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ためひとたけくま
4 years ago
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2 INDEX Resin Type Cation Exchanger Anion Exchanger Net Charge of Molecule of Interest Charge of Resin Running Conditions Run at ph units below the pi of the molecule of interest Run at ph units above the pi of the molecule of interest OH Ca +)) ((+ H2 N Ca +)) ((+ H2 N OH PO = H2 N PO = H2 N PO = 4 A HO + Ca COO + Ca COO OH B COO ))((= O 4 P COO )) ((= O4 P = O4 P OH HO A. B. Bead Ligand Sample A B C Bead Ligand Sample Eluant 2

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DBC polyclonal higg, 10% BT, mg/ml 140 120 100 80 60 40 20 0

8 DBC polyclonal higg, 10% BT, mg/ml Nuvia S media Media 1 Media 2 Media 3 Media 4 Media 5 Media 6 8

9 AU Flow through Buffer B wash Time, min Buffer C elution 1 N NaOH strip Conductivity, ms/cm Buffer B wash fractions AU Buffer C elution pooled fractions, M Time, min AU AU SEC-HPLC Analysis Load Buffer E Fractions elution AU N NaOH strip Min.Tenth Buffer D wash ms/cm 0.25 Buffer A equilibration Time, min 100 9

Frac# 0.500 0.450 0.400 0.350 0.300 0.250 0.200 0.150 0.100 0.

050 AU 2F 3F 4F 5F 6F 7F 8F 9F 10F 11F 12F mab in Flow-through

10 Frac# AU 2F 3F 4F 5F 6F 7F 8F 9F 10F 11F 12F mab in Flow-through OD280 OD260 ph impurities stripped by 1N NaOH Min.Tenth ph 10

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14 X Soybean trypsin inhibitor Conalbumin 10 Ovalbumin Myoglobin 10% Breakthrough, mg BSA/ml Lot 1 (0.1 N NaOH) 225 Lot 2 (1.0 N NaOH) ,000 4,000 6,000 8,000 10,000 Retention time, min ,000 4,000 6,000 8,000 10,000 Storage time, hr Storage time in 1 N NaOH, hr 14

15 25 20 Macro-Prep DEAE support TSK DEAE 650M DEAE Sepharose FF Percentage Particle size, μm

16 Fractions Fractions A NaCl concentration, M A % B Run time, min Run time, min 0% 16

17 A μm Pressure: 23 bar 20μm Pressure: 6 bar 40μm Pressure: 2 bar 80μm Pressure: 0.5 bar Time(min) Time(min) Time(min) Time(min) 17

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19 % B E OD280 ALBUMIN TRANSFERRIN I gg FIBRONECTIN t (min) Retention Time (min) 19

20 E 280 OD280 ALBUMIN TRANSFERRIN I gg FIBRONECTIN % B E 280 ALBUMIN TRANSFERRIN I gg FIBRONECTIN % B {Phosphate (mm)} IIm t (min) t (min) Retention Time (min) Retention Time (min) Phosphate vs Chloride 20

21 NaPO4 gradient 0.5M NaP SS DNA RNA DS DNA NaCl gradient 2.0M NaCl 0.5M NaP 21

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24 Recovery, % Binding [NaCl], mm10, 400 Binding capacity, mg/ml Binding ph4, 8 Binding ph4, 8 Binding capacity, mg/ml Binding [NaCl], mm10, 400 Elution [NaCl], mm10, 1,000 Recovery, % Elution ph4, 8 Elution ph4, 8 Elution [NaCl], mm10, 1,000 24

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29 For More Information Web: 29

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2289 com/econopaccolumns Request or download

36 For More Information Web: Request or download bulletin: 2289 For More Information Web: Request or download bulletin: 2289 For More Information Web: Request or download bulletin:

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40 A Column volume = ml 40

Waters Sigma-Aldrich 1.0 mg/ml LC Waters Alliance HPLC ACQUITY UPLC H-Class Bio 30 cm Alliance HPLC UV ACQUITY UPLC TUV mm nm XBridge Protein B

Waters Sigma-Aldrich 1.0 mg/ml LC Waters Alliance HPLC ACQUITY UPLC H-Class Bio 30 cm Alliance HPLC UV ACQUITY UPLC TUV mm nm XBridge Protein B HPLC 3. µm BEH Stephan Koza, Susan Serpa, Hua Yang, Edouard Bouvier, and Kenneth J. Fountain Waters Corporation, Milford, MA, USA HPLC 200 0 HPLC 2 2010 UPLC 200 SEC 1 UPLC SE-UPLC 2 µm BEH BEH 100% SE-UPLC