生物工程下游技術(shù)-第一章.ppt

1、第一章 生物反應(yīng)器Bioreactor,Section One,A 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 of sta

2、inless 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 disso

3、lved oxygen 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. Introduction,Crude medicinal preparatio

4、ns that were aqueous or alcoholic extracts of plant materials were known for centuries to practitioners of the indigenous methods of medicine.,The pain-killing salicylates and antimalarial compounds extracted from the bark of certain trees are notable examples of older medicines. Similarly, animal o

5、rgans such as the pancreas, placenta, and the urine of pregnant females have been a source of hormones for therapeutic use.,Until recently, human albumin was manufactured from pools of human placenta collected from Third World countries. But the high risk of virus contamination from unidentified don

6、ors of placenta and 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 inac

7、tivation steps to render the product safe for human use.,Although 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. The high level of drug safety and purity are demanded by the regulatory au

8、thorities in such countries 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.,The science of biotechnology covers the exploitation of microorganisms

9、 and cell cultures, which form the major source of high value compounds. More 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. The industry today manufactures on a large sc

10、ale compounds that would otherwise have been difficult, if not impossible, to produce in significant quantities for treating many diseases.,Whether produced from plants, animal tissue, microorganisms, or from cell culture, the desired products are present in rather complex process streams and need e

11、xtensive purification. A great majority 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 prese

12、nt.,Thus, in the biotechnology industry, there is quite a challenge to the biochemists and chemical engineers in the downstream processing departments of the companies. They employ diverse purification methods in the research laboratory at the bench scale and these are eventually scaled up to the pr

13、oduction floor. The methods are used in complementary fashion to develop cost-effective methods in quick time and enable the companies to bring the products to market ahead of their competitors.,This chapter attempts to give the reader an overview of the techniques available for downstream purificat

14、ion of biotechnology products. Readers are advised to refer to specific chapters in later sections of this volume where these techniques are described in detail.,As stated before, the industry manufactures products from a number of sources and their downstream processing varies not only from product

15、 to product, but also varies depending on the 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.,2. Manufacturing Processes in the Industry,2.1. Products of Recombinant

16、 Bacterial Fermentation,The first step in these processes 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.”,T

17、he recovery of the biomass is sometimes performed by preparative centrifugation, but the preferred method today is by means of tangential-flow filtration systems using microporous membranes of appropriate pore diameters.,The different filter manufacturers like Millipore Corporation (Bedford, MA), Pa

18、ll Corp. (Port Washington, NY) and other companies offer membranes 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.,The particulates from the process fluids can get into the membrane pores and

19、cause a significant drop in filtration rates. The phenomenon can be controlled by fine tuning the process conditions to obtain the optimum feed and permeate flow rates and transmembrane pressures.,The composition of the fermentation broths can have significant effects on the filtration rates. One co

20、mponent that has such an impact is the antifoam used to control foaming during fermentation. These hydrophobic chemicals are quickly absorbed to the surface of the membrane and cause a drop in flux.,Under certain process conditions such as temperature for example, some antifoams come out of solution

21、 to form insoluble micelles, which can easily adsorb to the membrane surface. Therefore, an appropriate antifoam is selected for the fermentation process bearing in mind the downstream processing steps.,During cell harvesting, simultaneous cell washing (also referred to as diafiltration) can be perf

22、ormed by adding a suitable solution to the cell concentrate, which also helps to maintain the desired pH or ionic strength of the cell suspension to avoid cell lysis.,Diafiltration also helps to wash away the soluble impurities from the process stream. This step is usually started when the cell conc

23、entration reaches a specific point where rapid flux decay is observed.,2.2. Cell Lysis and Clarification of the Lysate,The 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 processin

24、g.,The lysate, which consists of both soluble and insoluble components, notably the cell debris, is then clarified by a tangential flow filtration step with an appropriate membrane device.,Here, once again, the choice of the right microporous membrane is critical. The smaller pore diameters, such as

25、 the 0.22 m, perform better. The larger pores can get plugged by the cell debris or other particulate contaminants.,However, ultrafiltration membranes with even smaller pore diameters most often perform better than the microporous membranes because the debris cannot get lodged in the pores.,One can,

26、 therefore, avoid flow decay. However, the fluxes through the ultrafiltration membranes are, 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

27、 to the next purification step.,If the product is present as inclusion bodies, it is present in the 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. T

28、he selected membrane should permit the passage of the solubilized protein while retaining the debris and particulates in the retentate.,2.3. Harvesting Mammalian Cell Cultures,The desired products in these fermentation processes are in the extracellular fraction. If the cells are lysed during the ce

29、ll concentration, the intracellular proteins can spill out of the cells and contaminate the extracellular product.,The extremely fragile mammalian cells, therefore, need careful handling. An elevated transmembrane pressure and high filtration rates can damage the cells. An excellent membrane-based t

30、angential-flow filtration system was developed by Millipore in the early 1980s.,The system contains a microporous 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 flo

31、w. The second pump helps to accurately control the permeate flux and to maintain a low transmembrane pressure. 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.

32、Diafiltration helps to further improve product recovery.,Aggregates of proteins and colloidal material are also retained, 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 protei

33、n recovery in the permeate. The product is then sent for further purification.,Section Two,第一節(jié) 概述 各種細胞及其代謝產(chǎn)物的生產(chǎn)過程都要通過細胞的培養(yǎng)，而細胞培養(yǎng)所用的裝置就是反應(yīng)器。 生物反應(yīng)器的作用：就是要為細胞代謝提供一個優(yōu)化的物理及化學環(huán)境，使細胞能更快更好地生長，得到更多的需要的生物量或代謝產(chǎn)物。 生物反應(yīng)器：生物反應(yīng)器是利用酶或生物體（如微生物）所具有的生物功能，在體外進行生化反應(yīng)的裝置系統(tǒng)，是一種生物功能模擬機，如發(fā)酵罐、固定化酶或固定化細胞反應(yīng)器等。,如何使細胞生長的更快更好？,一、好

34、的細胞株系 二、良好的環(huán)境條件 1、良好的物理環(huán)境：最主要的有溫度、pH、溶氧量、合適的混合強度以保證細胞與營養(yǎng)物的接觸及細胞的懸浮等。 2、合適的化學環(huán)境：要求有合適的各種營養(yǎng)物的濃度，并限制各種妨礙生長代謝的有毒物質(zhì)的濃度。,研究生物反應(yīng)器的目的,1、確定為達到一定的生產(chǎn)目的需要多大的生物反應(yīng)器，什么樣的結(jié)構(gòu)更好。 2、對已有的生物反應(yīng)器進行分析，達到優(yōu)化的目的。 3、分析各種生物反應(yīng)器的數(shù)據(jù)，從而對細胞的生長、代謝等過程有更加深入的理解。 （生物反應(yīng)器是工程學的一部分也是化學工程的一個分支）,化學工程還包括下面幾個重要的內(nèi)容,1、流體的輸送及混合。核心問題是流體之間動量的傳遞、機械能的轉(zhuǎn)

35、化。 2、熱量的傳遞。生物反應(yīng)器要考慮發(fā)酵熱的傳出以及發(fā)酵罐溫度的控制。 3、物質(zhì)的傳遞。生物反應(yīng)器內(nèi)進行著各種物質(zhì)傳遞過程，這些傳遞過程的強度主要由濃度差以及擴散的面積決定。,第二節(jié) 細胞生長及代謝過程動力學,一、細胞生長的特點、描述方法的分類 二、細胞的濃度及其測量 三、均衡生長模型 四、其它模型,一、細胞生長的特點、描述方法的分類,（一）細胞培養(yǎng) 1、細胞培養(yǎng)的一般條件 溫度 pH 滲透壓 營養(yǎng)物 水 無菌條件 光 氣體,2、動物細胞培養(yǎng)的特殊條件,(1)血清：動物細胞離體培養(yǎng)常常需要血清。最常用的是小牛血清。血清提供生長必需因子，如激素、微量元素、礦物質(zhì)和脂肪。 (2)支持物：大多數(shù)動

36、物細胞有貼壁生長的習慣。離體培養(yǎng)常用玻璃，塑料等作為支持物。 (3)氣體交換：二氧化碳和氧氣的比例要在細胞培養(yǎng)過程中不斷進行調(diào)節(jié)，不斷維持所需要的氣體條件。,3、植物細胞培養(yǎng)的特殊條件,(1)光照：離體培養(yǎng)的植物細胞對光照條件不嚴格，因為細胞生長所需要的物質(zhì)主要是靠培養(yǎng)基供給，但光照不但與光合作用有關(guān)，而且與細胞分化有關(guān)。 (2)激素：植物細胞的分裂和生長特別需要植物激素的調(diào)節(jié)，促進生長的生長素和促進細胞分裂的分裂素是最基本的激素。,4、微生物細胞培養(yǎng)的特殊條件,微生物多為單細胞生物，野生生存條件比較簡單。 所以微生物人工培養(yǎng)的條件比動植物細胞簡單得多。其中厭氧微生物培養(yǎng)比好氧微生物復雜。 微

37、生物對培養(yǎng)條件要求不如動植物細胞那樣苛刻，玉米漿、蛋白胨、麥芽汁、酵母膏等成為良好的微生物天然培養(yǎng)基。,（二）描述方法,常用的有：,反應(yīng)速率：單位時間物質(zhì)濃度的變化量。如：細胞的生長速率、代謝產(chǎn)物的生成速率等。 得率系數(shù)：兩種物質(zhì)得失之間的計量比。如：菌體的生成量對基質(zhì)消耗量的得率系數(shù)。 比速率：單位濃度的菌體、單位時間引起某物質(zhì)濃度的變化量。如：菌體的比生長速率、基質(zhì)的比消耗速率、產(chǎn)物的比生成速率。,理想流動和非理想流動,兩種理想流動模式 全混式，即反應(yīng)器內(nèi)各點濃度及其它條件均一。 活塞流式，即反應(yīng)器內(nèi)物質(zhì)沿一定方向流動，完全沒有反向混合。 實際反應(yīng)裝置常常介于兩者之間。,細胞生長的特點及細

38、胞群體的描述,細胞的生長、代謝是一個復雜的生物化學過程 與一般的化學過程不同，這個反應(yīng)體系的特點是，它是一種多相、多組分、非線性的體系。 細胞的培養(yǎng)和代謝還是一個復雜的群體的生命活動，通常每毫升培養(yǎng)液中含104-108個細胞。而且，像任何有生命的東西一樣，細胞也經(jīng)歷著新生、成長、成熟直至衰老的過程，在其生命的循環(huán)中，也存在退化與變異的問題。,細胞群體進行簡化假設(shè),是否考慮細胞內(nèi)部復雜的結(jié)構(gòu) 是否考慮細胞之間的差別,4種模型,非離散的結(jié)構(gòu)模型,文獻上簡稱結(jié)構(gòu)模型。這種模型把細胞分為具有不同生理功能的組分。 這種模型考慮到胞內(nèi)不同的結(jié)構(gòu)單元，對更精細地分析細胞的代謝調(diào)控是很重要的，其分析結(jié)果對于過程的優(yōu)化往往具有指導作用。 結(jié)構(gòu)模型考慮了胞內(nèi)各結(jié)構(gòu)單元的代謝及相互作用，因此列出的方程參數(shù)多、復雜，不容易解，即使用計算機求解也要花費相當?shù)臅r間，因此在過程控制中較少用這種模型。,離散型非結(jié)構(gòu)模型,把細胞分為幾種不同形態(tài)或功能的類別。總的細胞量是各類細胞量的和，各類細胞有不同的生理功能。 對于培養(yǎng)中細胞有明顯差別（形態(tài)、功能）的過程用此種離散模型最好。 缺點：分別測出各類細胞量是有困難的。,離散型結(jié)構(gòu)模型,細胞培養(yǎng)的實際情況。細胞之間不均一，細胞內(nèi)部