生物工程下游技术-第一章.ppt

1、第一章生物反应器Bioreactor,SectionOne,Abioreactorisavesselinwhichiscarriedoutachemicalprocesswhichinvolvesorganismsorbiochemicallyactivesubstancesderivedfromsuchorganisms.Bioreactorsarecommonlycylindrical,ranginginsizefromsomelitertocubemeters,andareoftenmadeofstainlesssteel.,Bioreactor,Bioreactordesignisqu

2、iteacomplexengineeringtask.Underoptimumconditionsthemicroorganismsorcellswillreproduceatanastoundingrate.Thevesselsenvironmentalconditionslikegas(i.e.,air,oxygen,nitrogen,carbondioxide)flowrates,temperature,pHanddissolvedoxygenlevels,andagitationspeedneedtobecloselymonitoredandcontrolled.,Onebioreac

3、tormanufacturer,Broadley-JamesCorporation,usesvessels,sensors,controllers,andacontrolsystem,digitallynetworkedtogetherfortheirbioreactorsystem.,1.Introduction,Crudemedicinalpreparationsthatwereaqueousoralcoholicextractsofplantmaterialswereknownforcenturiestopractitionersoftheindigenousmethodsofmedic

4、ine.,Thepain-killingsalicylatesandantimalarialcompoundsextractedfromthebarkofcertaintreesarenotableexamplesofoldermedicines.Similarly,animalorganssuchasthepancreas,placenta,andtheurineofpregnantfemaleshavebeenasourceofhormonesfortherapeuticuse.,Untilrecently,humanalbuminwasmanufacturedfrompoolsofhum

5、anplacentacollectedfromThirdWorldcountries.Butthehighriskofviruscontaminationfromunidentifieddonorsofplacentaandtheimpracticalityofidentifyingthedonors,forcedthediscontinuationofthisprocess.Today,plasmafromunpaiddonorsisthemajorsourceofalbuminandtheriskoftransmissionofvirusescallsforextensivepurific

6、ationincludingtheuseofdedicatedvirusremovalandinactivationstepstorendertheproductsafeforhumanuse.,Althoughcrudepreparationsfromplantoranimalsourcesarestillusedasmedicinalsinsomepartsoftheworld,modernmedicinesinmostcountriesareextremelypure.Thehighlevelofdrugsafetyandpurityaredemandedbytheregulatorya

7、uthoritiesinsuchcountriesastheUnitedStates,Europe,andJapan.Fortunately,thebiopharmaceuticalindustriesareabletomeetthestringentdemandsbecausetheyhaveaccesstoavarietyofexcellentpurificationtechniques.,Thescienceofbiotechnologycoverstheexploitationofmicroorganismsandcellcultures,whichformthemajorsource

8、ofhighvaluecompounds.Morerecently,geneticistshavesucceededinbreedingtransgenicsheepandgoats,andmethodshavebeendevelopedtogettheseanimalstoexpressthedesiredproductsintheirmilk.Theindustrytodaymanufacturesonalargescalecompoundsthatwouldotherwisehavebeendifficult,ifnotimpossible,toproduceinsignificantq

9、uantitiesfortreatingmanydiseases.,Whetherproducedfromplants,animaltissue,microorganisms,orfromcellculture,thedesiredproductsarepresentinrathercomplexprocessstreamsandneedextensivepurification.Agreatmajorityoftheseproductsareproteins,whichmakesthistaskevenmoredifficult.Ifthesewerenonproteinmolecules,

10、suchasantibioticsforexample,onecouldusesimplersolventextractionmethodstoisolatethecompoundsfromthesolutionsinwhichtheyarepresent.,Thus,inthebiotechnologyindustry,thereisquiteachallengetothebiochemistsandchemicalengineersinthedownstreamprocessingdepartmentsofthecompanies.Theyemploydiversepurification

11、methodsintheresearchlaboratoryatthebenchscaleandtheseareeventuallyscaleduptotheproductionfloor.Themethodsareusedincomplementaryfashiontodevelopcost-effectivemethodsinquicktimeandenablethecompaniestobringtheproductstomarketaheadoftheircompetitors.,Thischapterattemptstogivethereaderanoverviewofthetech

12、niquesavailablefordownstreampurificationofbiotechnologyproducts.Readersareadvisedtorefertospecificchaptersinlatersectionsofthisvolumewherethesetechniquesaredescribedindetail.,Asstatedbefore,theindustrymanufacturesproductsfromanumberofsourcesandtheirdownstreamprocessingvariesnotonlyfromproducttoprodu

13、ct,butalsovariesdependingonthesourceoftheproduct.Eachprocess,therefore,needstobefinelytailoreddependingonthepropertiesoftheproductandtheprocessstreamfromwhichitisrecoveredandpurified.,2.ManufacturingProcessesintheIndustry,2.1.ProductsofRecombinantBacterialFermentation,Thefirststepintheseprocessesist

14、heseparationofthebiomassfromitssurroundingbroth.Theproteinofinterestisexpressedwithinthecellasasolubleprotein,butitisquiteoftenpresentintheformofaninsolublerefractivemasscalledthe“inclusionbodies.”,Therecoveryofthebiomassissometimesperformedbypreparativecentrifugation,butthepreferredmethodtodayisbym

15、eansoftangential-flowfiltrationsystemsusingmicroporousmembranesofappropriateporediameters.,ThedifferentfiltermanufacturerslikeMilliporeCorporation(Bedford,MA),PallCorp.(PortWashington,NY)andothercompaniesoffermembraneswith0.22,0.45,and0.65mporesizes,andthescientistsdevelopingtheprocessselectthemembr

16、anesbestsuitedtotheirneedsofbiomassconcentration.,Theparticulatesfromtheprocessfluidscangetintothemembraneporesandcauseasignificantdropinfiltrationrates.Thephenomenoncanbecontrolledbyfinetuningtheprocessconditionstoobtaintheoptimumfeedandpermeateflowratesandtransmembranepressures.,Thecompositionofth

17、efermentationbrothscanhavesignificanteffectsonthefiltrationrates.Onecomponentthathassuchanimpactistheantifoamusedtocontrolfoamingduringfermentation.Thesehydrophobicchemicalsarequicklyabsorbedtothesurfaceofthemembraneandcauseadropinflux.,Undercertainprocessconditionssuchastemperatureforexample,somean

18、tifoamscomeoutofsolutiontoforminsolublemicelles,whichcaneasilyadsorbtothemembranesurface.Therefore,anappropriateantifoamisselectedforthefermentationprocessbearinginmindthedownstreamprocessingsteps.,Duringcellharvesting,simultaneouscellwashing(alsoreferredtoasdiafiltration)canbeperformedbyaddingasuit

19、ablesolutiontothecellconcentrate,whichalsohelpstomaintainthedesiredpHorionicstrengthofthecellsuspensiontoavoidcelllysis.,Diafiltrationalsohelpstowashawaythesolubleimpuritiesfromtheprocessstream.Thisstepisusuallystartedwhenthecellconcentrationreachesaspecificpointwhererapidfluxdecayisobserved.,2.2.Ce

20、llLysisandClarificationoftheLysate,Therecoveredbacterialcellmassisnextlysedbymechanicalcelldisruptionunderhighpressure.Thisstepreleasesthedesiredproductfrominsidethecellsforfurtherprocessing.,Thelysate,whichconsistsofbothsolubleandinsolublecomponents,notablythecelldebris,isthenclarifiedbyatangential

21、flowfiltrationstepwithanappropriatemembranedevice.,Here,onceagain,thechoiceoftherightmicroporousmembraneiscritical.Thesmallerporediameters,suchasthe0.22m,performbetter.Thelargerporescangetpluggedbythecelldebrisorotherparticulatecontaminants.,However,ultrafiltrationmembraneswithevensmallerporediamete

22、rsmostoftenperformbetterthanthemicroporousmembranesbecausethedebriscannotgetlodgedinthepores.,Onecan,therefore,avoidflowdecay.However,thefluxesthroughtheultrafiltrationmembranesare,ingeneral,lowerthanthosewiththemicroporousmembranes.Ifthedesiredproteinisinthesolublefractionofthelysate,itpassesthemem

23、braneinthissteptoendupinthepermeateanditisthensenttothenextpurificationstep.,Iftheproductispresentasinclusionbodies,itispresentintheretentateoftheabovestepandhastobefirstsolubilizedbytheadditionofanagentsuchasguanidineorurea.Thesolubilizedproteinisthenseparatedfromtheparticulatesbyultrafiltration.Th

24、eselectedmembraneshouldpermitthepassageofthesolubilizedproteinwhileretainingthedebrisandparticulatesintheretentate.,2.3.HarvestingMammalianCellCultures,Thedesiredproductsinthesefermentationprocessesareintheextracellularfraction.Ifthecellsarelysedduringthecellconcentration,theintracellularproteinscan

25、spilloutofthecellsandcontaminatetheextracellularproduct.,Theextremelyfragilemammaliancells,therefore,needcarefulhandling.Anelevatedtransmembranepressureandhighfiltrationratescandamagethecells.Anexcellentmembrane-basedtangential-flowfiltrationsystemwasdevelopedbyMilliporeintheearly1980s.,Thesystemcon

26、tainsamicroporousmembrane,usuallywithporediametersof0.45m,afeedpumpmuchlikeintheconventionalTFFsystems,butapermeatepumpreplacestheusualvalveusedforrestrictingthepermeateflow.Thesecondpumphelpstoaccuratelycontrolthepermeatefluxandtomaintainalowtransmembranepressure.Underhightransmembranepressures,the

27、fragilecellscanbepushedintothemembraneporesandgetdamaged.Withthesesystems,ahighproductrecoverycanbeachievedwithoutcelllysis.Diafiltrationhelpstofurtherimproveproductrecovery.,Aggregatesofproteinsandcolloidalmaterialarealsoretained,andcaremustbetakentomakesurethatthedesiredproteinisrecoveredingoodyie

28、ldsinthepermeate.Washingtheretentatewithasuitablebufferhelpstoimprovetheproteinrecoveryinthepermeate.Theproductisthensentforfurtherpurification.,SectionTwo,第一节概述各种细胞及其代谢产物的生产过程都要通过细胞的培养，而细胞培养所用的装置就是反应器。生物反应器的作用：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。生物反应器：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

29、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？,一、好的细胞株系二、良好的环境条件1、良好的物理环境：最主要的有温度、pH、溶氧量、合适的混合强度以保证细胞与营养物的接触及细胞的悬浮等。2、合适的化学环境：要求有合适的各种营养物的浓度，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,1、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。2、对已有的生物反应器进行分析，达到优化的目的。3、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。（生物反应器是工程学的一部分也是化学工程

30、的一个分支）,化学工程还包括下面几个重要的内容,1、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、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、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定。,第二节细胞生长及代谢过程动力学,一、细胞生长的特点、描述方法的分类二、细胞的浓度及其测量三、均衡生长模型四、其它模型,一、细胞生长的特点、描述方法的分类,（一）细胞培养1、细胞培养的一般条件温度pH渗透压营养物水无菌条件光气体,2、动物细胞培养的特殊条件,(1)血清：动物细胞离体培养常常需要血清。最常用的是小牛血清

31、。血清提供生长必需因子，如激素、微量元素、矿物质和脂肪。(2)支持物：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。(3)气体交换：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、植物细胞培养的特殊条件,(1)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。(2)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本的激素。,4、微生物细胞培养的特殊条件,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

32、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。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,常用的有：,反应速率：单位时间物质浓度的变化量。如：细胞的生长速率、代谢产物的生成速率等。得率系数：两种物质得失之间的计量比。如：菌体的生成量对基质消耗量的得率系数。比速率：单位浓度的菌体、单位时间引起某物质浓度的变化量。如：菌体的比生长速率、基质的比消耗速率、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，即反应器内各点浓度及其它条件均一。活塞流式，即反应器内物质沿一定方向流动，完全没有

33、反向混合。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,是否考虑细胞内部复杂的结构是否考虑细胞之间的差别,4种模型,非离散的结构模型,文献上简称结构模型。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

34、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：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,简称均衡生长模型。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