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1、第一章 生物反应器BioreactorBioreactorSection OneA bioreactor is a vessel in which is carried out a chemical process which involves organisms or biochemically active substances derived from such organisms.Bioreactors are commonly cylindrical,ranging in size from some liter to cube meters,and are often made o
2、f stainless steel.Bioreactor Bioreactor design is quite a complex engineering task.Under optimum conditions the microorganisms or cells will reproduce at an astounding rate.The vessels environmental conditions like gas(i.e.,air,oxygen,nitrogen,carbon dioxide)flow rates,temperature,pH and dissolved o
3、xygen levels,and agitation speed need to be closely monitored and controlled.One bioreactor manufacturer,Broadley-James Corporation,uses vessels,sensors,controllers,and a control system,digitally networked together for their bioreactor system.1.Introductionn Crude medicinal preparations that were aq
4、ueous or alcoholic extracts of plant materials were known for centuries to practitioners of the indigenous methods of medicine.n The pain-killing salicylates and antimalarial compounds extracted from the bark of certain trees are notable examples of older medicines.Similarly,animal organs such as th
5、e pancreas,placenta,and the urine of pregnant females have been a source of hormones for therapeutic use.nUntil recently,human albumin was manufactured from pools of human placenta collected from Third World countries.nBut the high risk of virus contamination from unidentified donors of placenta and
6、 the impracticality of identifying the donors,forced the discontinuation of this process.Today,plasma from unpaid donors is the major source of albumin and the risk of transmission of viruses calls for extensive purification including the use of dedicated virus removal and inactivation steps to rend
7、er the product safe for human use.nAlthough crude preparations from plant or animal sources are still used as medicinals in some parts of the world,modern medicines in most countries are extremely pure.nThe high level of drug safety and purity are demanded by the regulatory authorities in such count
8、ries as the United States,Europe,and Japan.Fortunately,the biopharmaceutical industries are able to meet the stringent demands because they have access to a variety of excellent purification techniques.nThe science of biotechnology covers the exploitation of microorganisms and cell cultures,which fo
9、rm the major source of high value compounds.nMore recently,geneticists have succeeded in breeding transgenic sheep and goats,and methods have been developed to get these animals to express the desired products in their milk.nThe industry today manufactures on a large scale compounds that would other
10、wise have been difficult,if not impossible,to produce in significant quantities for treating many diseases.nWhether produced from plants,animal tissue,microorganisms,or from cell culture,the desired products are present in rather complex process streams and need extensive purification.nA great major
11、ity of these products are proteins,which makes this task even more difficult.If these were nonprotein molecules,such as antibiotics for example,one could use simpler solvent extraction methods to isolate the compounds from the solutions in which they are present.nThus,in the biotechnology industry,t
12、here is quite a challenge to the biochemists and chemical engineers in the downstream processing departments of the companies.n They employ diverse purification methods in the research laboratory at the bench scale and these are eventually scaled up to the production floor.The methods are used in co
13、mplementary fashion to develop cost-effective methods in quick time and enable the companies to bring the products to market ahead of their competitors.nThis chapter attempts to give the reader an overview of the techniques available for downstream purification of biotechnology products.Readers are
14、advised to refer to specific chapters in later sections of this volume where these techniques are described in detail.nAs stated before,the industry manufactures products from a number of sources and their downstream processing varies not only from product to product,but also varies depending on the
15、 source of the product.Each process,therefore,needs to be finely tailored depending on the properties of the product and the process stream from which it is recovered and purified.n2.Manufacturing Processes in the Industry2.1.Products of Recombinant Bacterial FermentationnThe first step in these pro
16、cesses is the separation of the biomass from its surrounding broth.The protein of interest is expressed within the cell as a soluble protein,but it is quite often present in the form of an insoluble refractive mass called the“inclusion bodies.”nThe recovery of the biomass is sometimes performed by p
17、reparative centrifugation,but the preferred method today is by means of tangential-flow filtration systems using microporous membranes of appropriate pore diameters.nThe different filter manufacturers like Millipore Corporation(Bedford,MA),Pall Corp.(Port Washington,NY)and other companies offer memb
18、ranes with 0.22,0.45,and 0.65 m pore sizes,and the scientists developing the process select the membranes best suited to their needs of biomass concentration.nThe particulates from the process fluids can get into the membrane pores and cause a significant drop in filtration rates.The phenomenon can
19、be controlled by fine tuning the process conditions to obtain the optimum feed and permeate flow rates and transmembrane pressures.nThe composition of the fermentation broths can have significant effects on the filtration rates.One component that has such an impact is the antifoam used to control fo
20、aming during fermentation.These hydrophobic chemicals are quickly absorbed to the surface of the membrane and cause a drop in flux.nUnder certain process conditions such as temperature for example,some antifoams come out of solution to form insoluble micelles,which can easily adsorb to the membrane
21、surface.Therefore,an appropriate antifoam is selected for the fermentation process bearing in mind the downstream processing steps.nDuring cell harvesting,simultaneous cell washing(also referred to as diafiltration)can be performed by adding a suitable solution to the cell concentrate,which also hel
22、ps to maintain the desired pH or ionic strength of the cell suspension to avoid cell lysis.nDiafiltration also helps to wash away the soluble impurities from the process stream.This step is usually started when the cell concentration reaches a specific point where rapid flux decay is observed.2.2.Ce
23、ll Lysis and Clarification of the LysatenThe recovered bacterial cell mass is next lysed by mechanical cell disruption under high pressure.This step releases the desired product from inside the cells for further processing.nThe lysate,which consists of both soluble and insoluble components,notably t
24、he cell debris,is then clarified by a tangential flow filtration step with an appropriate membrane device.nHere,once again,the choice of the right microporous membrane is critical.The smaller pore diameters,such as the 0.22 m,perform better.The larger pores can get plugged by the cell debris or othe
25、r particulate contaminants.nHowever,ultrafiltration membranes with even smaller pore diameters most often perform better than the microporous membranes because the debris cannot get lodged in the pores.n One can,therefore,avoid flow decay.However,the fluxes through the ultrafiltration membranes are,
26、in general,lower than those with the microporous membranes.If the desired protein is in the soluble fraction of the lysate,it passes the membrane in this step to end up in the permeate and it is then sent to the next purification step.nIf the product is present as inclusion bodies,it is present in t
27、he retentate of the above step and has to be first solubilized by the addition of an agent such as guanidine or urea.The solubilized protein is then separated from the particulates by ultrafiltration.The selected membrane should permit the passage of the solubilized protein while retaining the debri
28、s and particulates in the retentate.2.3.Harvesting Mammalian Cell CulturesnThe desired products in these fermentation processes are in the extracellular fraction.If the cells are lysed during the cell concentration,the intracellular proteins can spill out of the cells and contaminate the extracellul
29、ar product.nThe extremely fragile mammalian cells,therefore,need careful handling.An elevated transmembrane pressure and high filtration rates can damage the cells.An excellent membrane-based tangential-flow filtration system was developed by Millipore in the early 1980s.nThe system contains a micro
30、porous membrane,usually with pore diameters of 0.45 m,a feed pump much like in the conventional TFF systems,but a permeate pump replaces the usual valve used for restricting the permeate flow.nThe second pump helps to accurately control the permeate flux and to maintain a low transmembrane pressure.
31、Under high transmembrane pressures,the fragile cells can be pushed into the membrane pores and get damaged.With these systems,a high product recovery can be achieved without cell lysis.Diafiltration helps to further improve product recovery.nAggregates of proteins and colloidal material are also ret
32、ained,and care must be taken to make sure that the desired protein is recovered in good yields in the permeate.Washing the retentate with a suitable buffer helps to improve the protein recovery in the permeate.The product is then sent for further purification.Section Two第一节第一节 概述概述 各种细胞及其代谢产物的生产过程都要
33、通过细胞的培养，而细胞培养所用的装置就是反应器反应器。生生物物反反应应器器的的作作用用：就是要为细胞代谢提供一个优化的物理及化学环境，使细胞能更快更好地生长，得 到 更 多 的 需 要 的 生 物 量 或 代 谢 产 物。生生物物反反应应器器：生物反应器是利用酶或生物体（如微生物）所具有的生物功能，在体外进行生化反应的装置系统，是一种生物功能模拟机，如发酵罐、固 定 化 酶 或 固 定 化 细 胞 反 应 器 等。如何使细胞生长的更快更好？如何使细胞生长的更快更好？一、好的细胞株系一、好的细胞株系二、良好的环境条件二、良好的环境条件1 1、良良好好的的物物理理环环境境：最最主主要要的的有有温温
34、度度、pHpH、溶溶氧氧量量、合合适适的的混混合合强强度度以以保保证证细细胞胞与与营营养养物物的的接接触触及及细细胞胞的的悬悬浮等。浮等。2 2、合合适适的的化化学学环环境境：要要求求有有合合适适的的各各种种营营养养物物的的浓浓度度，并限制各种妨碍生长代谢的有毒物质的浓度。并限制各种妨碍生长代谢的有毒物质的浓度。研究生物反应器的目的研究生物反应器的目的1 1、确定为达到一定的生产目的需要多大的生物反应、确定为达到一定的生产目的需要多大的生物反应器，什么样的结构更好。器，什么样的结构更好。2 2、对已有的生物反应器进行分析，达到优化的目的。、对已有的生物反应器进行分析，达到优化的目的。3 3、分
35、析各种生物反应器的数据，从而对细胞的生长、分析各种生物反应器的数据，从而对细胞的生长、代谢等过程有更加深入的理解。代谢等过程有更加深入的理解。（生物反应器是工程学的一部分也是化学工程的一（生物反应器是工程学的一部分也是化学工程的一个分支）个分支）化学工程还包括下面几个重要的内容化学工程还包括下面几个重要的内容1 1、流体的输送及混合。、流体的输送及混合。核心问题是流体之间动量的传递、机械能的转化。2 2、热量的传递。、热量的传递。生物反应器要考虑发酵热的传出以及发酵罐温度的控制。3 3、物质的传递。、物质的传递。生物反应器内进行着各种物质传递过程，这些传递过程的强度主要由浓度差以及扩散的面积决
36、定。第二节第二节 细胞生长及代谢过程动力学细胞生长及代谢过程动力学n一、细胞生长的特点、描述方法的分类一、细胞生长的特点、描述方法的分类n二、细胞的浓度及其测量二、细胞的浓度及其测量n三、均衡生长模型三、均衡生长模型n四、其它模型四、其它模型一、细胞生长的特点、描述方法的分类一、细胞生长的特点、描述方法的分类（一）细胞培养（一）细胞培养 1 1、细胞培养的一般条件、细胞培养的一般条件n温度温度npHpHn渗透压渗透压n营养物营养物 n水水 n无菌条件无菌条件 n光光 n气体气体 2 2、动物细胞培养的特殊条件、动物细胞培养的特殊条件n (1)(1)血血清清：动物细胞离体培养常常需要血清。最常用
37、的是小牛血清。血清提供生长必需因子，如激素、微量元素、矿物质和脂肪。n (2)(2)支支持持物物：大多数动物细胞有贴壁生长的习惯。离体培养常用玻璃，塑料等作为支持物。n (3)(3)气气体体交交换换：二氧化碳和氧气的比例要在细胞培养过程中不断进行调节，不断维持所需要的气体条件。3 3、植物细胞培养的特殊条件、植物细胞培养的特殊条件n(1)(1)光照：光照：离体培养的植物细胞对光照条件不严格，因为细胞生长所需要的物质主要是靠培养基供给，但光照不但与光合作用有关，而且与细胞分化有关。n (2)(2)激素：激素：植物细胞的分裂和生长特别需要植物激素的调节，促进生长的生长素和促进细胞分裂的分裂素是最基
38、本的激素。4 4、微生物细胞培养的特殊条件、微生物细胞培养的特殊条件n微生物多为单细胞生物，野生生存条件比较简单。微生物多为单细胞生物，野生生存条件比较简单。所以微生物人工培养的条件比动植物细胞简单得多。其中厌氧微生物培养比好氧微生物复杂。n微生物对培养条件要求不如动植物细胞那样苛刻微生物对培养条件要求不如动植物细胞那样苛刻，玉米浆、蛋白胨、麦芽汁、酵母膏等成为良好的微生物天然培养基。（二）描述方法（二）描述方法常用的有常用的有：反反应应速速率率：单单位位时时间间物物质质浓浓度度的的变变化化量量。如如：细细胞胞的生长速率、代谢产物的生成速率等。的生长速率、代谢产物的生成速率等。得得率率系系数数
39、：两两种种物物质质得得失失之之间间的的计计量量比比。如如：菌菌体体的生成量对基质消耗量的得率系数。的生成量对基质消耗量的得率系数。比比速速率率：单单位位浓浓度度的的菌菌体体、单单位位时时间间引引起起某某物物质质浓浓度度的的变变化化量量。如如：菌菌体体的的比比生生长长速速率率、基基质质的的比比消消耗速率、产物的比生成速率。耗速率、产物的比生成速率。理想流动和非理想流动两种理想流动模式全混式全混式，即反应器内各点浓度及其它条件均一。活活塞塞流流式式，即反应器内物质沿一定方向流动，完全没有反向混合。u实际反应装置常常介于两者之间。细胞生长的特点及细胞群体的描述细胞的生长、代谢是一个复杂的生物化学过程
40、与一般的化学过程不同，这个反应体系的特点是，它是一种多相、多组分、非线性的体系。细胞的培养和代谢还是一个复杂的群体的生命活动，通常每毫升培养液中含104-108个细胞。而且，像任何有生命的东西一样，细胞也经历着新生、成长、成熟直至衰老的过程，在其生命的循环中，也存在退化与变异的问题。细胞群体进行简化假设u是否考虑细胞内部复杂的结构u是否考虑细胞之间的差别4种模型（最理想情况）（最理想情况）把细胞群体处理把细胞群体处理为一种溶质为一种溶质非结构模型非结构模型非离散模型非离散模型细胞之间无差异，细胞之间无差异，细胞内有多个组分细胞内有多个组分（结构）（结构）结构模型结构模型均衡生长（假设）均衡生长
41、（假设）细胞之间不均一，细胞之间不均一，细胞内部多组分细胞内部多组分（实际情况）（实际情况）“平均细胞平均细胞”近似近似不考虑细胞结不考虑细胞结构，但各种细构，但各种细胞不均一胞不均一“平均细胞平均细胞”近似近似离散模型离散模型均衡生长（假设）均衡生长（假设）非离散的结构模型文献上简称结构模型简称结构模型。这种模型把细胞分为具有不同生理功能的组分。这种模型考虑到胞内不同的结构单元，对更精细地分析细胞的代谢调控是很重要的，其分析结果对于过程的优化往往具有指导作用。结构模型考虑了胞内各结构单元的代谢及相互作用，因此列出的方程参数多、复杂，不容易解，即使用计算机求解也要花费相当的时间，因此在过程控制
42、中较少用这种模型。离散型非结构模型把细胞分为几种不同形态或功能的类别。总的细胞量是各类细胞量的和，各类细胞有不同的生理功能。对于培养中细胞有明显差别（形态、功能）的过程用此种离散模型最好。缺点缺点：分别测出各类细胞量是有困难的。离散型结构模型细胞培养的实际情况。细胞之间不均一，细胞内部多组分。在求解和分析中最繁杂，应用较少。非结构非离散模型u简称均衡生长模型。u这种模型没有考虑细胞内部的结构，又不考虑细胞之间有任何差异。因此，可以把细胞用“浓度”这一个量来描述，即把细胞看成一种“溶质”，从而简化了胞内外的传递过程分析，也简化了过程的数学描述。u对于相当多的微生物过程分析，特别是过程控制来说，均
43、衡生长模型是可以满足要求的。二、细胞浓度及其测量二、细胞浓度及其测量细胞浓度在培养过程中是一个十分重要的参数。在定量研究生物反应之前，首先需要说明微生物的浓度即菌体浓度的表示方法。（g/l,kg/m3)n细胞干重法：细胞干重法：测量细胞浓度的最基本方法。n显微计数法：显微计数法：显微镜和血球计数器。n平板计数法：平板计数法：生理盐水稀释，记录菌斑。n浊度法：浊度法：波长600-700nm范围测量。直接测定法直接测定法间接测定法：间接测定法：测定构成细胞的大测定构成细胞的大分子物质来确定细分子物质来确定细胞浓度。胞浓度。三、均衡生长模型三、均衡生长模型1 1、细胞生长模型：、细胞生长模型：均衡生
44、长模型只用一个量来描述细胞的量，即生物量或细胞的浓度。通常用每毫升培养液中菌体个数或干菌重来描述。（1）细胞生长动力学曲线：p 将微生物在一个封闭体系中培养，测定培养过程中细胞浓度的变化，可得到细胞生长的动力学曲线。p 以间歇培养微生物为例，在培养过程中动力学曲线包括延迟期、指数生长期、禁止期、自溶期等阶段。（2）细胞生长动力学描述：u细胞生长速率的一个重要参数重要参数是比生长速率。u比生长速率比生长速率：比生长速率表示在单位体积内单位量细胞经过单位时间增加的细胞量。这种增加包括生长和繁殖两个部分。u比生长速率表示菌体增长的能力，它也受菌株菌株及各种物理化物理化学环境因素学环境因素的影响。2
45、2、基质消耗的模型、基质消耗的模型 基质包括细胞生长所需各种营养成分，其消耗主要基质包括细胞生长所需各种营养成分，其消耗主要有有三个方面三个方面：一一.是细胞的生长，合成新的细胞；是细胞的生长，合成新的细胞；二二.是细胞维持生命要消耗的能源物质；是细胞维持生命要消耗的能源物质；三三.是合成次级代谢产物。是合成次级代谢产物。3 3、产物生成动力学模型、产物生成动力学模型u 产物主要指的是细胞培养过程中产物主要指的是细胞培养过程中代谢生成的除代谢生成的除细胞量以外的产品。细胞量以外的产品。u 按照其生成特点，产物可分为按照其生成特点，产物可分为两类：两类：生长偶联生长偶联型型及及非生长偶联型非生长
46、偶联型。u 这这两者的区别两者的区别在于在于前者前者的生成只是在细胞生长的生成只是在细胞生长时才能生成，而时才能生成，而后者后者则只要有细胞存在就能生则只要有细胞存在就能生成。成。4 4、均衡生长动力学模型应用实例、均衡生长动力学模型应用实例u青霉素球状菌发酵青霉素球状菌发酵u实际在生物培养过程中，菌体的生长、基质的菌体的生长、基质的消耗及产物的生成三个方面是交织在一起的消耗及产物的生成三个方面是交织在一起的。菌体的生长消耗了基质，而基质浓度的变化又影响菌体的生长速度，对于产物也是这样。u因此，细胞培养整体的动力学模型是上述几个细胞培养整体的动力学模型是上述几个微分方程联立的结果。微分方程联立
47、的结果。已知初始条件即可以应用合适的数学方法对过程求解及分析。四、其它模型四、其它模型n均衡生长模型把细胞看成一个溶质，没有考虑胞内的结构和细胞之间的差别。在分析胞内的在分析胞内的诱导作用诱导作用、对工程菌进行动力学描述对工程菌进行动力学描述以及细胞的细胞的形态和功能有较大差异形态和功能有较大差异时，应用这个模型是不合适的。n在实际过程中，有时还需使用其它模型。比如，离散模型、结构模型等。Genetic engineering medicine project Interferon productionPlant tissue cultureGinseng tissue cultureQuestion1、What is bioreactor?