红豆杉悬浮细胞反应器深层培养的过程工程研究
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摘要
紫杉醇为红豆杉的二萜类次级代谢物，是一种有效的抗癌药物，红豆杉细胞培养生产紫杉醇被认为是获取紫杉醇最有效的途径之一。目前，虽然红豆杉细胞培养生产紫杉醇研究取得了很大进展，但红豆杉细胞反应器培养的过程工程研究甚少，已成为限制其产业化水平进一步提高的因素之一。本论文将细胞培养和过程工程理论结合起来，研究生物反应器内的流体流动、传递规律和细胞生长及生化反应，建立细胞如何适应各种环境变化的数学模型，为指导反应器设计、放大和操作优化提供理论依据。主要研究结果如下：
（1）在基质充分条件下，细胞中基质的消耗不能有效地调整到与细胞外底物水平相适合，过量的基质通过形成代谢物和能量用于细胞生长、维持、形成产物和能量损益，如膜电位损耗、ATP水解作用、无效的循环、形成储存的化合物或胞外产品、代谢解耦、呼吸链的改变等等，因此建立了适用于基质充分条件下15L搅拌生物反应器中红豆杉细胞悬浮培养整合了能量损益的基质消耗动力学模型、产物合成动力学模型和系统流变特性变化动力学模型，同时发现logistic方程能更好地描述细胞生长过程。
内部交叉验证和外部验证表明：所建立的细胞生长模型、基质消耗模型和产物合成模型适用于模拟细胞培养的实际过程，模型可以用于指导反应器培养；能量损益是影响基质充分条件下搅拌反应器中红豆杉细胞生长和生化反应的重要因素，红豆杉细胞反应器放大培养应尽量维持限制基质过量的条件下进行。
（2）研究了反应器初始培养液和反应器培养过程的流变特性。发现：1）当接种量较低时，反应器初始培养液呈现牛顿流体特性；当接种量大于8%时，反应器初始培养液呈现出假塑性流体特性。2）在反应器培养过程中不同时期红豆杉细胞悬浮培养液均为非牛顿流体，细胞浓度是构成培养液表观粘度的主要因素。3）建立了表观粘度及其影响因素的定量关系。结果表明：可以通过测定反应器培养液表观粘度或电导率来表征细胞的生长状况，并实现快速对红豆杉细胞生长进行监测与控制；单位细胞浓度培养液表观粘度主要是受粒径≥1700m（10目）的细胞聚集体影响，其组成比越大，单位浓度细胞悬浮培养液的表观粘度越大；粒径在1700～830m （10～20目）、380～180m（40～80目）的细胞聚集体对表观粘度的影响次之，830～380m（20～40目）的细胞聚集体对表观粘度的影响为零。
（3）建立了反应器培养流变动力学模型，提出能量损益是影响基质充分条件下搅拌反应器中流变特性的重要因素。
（4）提出了实际培养过程氧传质系数的测定方法，研究了细胞生物量X、表观粘度以及搅拌器型式、搅拌转速和通气量变化对氧传质系数kLa的影响。结果表明：可以通过在线测定k La来表征细胞生物量和表观粘度，对培养过程实施在线控制；本实验条件下通气量对值的影响大于搅拌转速对kLa的影响。因此，细胞培养的操作优化应优先考虑改变通气量。提高通气量，适当降低搅拌转速，既有利于增加反应器的供氧能力，又有利于减小搅拌对细胞造成的剪切伤害。
（5）研究了能量损益系数、能量损益速率随细胞生物量变化的规律：能量损益消耗的碳源和能量在细胞指数生长初期较大；与能量损益相关的产物合成速率在细胞浓度和产物浓度之间起着关键的调节作用。要提高产物合成的速率，就要使细胞浓度小于或大于与能量损益相关的产物合成速率最大值对应的细胞浓度，如采用二相培养、培养分离耦合、二阶段培养、补料培养。
（6）研究了能量损益系数、能量损益速率随细胞生物量变化的规律，提出了限制性分次加入碳源培养策略，与用含蔗糖30g·l-1的培养基进行的曼地亚红豆杉细胞培养相比，限制性分次加入碳源培养所得细胞生物量和紫杉醇产量分别增加了17.64%和14.88%，表明所提出的限制性分次加入碳源培养是减小基质消耗能量损益、提高紫杉醇产量的可行的途径和方法，所建立的模型对指导反应器培养、提高紫杉醇产量具有重要的意义。
Taxol, a diterpenoid secondary metabolite of the Taxus species, is an effectiveanticancer drug. Cell suspension cultures of Taxus media in a bioreactor is an effectivemethod for taxol production. Although signifcant progress on Taxus cell culture forproduction of Paclitaxel has been made recently，commercial applications of cell culturefor paclitaxel production is still limited by the productivity of paclitaxel， kineticsimulation and scale-up for cell culture in a reactor.
The objective of this work was to study fluid flow, mass transfer, cell growth andbiochemical reaction in a bioreactor, to establish the mathematical models suitable forsuspension cultures of Taxus media cells,and to provide a theoretical basis for the reactordesign, amplification and operation optimization by combining the cell culture andprocess engineering theory. The main research conclusions in this paper are as follows:
(1) This work constituted a set of unstructured kinetic models suitable for suspensioncultures of Taxus media cells in bioreactors under substrate-sufficient conditions，including a logistic model for cell growth and models incorporating energy spilling forsubstrate consumption and product synthesis.
Results of cross-validation and external validation demonstrated that a set ofunstructured kinetic models constituted.in this paper should be suitable for suspensioncultures of Taxus media cells in a15-L bioreactor under substrate-sufficient conditions,and helpful in the process prediction and operation optimization to guide the productionand amplification of Taxus media cell suspension cultures in bioreactors. Energy spillingwas a key factor that must be considered in a stirred bioreactor under substrate-sufficientconditions. The influences of energy spilling on cell growth and biochemical reactions in astirred bioreactor under substrate-sufficient conditions were so significant that limitingsubstrate excess conditions should be maintained for scaled-up cultures of Taxus cells in abioreactor to reduce energy spilling.
(2) The determination method of apparent viscosity of Taxus media cell suspensionculture broth was proposed. Results showed that the initial cultures broth of Taxus media suspension cells were rheologically Newtonian fluid when cell inoculum size was equal orless than8%and the initial broth rheology exhibited non-Newtonian behavior when cellinoculum size was more than8%. Results also demonstrated that the suspension culturesbroth of Taxus media cells at different culture time exhibited pseudoplastic non-Newtonianfluid, and the cell concentration was the main factor of apparent viscosity of culture broth.This work established quantitative relation between the apparent viscosity and influencingfactors.
(3) This work constituted a unstructured kinetic models of rheological behavior，proposed that energy spilling was an important factor which influenced the rheologicalbehavior.
(4) The determination method of the oxygen transfer coefficient of cell suspensionculture broth was proposed，and the influences of cell biomass, apparent viscosity, agitatortype, stirring speed and ventilation on oxygen transfer coefficient of cell suspensionculture broth were studied.
(5) The changes of the energy spilling-associated coefficients，the rates of the energyspilling-associated coefficient with culture time were studied，Results showed that carbonand energy consumed by energy spilling were larger during the early stage of theexponential growth phase，the energy spilling-associated rate of taxol synthesis played akey regulatory role between cell biomass and product concentration. To make larger therate of product synthesis and improve product concentration，it was necessary to try toreduce or increase the cell concentration in the culture broth，for example，two phaseculture， culture separation coupling，two stage culture，fed-batch culture.
(6) An optimized operation strategy of limiting added carbon several times culture wasproposed. A17.64%increase in cell growth was obtained，and a14.88%increase in taxolproduction was also obtained， while limiting added carbon several times wasexperimentally implemented in the15-L bioreactor.
（1）在基质充分条件下，细胞中基质的消耗不能有效地调整到与细胞外底物水平相适合，过量的基质通过形成代谢物和能量用于细胞生长、维持、形成产物和能量损益，如膜电位损耗、ATP水解作用、无效的循环、形成储存的化合物或胞外产品、代谢解耦、呼吸链的改变等等，因此建立了适用于基质充分条件下15L搅拌生物反应器中红豆杉细胞悬浮培养整合了能量损益的基质消耗动力学模型、产物合成动力学模型和系统流变特性变化动力学模型，同时发现logistic方程能更好地描述细胞生长过程。
内部交叉验证和外部验证表明：所建立的细胞生长模型、基质消耗模型和产物合成模型适用于模拟细胞培养的实际过程，模型可以用于指导反应器培养；能量损益是影响基质充分条件下搅拌反应器中红豆杉细胞生长和生化反应的重要因素，红豆杉细胞反应器放大培养应尽量维持限制基质过量的条件下进行。
（2）研究了反应器初始培养液和反应器培养过程的流变特性。发现：1）当接种量较低时，反应器初始培养液呈现牛顿流体特性；当接种量大于8%时，反应器初始培养液呈现出假塑性流体特性。2）在反应器培养过程中不同时期红豆杉细胞悬浮培养液均为非牛顿流体，细胞浓度是构成培养液表观粘度的主要因素。3）建立了表观粘度及其影响因素的定量关系。结果表明：可以通过测定反应器培养液表观粘度或电导率来表征细胞的生长状况，并实现快速对红豆杉细胞生长进行监测与控制；单位细胞浓度培养液表观粘度主要是受粒径≥1700m（10目）的细胞聚集体影响，其组成比越大，单位浓度细胞悬浮培养液的表观粘度越大；粒径在1700～830m （10～20目）、380～180m（40～80目）的细胞聚集体对表观粘度的影响次之，830～380m（20～40目）的细胞聚集体对表观粘度的影响为零。
（3）建立了反应器培养流变动力学模型，提出能量损益是影响基质充分条件下搅拌反应器中流变特性的重要因素。
（4）提出了实际培养过程氧传质系数的测定方法，研究了细胞生物量X、表观粘度以及搅拌器型式、搅拌转速和通气量变化对氧传质系数kLa的影响。结果表明：可以通过在线测定k La来表征细胞生物量和表观粘度，对培养过程实施在线控制；本实验条件下通气量对值的影响大于搅拌转速对kLa的影响。因此，细胞培养的操作优化应优先考虑改变通气量。提高通气量，适当降低搅拌转速，既有利于增加反应器的供氧能力，又有利于减小搅拌对细胞造成的剪切伤害。
（5）研究了能量损益系数、能量损益速率随细胞生物量变化的规律：能量损益消耗的碳源和能量在细胞指数生长初期较大；与能量损益相关的产物合成速率在细胞浓度和产物浓度之间起着关键的调节作用。要提高产物合成的速率，就要使细胞浓度小于或大于与能量损益相关的产物合成速率最大值对应的细胞浓度，如采用二相培养、培养分离耦合、二阶段培养、补料培养。
（6）研究了能量损益系数、能量损益速率随细胞生物量变化的规律，提出了限制性分次加入碳源培养策略，与用含蔗糖30g·l-1的培养基进行的曼地亚红豆杉细胞培养相比，限制性分次加入碳源培养所得细胞生物量和紫杉醇产量分别增加了17.64%和14.88%，表明所提出的限制性分次加入碳源培养是减小基质消耗能量损益、提高紫杉醇产量的可行的途径和方法，所建立的模型对指导反应器培养、提高紫杉醇产量具有重要的意义。
Taxol, a diterpenoid secondary metabolite of the Taxus species, is an effectiveanticancer drug. Cell suspension cultures of Taxus media in a bioreactor is an effectivemethod for taxol production. Although signifcant progress on Taxus cell culture forproduction of Paclitaxel has been made recently，commercial applications of cell culturefor paclitaxel production is still limited by the productivity of paclitaxel， kineticsimulation and scale-up for cell culture in a reactor.
The objective of this work was to study fluid flow, mass transfer, cell growth andbiochemical reaction in a bioreactor, to establish the mathematical models suitable forsuspension cultures of Taxus media cells,and to provide a theoretical basis for the reactordesign, amplification and operation optimization by combining the cell culture andprocess engineering theory. The main research conclusions in this paper are as follows:
(1) This work constituted a set of unstructured kinetic models suitable for suspensioncultures of Taxus media cells in bioreactors under substrate-sufficient conditions，including a logistic model for cell growth and models incorporating energy spilling forsubstrate consumption and product synthesis.
Results of cross-validation and external validation demonstrated that a set ofunstructured kinetic models constituted.in this paper should be suitable for suspensioncultures of Taxus media cells in a15-L bioreactor under substrate-sufficient conditions,and helpful in the process prediction and operation optimization to guide the productionand amplification of Taxus media cell suspension cultures in bioreactors. Energy spillingwas a key factor that must be considered in a stirred bioreactor under substrate-sufficientconditions. The influences of energy spilling on cell growth and biochemical reactions in astirred bioreactor under substrate-sufficient conditions were so significant that limitingsubstrate excess conditions should be maintained for scaled-up cultures of Taxus cells in abioreactor to reduce energy spilling.
(2) The determination method of apparent viscosity of Taxus media cell suspensionculture broth was proposed. Results showed that the initial cultures broth of Taxus media suspension cells were rheologically Newtonian fluid when cell inoculum size was equal orless than8%and the initial broth rheology exhibited non-Newtonian behavior when cellinoculum size was more than8%. Results also demonstrated that the suspension culturesbroth of Taxus media cells at different culture time exhibited pseudoplastic non-Newtonianfluid, and the cell concentration was the main factor of apparent viscosity of culture broth.This work established quantitative relation between the apparent viscosity and influencingfactors.
(3) This work constituted a unstructured kinetic models of rheological behavior，proposed that energy spilling was an important factor which influenced the rheologicalbehavior.
(4) The determination method of the oxygen transfer coefficient of cell suspensionculture broth was proposed，and the influences of cell biomass, apparent viscosity, agitatortype, stirring speed and ventilation on oxygen transfer coefficient of cell suspensionculture broth were studied.
(5) The changes of the energy spilling-associated coefficients，the rates of the energyspilling-associated coefficient with culture time were studied，Results showed that carbonand energy consumed by energy spilling were larger during the early stage of theexponential growth phase，the energy spilling-associated rate of taxol synthesis played akey regulatory role between cell biomass and product concentration. To make larger therate of product synthesis and improve product concentration，it was necessary to try toreduce or increase the cell concentration in the culture broth，for example，two phaseculture， culture separation coupling，two stage culture，fed-batch culture.
(6) An optimized operation strategy of limiting added carbon several times culture wasproposed. A17.64%increase in cell growth was obtained，and a14.88%increase in taxolproduction was also obtained， while limiting added carbon several times wasexperimentally implemented in the15-L bioreactor.
引文
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