basic theoretical research,cognitive deficiency on cells’ shear sensitivity semi-empirical method design, lowefficiency reactor, foreign low-end products imitation, etc. CHO cells, also knownas Chinese hamster ovary cells, are one of the most widely used cells in the animalcell engineering. For these problems, the dissertation studied the mechanicalcharacters of CHO cells from the basis; simulation optimized bioreactor’s stirrertank structure, temperature maintenance and feeding mode. The bubble distributoris critical as the supply of oxygen in the bioreactor device, however, the generatedbubble diameter is hard to measure and the high-speed shear can damage cells inthe process of rupture. Thus, these problems can be solved well through simulationand experiments. The dissertation put forward a new method--Shear RateParameter Method (SRPM). It uses existing equipment to verify and evaluate thereactor’s newly developed shear environment in the precondition of non-manufacturing mockup. The SRPM is simple and reliable; it not only shortens thedevelopment cycle, but saves cost.
In order to provide the fundamental basis for bioreactor design, it appliesatomic force microscope to have imaging and mechanical measurement on CHOcells. The measured modulus of elasticity of physiological state CHO cells is5.278±0.395kPa. With the numerical calculation and cell culture experiments, toanalyze the force environment of CHO cells in the bioreactor. It shows that theCHO cells can endure the reasonable shear force is approximately0.392Pa,meanwhile, the shearing rate of flow field is391.41(1/s), the maximumdisplacement of U-direction for20μm cell under such shear force is12.9μm. Thus,it offers the basis for the problems solving of fluid shear in the bioreactor design.
The homogenization of mixed flow fields and the stirring shear force inbioreactor are the two key factors in process of the cell scale culture. Based on the CH cells’ enduring reasonable shear force in the condition of reducing flow filedshear force and satisfying mixed effect, the flow field effect of optimizing structureis the best, when bioreactor adopts the Elephant Ear stirrer with45oblade includedangle by downdraft installation, the tank high-diameter ratio H/T=1, the radium ofthe tank bottom, the installation height of stirrer C/H=1/3and the number of baffleis3pairs. Temperature in the cell culture process requires strict control. It buildsthe bioreactor’s simulation model of temperature field aiming to have comparativesimulation analyzing for the three heating structure. It indicates that when theoverall wall heating, the fluid within the reactor can be faster to reach temperatureequilibrium. The bioreactor feeding position has an obvious influence on materialmixed effect. It can use tracer method to find better feeding position of the reactoras well as to shorten the mixing time.
The bubble distributor as the oxygen supply device of the reactor used togenerate bubble. In process of the cell culture, bubble at reactor orifices from risingto bursting is concerned with three parameters. Study supports, within a certainrange the relation between bubble’s average diameter and orifice ventilation speed,surface coefficient of tension and orifice diameter. It analyzes from the quantitativeangle, the produced in the process of bursting with bubble diameters from4mm-10mm varies from0.97-1.91Pa. It is clear that cell damage happens in the processin terms of the maximum shear force the CHO cell enduring is about0.392Pa.However, the smaller the bubble, the higher the gas-liquid surface area ratio and thebetter the dissolved oxygen in the reactor, in order to meet the dissolved oxygenrequirements in the process of cell culture, the bubble diameter should be controlledat about4mm. During the bubble distributor installation, the distance from orificeto bottom of the bioreactor has little effect on gas holdup. And bubble distributorwith larger distribution ring can balance gas holdup better. Moreover, largedistribution ring ventilation structure can effectively prevent the gas overflowingfrom the area of stirrer raise directly up to the fluid level. It decreases gas holdup;thus, try to choose the large bubble distributor as required in practical application.
One of the key problems needs to be solved in bioreactor design andmanufacture is the damage caused by shear force to cell in the process of flowfields mixed. It is hard to measure and verify the problem at the early stage of thebioreactor’s research and development It normally uses mockup to experiment Anew verification method is developed, namely, shear rate parameter method. It can solve the problem through experiments and simulation based on the existingequipments. The dissertation presented a number of techniques, designed reactorexperiment system and verified the SRPM by it. When adopt upper ventilation androtate speed is180r/min, the increment of cell density is the maximum, meanwhile,the average shear rate in main flow field is14.5. It fits the appropriate cell growthshear rate range from11.6-17.4(1/s) presented in the SRPM. The stirrer, tankstructure, heating insulation device in the experiment system all use theoptimization results. It can get better culture effect with such structural parameterswhen adopts upper ventilation and rotate speed is180r/min. It also can get betterculture effect when the bubble diameter is4-8mm, the rotate speed is80r/min andventilate down. The experiment system and commercialization bioreactor havesimilar performance.
The dissertation studied the key technology of CHO cell bioreactor; the majorfindings including the mechanical characters of CHO cell, bioreactor structure andthe effect of flow field in bioreactor, bubble distributor structure and the process ofits generation to rupture, verification methods for SRPM in bioreactor, etc, whichlaid a foundation for the practicality of CHO cell scale culture bioreactor.
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