4-5 6-33 34-36 37-40

INDEX Resin Type Cation Exchanger Anion Exchanger Net Charge of Molecule of Interest Charge of Resin Running Conditions + -- + -- Run at 0.5 1.5 ph units below the pi of the molecule of interest Run at 0.5 1.5 ph units above the pi of the molecule of interest OH Ca +)) ((+ H2 N Ca +)) ((+ H2 N OH PO =... + 4 H2 N PO =... + 4 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

3

4

5

6

7

DBC polyclonal higg, 10% BT, mg/ml 140 120 100 80 60 40 20 0 Nuvia S media Media 1 Media 2 Media 3 Media 4 Media 5 Media 6 8

AU 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 0.50 Flow through 1 2 3 4 5 6 7 8 9 10 111213141516 17 18 Buffer B wash 30 60 90 Time, min Buffer C elution 1 N NaOH strip 350 300 250 200 150 100 50 0 50 Conductivity, ms/cm Buffer B wash fractions 2 3 4 5 6 7 8 9 AU 0.01 0.008 0.006 0.004 0.002 0 Buffer C elution pooled fractions, 11 15 M 250 190 100 75 50 37 25 20 15 10 12 13 14 15 16 Time, min AU 2.00 1.75 1.50 1.25 1.00 0.75 0.50 AU 0.0125 0.0100 0.0075 0.0050 0.0025 0.0000 0.0025 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 SEC-HPLC Analysis 0.0125 Load Buffer E Fractions 3-8 0.0100 elution 0.0075 0.0050 AU 0.0025 0.0000 0.0025 1N NaOH 11.00 12.00 13.00 strip Min.Tenth Buffer D wash 500 400 300 200 100 ms/cm 0.25 Buffer A equilibration 0.00 0 0.25 0 10 20 30 40 Time, min 100 9

Frac# 0.500 0.450 0.400 0.350 0.300 0.250 0.200 0.150 0.100 0.050-0.000-0.050 AU 2F 3F 4F 5F 6F 7F 8F 9F 10F 11F 12F mab in Flow-through OD280 OD260 ph impurities stripped by 1N NaOH 10.00 20.00 30.00 40.00 Min.Tenth 12.5 10.0 7.5 5.0 2.5 0.0-2.5 ph 10

11

12

13

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) 200 175 150 125 100 0 2,000 4,000 6,000 8,000 10,000 Retention time, min 8 6 4 2 0 0 2,000 4,000 6,000 8,000 10,000 Storage time, hr Storage time in 1 N NaOH, hr 14

25 20 Macro-Prep DEAE support TSK DEAE 650M DEAE Sepharose FF Percentage 15 10 5 0 0 50 100 Particle size, μm 150 15

Fractions Fractions A280 2 4 6 8 11 17 23 29 35 41 47 53 59 65 1.0 0.5 0.0 1.0 NaCl concentration, M A280 0.4 0.3 0.2 0.1 1 2 3 4 6 8 10 12 14 16 18 20 22 24 26 100% B 0 30 60 90 Run time, min 0.0 0.0 0 5 10 15 Run time, min 0% 16

A 280 10μm Pressure: 23 bar 20μm Pressure: 6 bar 40μm Pressure: 2 bar 80μm Pressure: 0.5 bar 0 4 8 12 0 4 8 12 0 4 8 12 0 4 8 12 Time(min) Time(min) Time(min) Time(min) 17

18

% B E 280 100 OD280 ALBUMIN TRANSFERRIN I gg FIBRONECTIN 50 0 0 10 20 t (min) Retention Time (min) 19

E 280 OD280 ALBUMIN TRANSFERRIN I gg FIBRONECTIN % B 100 50 0 E 280 ALBUMIN TRANSFERRIN I gg FIBRONECTIN % B 100 50 0 {Phosphate (mm)} IIm 0 10 20 t (min) 0 10 20 t (min) Retention Time (min) Retention Time (min) Phosphate vs Chloride 20

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

22

23

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

25

26

27

28

For More Information Web: www.bio-rad.com/biobeads 29

30

31

32

33

34

35

For More Information Web: www.bio-rad.com/spincolumns Request or download bulletin: 2289 For More Information Web: www.bio-rad.com/econopaccolumns Request or download bulletin: 2289 For More Information Web: www.bio-rad.com/econopaccolumns Request or download bulletin: 2289 36

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700 600 500 A 280 400 300 200 100 0 0 0.16 0.32 0.48 0.64 0.80 0.96 1.12 1.28 Column volume = 23.56 ml 40