BPG層析柱標(biāo)準(zhǔn)裝柱流程規(guī)范

1、操作標(biāo)準(zhǔn)目的：為了使BPG300層析柱裝柱過程可重復(fù)，并能夠得到較好的柱效，故建立此操作規(guī)X。X圍：配合？KTAprocess系統(tǒng)進(jìn)展有效的蛋白質(zhì)純化操作。責(zé)任人：層析工序操作人員標(biāo)準(zhǔn)操作程序：1根本術(shù)語概念填料因子PackingFactor,PF：指在在裝柱過程中，用低流速如60cm/h對(duì)填料進(jìn)展沉降壓縮得到的調(diào)料高度和最后裝柱完成時(shí)填料高度柱高的比值，consolidatedbedheightPackingFactor=二工=丁匚*packedbedheightGEHC生產(chǎn)的各種常見填料的PF參見？GEHCAxiChrom300-1000columnsUserManual28-9562-8

2、9EditionAA?;packingfactorPF=(consolidatedbedheight/(packedbedheightThePackingFactortakesthesettlingspeedofmediaintoaccount.TheCompressionFactor(CF)pressionfactorCF=(gravitysettledbedheight)/(packedbedheight)ThereisadifferencebetweenPackingFactor

3、andCompressionFactor.ThedefinitionofPackingFactorinvolvesflowthroughthebedwhichinsomecasesleadstogmorecompressedbedandlowerconsolidatedbedheight,whileinothercasesthegravitysettledbedheightislowersincethebedpacksmoredenselyifgivenmoretimetodoso.MabselectSuRe,forexample,hasasignificantlylowergravityse

4、ttledheightthanitsconsolidatedheightandthishastobeconsideredwhencalculatingtheamountofmediatobepackedinthecolumn.線性流速linearflowrate：?jiǎn)挝粫r(shí)間內(nèi)液體流經(jīng)的直線距離，常用cm/h表示；體積流速volumeflowrate：?jiǎn)挝粫r(shí)間內(nèi)液體流經(jīng)的空間容積，常用ml/min表不；換算:ml線性流速(午)體枳流速一)=一6/X柱橫截面面積(門爐)min62準(zhǔn)備工作2.1 檢查層析柱的完整性，所帶配件，是否水平用水平儀矯正,是否清潔如有污染應(yīng)進(jìn)展清洗。2.2 確定所用填料及其特

5、性、濃度等以DEAESepharoseFastFlow填料為例，其填料因子PF為1.15,新填料slurry濃度為75%保存于20%乙醇中，耐壓3ban2.3 確定放大后的工藝參數(shù)以XX生化制藥股份XX的放大工藝為例，中試工藝參數(shù)：柱高h(yuǎn)=10cm；體積流速v1=3ml/min;柱內(nèi)徑d=1.6cm.即，線性流速v2=v1/s柱=3/(3.14X0.82)cm/min=1.5cm/min;樣品與填料接觸時(shí)間t=h/v2=10/1.5min=6.67min;在工藝的線性放大中h、v2和t均不變，那么可保證樣品出峰的位置洗脫時(shí)間和形狀不變。使用BPG300層析柱進(jìn)展放大的工藝參數(shù)：裝柱體積V柱=S

6、柱h=3.14X1.52X1L=7L;裝柱需要slurry體積Vslurry=V柱XPF/Cslurry以DEAESepharoseFastFlowW料，刃B么，Vslurry=7X1.15/0.75L=10.73L;裝入柱中的slurry高度1皿叮=丫皿叮/$柱=10.73/(3.14義1.52)dm=1.52dm=15.2cm.3管路安裝3.1 層析柱管道和配件安裝3.1.1 卸下柱子頂部和底部的盲堵墊圈，旋松密封調(diào)節(jié)器，用純水或20%乙醇充分浸潤柱內(nèi)壁和柱頭O型圈；3.1.2 用扳手對(duì)稱卸下支持桿連接到法蘭的螺絲，并通過旋轉(zhuǎn)高度調(diào)節(jié)手柄將柱頭旋至最高處，在柱頭頂部依次安裝壓力表和四路二通

7、閥，在柱底安裝四路二?KTAprocess 上端連3.1.3 在頂部四路二通閥上橫向連接一根細(xì)管，另一端連至接口COLUMNTOP,同時(shí)從柱底四路二通閥橫向接出一根細(xì)管至?KTAprocess的下端連接口COLUMNBOTTOM。圖1、BPG300層析柱安裝示意圖Tabi#1,Overviewof8P6columns日曲dhalghUcm)"Num白糖ColtimnDiumeter4mmiCollumnArea(cm1)ColumnRuckingv>ithoatcfi用口加nmngwitho»tentk>nJMlhMujmjRackingwithCKin&

8、;lonRunnmgwithcjitoniion9Man.pressurefbargl11TOtllweightAdapter阿CMerulldimehfi»h&cmjDxWMMHeightkJinMllnMa»L1007S.5州D2b344ao3.0工73£&1&7白恥4前1”IDO7&5752S415565?01?4.351B167在AM父1007&59545?Z7835425.76.1B177山覦AH熄花MO1鈉§0D?634妁00丸口136.95尊11如盛舞127MOIV75否415565J96J851D.

9、06?611的在異15?140154954554727E69白311.11?.D&?71159的加L72314葡0Z6M&&DOB2JD.7141A34135&j69k127200314芯居41酩6s7.612917.J前4A違135。延力妁Z200214為72第14117.0江白溫36珀13狗生如戈拄29663850口跖咕0.0179打金J10468舞砂工田MA打296683752SM55651722827&44.7473-勢(shì)69&Axl5829663895第547B31口RW旗.55"A7B勢(shì)69&9xl78446156?5

10、011?30曬172如46.970.?5?QDICT80wBDwl4D*4156Z75363B5162562S9479.7R6EZ52151DD30kEDxl&54照15K1DD6164727E953100011251Z1325一口1DDSDkEDkIW白山ekiiTHRd111dbsdM初出也瞳MdNGi*idilii”岫心Mi修詁一司TEKaftdla2加銀桿1即131104MifN.嗝吊七上加川比以1ilEl'sditiiti'iaifevkiBU白海占吊5艮日6上&巾+MLdti近LU'akaiachimaiihwri(kubapockinghM

11、niHri"VdIjihwht»*pactIr>9«rikused汩s>bdlnitii44moftbrbedf75司1出sbnumudfriInia1ht«4unnardFhhdwhmtht口修改ifc&y'ahiuwhKvahbwHerI&cptE.kntg«?dDii|anismaraulfiadiidlx*Llw6Dkirtw修Eh«duatibnlcUixfinuifl.T(KaclaptisrraialMiQl«EIcaiEmihIdtfiEcalumntube七sivdhql

12、BnuaminIhs-gLmikibi"Umamamrrdvlar口nKruhn*!%pimxuni.nTabluSind>3.2 層析柱下端篩網(wǎng)排氣泡3.2.1 連接兩根足夠長的細(xì)管到？KTAprocess進(jìn)樣泵的A1和B1進(jìn)口，兩管另一端均放入純水中；3.2.2 從A1泵入純水：翻開UNICORN軟件，在systemcontrol窗口點(diǎn)擊manualfvalvesInletA,選擇inletA1,insert;InletB,選擇closed,insert;Column,選擇upflow,insert；Pump-Flow,設(shè)定流速60L/h,點(diǎn)擊execute運(yùn)行程序充水至B

13、PG柱中2-3cm左右，pause去除下端篩網(wǎng)中的空氣，用注射器吸出篩網(wǎng)中殘留的氣泡。4裝填料4.1 準(zhǔn)備填料所用填料為DEAESepharoseFastFlow,裝置的是保存于20%乙醇中的新slurry;4.2 卸下柱頭，用攪拌棍引流參加混勻的slurry至15.2cm處，再參加純水至40cm左右，充分?jǐn)嚢杌靹颍瑫r(shí)防止擠壓篩網(wǎng)和柱壁，靜置使填料界面至少低于38cm；4.3 在systemcontrol窗口點(diǎn)擊manua口valvesInletA,選擇inletA1,insert;InletB,選擇closed,insert;Column,選擇downflow,insert;PumprFl

14、ow,設(shè)定流速60L/h，點(diǎn)擊execute運(yùn)行程序?qū)⒅^上端篩網(wǎng)充滿水以除去氣泡，再裝上柱頭，用密封調(diào)節(jié)器固定柱頭，擰緊螺絲；4.4 在systemcontrol窗口點(diǎn)擊manualPump-Flow,設(shè)定流速為42L/h60cm/h，進(jìn)展填料的沉降，待填料高度降至14cm左右，將密封調(diào)節(jié)器旋至全松，將柱頭快速調(diào)至距離填料3-4cm處，將密封調(diào)節(jié)器旋至半松，將柱頭快速調(diào)至濾網(wǎng)距離填料1-2cm,旋嚴(yán)密封調(diào)節(jié)器，再設(shè)定高流速如150L/h，繼續(xù)壓縮填料，最后再調(diào)節(jié)柱頭至10cmi整個(gè)過程一直有流速，泵速不能停。5柱效測(cè)定紫外吸收體積圖2、柱效測(cè)定示意圖5.1 平衡將A1接口細(xì)管放入0.4MNa

15、Cl溶液中，將柱頂端四路二通閥調(diào)至管路與大氣相連狀態(tài)，在systemcontrol窗口點(diǎn)擊manuaRvalvesInletA,選擇inletAI,insert;InletB,選擇closed,insert;Column,選擇downflow,insert;PumprFlow,再設(shè)定高流速如150L/h，點(diǎn)擊execute運(yùn)行，排氣泡，再將閥調(diào)至管路與柱頭相連狀態(tài)，平衡3-5個(gè)柱體積至電導(dǎo)曲線穩(wěn)定，點(diǎn)擊END;5.2 上樣5.2.1 將B1接口細(xì)管放入2MNaCl溶液，柱頂端四路二通閥調(diào)至管路與大氣相連狀態(tài)，在systemcontrol窗口點(diǎn)擊manuaRvalvesInletA,選擇Clos

16、ed,insert;InletB,選擇InletB1,insert;Column,選擇downflow,insert;PumprFlow,設(shè)定高流速150L/h點(diǎn)擊execute運(yùn)行，排氣泡，pause5.2.2 在systemcontro窗口,點(diǎn)擊manualOtherfEnd_Timer,選擇Acc.Volumn,Timeout設(shè)上樣量為70mL1%柱體積；PumprFlow,設(shè)定上樣流速60L/h,點(diǎn)擊execute的同時(shí)將柱頂端四路二通閥調(diào)至管路與柱頭相連，完成Columnpacking檢測(cè)裝柱效果一般用<0.5%（30um介質(zhì)）或<1%（90ue介質(zhì)）柱體積的1%丙酮測(cè)柱

17、效和峰形Thelinearflowrateshouldbe30cm/hfar34pmand50mediaand20cm/hfor90pmmedia上樣；5.3 洗脫5.3.1 將柱頂端四路二通閥調(diào)至管路與大氣相連狀態(tài)，在systemcontrol窗口點(diǎn)擊manualvalvesInletA,選擇inletAI,insert;InletB,選擇closed,insert;Column,選擇downflow,insert;Pump-Flow,設(shè)定高流速150L/h點(diǎn)擊execute運(yùn)行，排氣泡，同時(shí)將管路充滿0.4MNaCl溶液，調(diào)流速至21L/h30cm/h；5.3.2 待流速穩(wěn)定后，在syst

18、emcontrol窗口點(diǎn)擊manuaRvalvesInjectionMark,insert,點(diǎn)擊execute的同時(shí)將柱頂端四路二通閥調(diào)至管路與柱頭相連，開場(chǎng)洗脫，在一個(gè)柱體積左右，電導(dǎo)檢測(cè)下將產(chǎn)生目的峰。5.4 測(cè)柱效在Evaluation窗口中，選擇實(shí)驗(yàn)保存的結(jié)果，在結(jié)果圖中點(diǎn)擊鼠標(biāo)右鍵，選擇Properties,點(diǎn)擊Clear,并選中Cond_102,點(diǎn)擊OK?；氐紼valuation窗口，選擇積分按鈕，在彈出窗口中，選擇Cond_102，peakwindow中調(diào)整積分的X圍，并在RejectPeak即，填Peakmustbeoneof1largest確定后，在積分結(jié)果窗口，點(diǎn)擊右鍵，選

19、擇Properties,在peaktable選項(xiàng)中，勾選PlateHeightHETP和Asymmetry。確定后即可計(jì)算得到計(jì)算理論塔板高度HETP和對(duì)稱因子As。AppendixBPackingbufferinformationPackingbuffersandpackingfactorsPackingvaluesNot艮ThePacingFccrarrangeusedinthevendedpachngprocedureint/?eMasterorUN忙ORNisPF±0.01TherangehastenwriHedtogiwstableeffioentpackedbeds.Ie

20、dianotlistedheremaybeavailableinUNiCORNorAxichromMasterTypeMediaPackingbufferPackingfactorPackingvelocityIcnfVhlAffinityBLgSepharoseFastFlowWater/0-20%EtOH1.15SOHeparinSepharose6FastFlowWater/0-20%EtOH1.1560MobSelectWater/0-20%EtOH1.1560MobSelectSuReWater/0-20%EtOHI.15160I'dabSelectSuReLXWater/0

21、-20%EtOH1.IS1SOiabSelectXtrcfgWater/0-20%Eton1.15601WhencalculatingtheamountofmediatobepackedusethepressionFactor1.1insteadofthePackingFactorabovewhichisusedduringpacking.TypeMediaPackingbufferPackingfactorPackingvelocity(cm/hlAnionexchangeCaptoadhereWater/0-20%EtOH11060CaptoDEAEWater/0-2QKEtOH1.106

22、0CaptODEAEAnysaltsob-tion11560CaptoQWater/0-20%EtOH1.1060CaptoQAnysaltsob-tion1.1560DEAESepharoseFastFlowWater/0-20%EtOH11560QSepharoseBigBeadsWater/0-?0%LtOH1IS60QSepharoseFastFlowWater/0-20%EtOHL1560QSepharoseHighPerformanceWater/0-20%EtOH11560QSepharoseXLWater/0-20%EtOH1.1560TypeMediaPackingbuffe

23、rPackingfactorPackingvelocitycm/hjCationewchdngeCaptoMMCWater/0-20%EtOH1.1360CaptoMMCMysaltsolu-lioii1.1560CaptoSWater/0-20%LtOH1.1060CapLoSAnysaltsolution1.2060CMSepharoseFastFlowWater/0-20%EtOH1.1560SPSepharo<&BigBeadsWater/0-20%EtOH1.1860SPSepharoseBigBeadsAnysaltsolution12060SPSepharoseFastFlowWater/0-2096EtOH1.1560SPSephaoseFustFlowAnysaltsolution1.1560SPSepharoseHighperformanceWater/0-20%FtOH1.1530S