因子层析纯化工艺的具体步骤

1、Purificati on of Factor VIII (Process example)(1) Pate nt: US 5,714,590Title: Process for recovering a high-purity virus-inactivatedfactor VIII by anionexcha nger chromatographyAuthor: OctapharmaPretreatme ntCryoprecipitate(1) in case of cryoprecipitate sample(thaw at room temperature, 3-4h)2U/mL He

2、pari n-sodium twice volumeAdjusted to pH 7.0-7.1 with 0.1M acetic acid (20-25 C, 30mi n) 108g of 2% AI(OH)3 suspension are added per 1kg of cryoprecipitateAdjusted to pH 6.5-6.6 with 0.1M aceticCentrifugationSupernatantFiltrati on(Pall AB-1 UO1OZP filter)(2) in case of Huma n plasma sampleHuma n pla

3、sma(thaw at 20C)4 / 4Diluted with 50% volume of waterFiltrati onEMD-TMAE-Fractogel(M)650Equilibrati on : 50mM NaCl, 10mM Sodium citrate, 120mM Glyc ine, 100U/L Hepari n, pH6.5-6.9Virus in activati onA virus in activati on by means of Twee n/TNBP has prove n to be particularly useful. ”/Suitable TOYO

4、PEARL resi n for Factor VIII purificati on is DEAE-650M.ChromatographyIn case of using DEAE-650M, it is assumed that prote in is eluted with 20-30% lower salt concen tratio n.Resi nEMD-TMAE-Fractogel(M)650Washi ng0.1M NaCl 5CVEquilibrati on120mM NaCl,10mM Sodium citrate,120mM Gly cine,1mM CaCI2(pH 6

5、.5-7.0 with 1MHCI)Wash ing after loadi ng180-200mM NaCl,10mM Sodium citrate, 120mM Gly cine,1mM CaCl2(pH 6.9-7.0)Eluti on400mM NaCl,1mM Sodium citrate, 120mM Gly cine,1mM CaCl2(pH 6.9-7.0)Washi ng1M NaCl 5CVRege nerati on0.1N NaOH3CV0.1N HCl3CV25% alcohol in water5CVLoadi ng1kg of cryoprecipitate pe

6、r at least 0.5L of resinDilution of fracti on ated sample20mM Sodium citrate,80mM Gly cine,2.5mM CaCl2(pH 6.9-7.1)Caution: These conditions might infringe patents filed.Please check the orig inal pate nt adequately. Journal of Chromatography, 662 (1994) 181-190Title: Purification of factor VIII and

7、von Willebrand factor from human plasma by anion-excha nge chromatographyAuthor: Dj. Josic, et al (Octapharma)CryoprecipitateDissolved in 10-50 mM sodium citrate (pH7.2)Filtered through a filter with 1 um pore sizeA large part of the fibri nogen, the fibron ect in and unwan ted clott ing factors are

8、 removed through adsorpti on to AI(OH)3.Virus in activati onS/D treatme nt (e.g. Twee n 80, tri-n-butylphosphate)ChromatographyColu mn: TOYOPEARL DEAE-650S250mmID*120mmLoading buffer: 10 mM citrate buffer, pH7.5 with 110-200 mM NaClWashi ng: Step gradie nt of NaCl solutio n (Osmolarity: 400-600 mOsm

9、ol)Elution: Step gradient of NaCl solution (Osmolarity: 550-900 mOsmol)Washi ng: 1M NaCl and 0.5 M NaOHVirus in activati onHeati ng in prese nee of sugars and amino acids(for removal of stabilizer)Colu mn: TOYOPEARL DEAE-650S26mmID*60mmLoading buffer: 10 mM citrate buffer, pH7.5 with 110-200 mM NaCl

10、Washi ng: Step gradie nt of NaCl solutio n (Osmolarity: 400-600 mOsmol)Elution: Step gradient of NaCl solution (Osmolarity: 550-900 mOsmol)Washi ng: 1M NaCl and 0.5 M NaOHSutable TOYOPEARL res in is DEAE-650M orSuperQ-650M.it is assumedIn case of using TOYOPEARL, that same con diti on is applicable.Chromatography(for separati on of FVIII and vWF)Colu mn: Fractogel EMD TMAE