Using Design of Experiments (DOE) with Nuvia cPrime to Elucidate
Capture Conditions of a Recombinant Protein
ProtocolProtein Purification

Bulletin 7128

Table of Contents
Introduction..................................................................................................................................................................................... 1
DOE with JMP Software.................................................................................................................................................................. 2
Sample and Spin Column Preparation............................................................................................................................................. 3
Spin Column Experiment................................................................................................................................................................. 3
Electrophoresis............................................................................................................................................................................... 4
Appendix 1: Spin Column Sample Buffers (100 mM)....................................................................................................................... 5
Appendix 2: Spin Column Binding and Elution Buffers (50 mM)....................................................................................................... 5
Appendix 3: Sample Preparation for Protein Electrophoresis........................................................................................................... 6
Appendix 4: Gel Analysis with Image Lab Software......................................................................................................................... 6
Appendix 5: Stain-Free Imaging Technology Example Images......................................................................................................... 7
Appendix 6: Results........................................................................................................................................................................ 7

Introduction
Here we describe the use of a Design of Experiment (DOE) approach using spin columns in the initial screening ofchromatographic conditions for purification of a recombinant protein expressed in E. coli on Nuvia cPrime Hydrophobic
1
Cation Exchange Resin. The ligand on this media has three major functionalities: a weak carboxylic acid end group, anaromatic hydrophobic ring, and an amide bond serving as a potential hydrogen bond donor/acceptor. Under specificpurification conditions, one or more such interaction modes may be involved in the binding or repulsion between thetarget protein and the chromatographic media. Therefore, the behavior of a protein during purification by mixed-mode
chromatography often cannot be predicted based on its isoelectric point or amino acid sequence.
JMP Software was used in these DOE studies to identify optimal binding and elution conditions on Nuvia cPrime for thetarget protein contained in the E. coli control lysate. The effects of four parameters were evaluated using a response
surface matrix custom design provided by JMP Software, including three center points and a total of 18 experiments:
pH binding conditions
conductivity binding conditions■■ pH elution conditions■■ conductivity elution conditions■■■■

Our studies show that, with a limited amount of protein sample and chromatography media, a simple DOE setup can beused to determine the effects of buffer pH and conductivity on selectivity, recovery, and robustness of purification on
Nuvia cPrime. Working conditions established by such scale-down studies can be used for the purification of a target
protein on a preparative scale.
1

Bio-Rad Bulletin 6418. Purification of Recombinant Proteins on Nuvia cPrime Hydrophobic Cation Exchange Media: A Simple Approach to Method Development

Using Design of Experiments (DOE) with Nuvia cPrime to Elucidate Capture Conditions of a Recombinant Protein

DOE with JMP Software
1. Open JMP Software (v10.0.2).
2.

3.

Open the DOE Custom Design dialog and add responses(binding, yield, purity) and factors as indicated in theimage. Adjust limits and values as needed. The values ofthe factors are as follows:n  pH range protein binding: 4.75–6.75
n  NaCl concentration protein binding: 0–400 mMn  pH range protein elution: pH 6–8.75
n  NaCl concentration protein elution: 0–1,000 mM

Continue and click RSM (response surface matrix). In
contrast to screening experiments, researchers use RSM
when they already know which factors are important.

© 2018 Bio-Rad Laboratories, Inc.

2

4.

Adjust the number of center points to three, limit the
number of runs to 18, and continue by clicking
Make Design.

5.

JMP Software now provides a table for the 18 spincolumn experiments.

6.

After the spin column experiments and SDS-PAGE
analysis of the individual fractions, add the corresponding
data to the JMP design table and begin data evaluation
with the JMP Software. A good start is to design contour
plots to display the influence of pH and conductivity on
target binding, yield, and purity (see example). For more
information, refer to the JMP Software manual.

Bulletin 7128

Using Design of Experiments (DOE) with Nuvia cPrime to Elucidate Capture Conditions of a Recombinant Protein

Sample and Spin Column Preparation

16. Seal the bottoms and tops of the spin columns.
17. Following centrifugation, the resin is now in thebinding buffer.

Materials
Item

Catalog #E. coli Control Lysate
Mini Bio-Spin Chromatography Columns

6200233
2

Nuvia cPrime Hydrophobic Cation Exchange Resin3

Microtube Racks

7326207

18. Prepare 2% SDS solution in water.

1563401

Spin Column Experiment

1660481

Binding buffers (see Appendix 2)–

1.5 ml microcentrifuge tubes–

2 ml microcentrifuge tubes, capless–

Materials
Item

Catalog #Sample buffers (see Appendix 1)–

Microtube racks

1660481

Binding buffers (see Appendix 2)

Additional Materials Required–

Elution buffers (see Appendix 2)

Vortex mixer
Centrifuge
Pipets and pipet tips
Laboratory gloves
Deionized water–

10% SDS solution, 250 ml

1610416

1.5 ml microcentrifuge tubes–

2 ml microcentrifuge tubes–

Additional Materials Required1.

Add 15 ml of deionized water to the E. coli control
lysate4 and shake well until all lyophilized material isdissolved. Set the sample aside at room temperatureand do not put on ice. The total protein concentration isabout 2 mg/ml.

2.

Snap off the tips of the 18 spin columns and seal thebottoms again.

Overview of the spin column experiment
1. Add 500 µl of sample solution

Incubate for 10 min and centrifuge.

3.

Place the columns in 2 ml microcentrifuge tubes.

2. Add 167 µl of binding buffer

4.

Remove the top caps of the microcentrifuge tubes.

Vortex and centrifuge.
Repeat twice.

3. Add 500 µl of binding buffer

5.

Shake the Nuvia cPrime bottle to obtain ahomogenous suspension.

Vortex and centrifuge.
Discard solution.

4. Add 167 µl of elution buffer

Vortex and centrifuge.
Repeat twice.

6.

Carefully apply 200 µl of the resin suspension to thecenter of all spin columns.

5. Add 250 µl of elution buffer

Vortex and centrifuge.

6. Add 167 µl of 2% SDS solution

Vortex and centrifuge.
Repeat twice.

7.

Remove the bottom seal from the columns and then put
the columns back into the microcentrifuge tubes.

8.

Centrifuge for 1 min in a microcentrifuge at 1,000 x g.

9.

Discard the buffer and seal the bottoms of the columns.

End-over-end shaker
Vortex mixer
Centrifuge
Pipets and pipet tips
Laboratory gloves
Deionized water

1.

Pipet 300 µl of each sample buffer into a 1.5 ml
microcentrifuge tube and add 300 µl of E. coli sample
dissolved in water. Prepare the samples just before
beginning the spin column experiments. Close the tubes
and mix well.

11. Apply 500 µl of appropriate binding buffer to the columns
and apply the top seal (see Appendix 1).

2.

Open the top cap of the spin columns and add 500 µl of
the sample solution to the center of all spin columns.

12. Carefully vortex the columns for 1 min.

3.

13. Remove the top and bottom seals and place the columns
in a 2 ml microcentrifuge tube.

Seal all columns and mix well for 10 min using an
end-over-end rotator.

4.

Place columns in 2 ml microcentrifuge tubes and remove
the top and bottom seals.

5.

Centrifuge for 1 min in a microcentrifuge at 1,000 x g.

6.

Keep flowthrough (U) after centrifugation.

7.

Seal the bottom of all columns and place columns in 2 ml
microcentrifuge tubes.

8.

Add 167 µl of binding buffer to wash the resin and apply
the top seal.

10. Place the columns in a 2 ml microcentrifuge tube.

14. Centrifuge for 1 min in a microcentrifuge at 1,000 x g.
15. Repeat buffer exchange twice.

2
3
4

Note: 1.2 ml; bed height: 2.8 cm; overall length: 3.3 cm.
Note: 50% v/v slurry in 20% ethanol.
Note: Quantity per bottle: 30 mg lyophilized total protein, including 10 mg oftarget protein. pI target protein: 5.47 (calculated) and 5.9 (isoelectric focusing);
MW target protein: 51 kD. Grand average of the hydropathicity (GRAVY) of thetarget protein: –0.275.

© 2018 Bio-Rad Laboratories, Inc.

3

Bulletin 7128

Using Design of Experiments (DOE) with Nuvia cPrime to Elucidate Capture Conditions of a Recombinant Protein

9.

Vortex three times for 5 sec each and remove the top andbottom seals.

35. Centrifuge for 1 min in a microcentrifuge at 1,000 x g.
36. Keep the elution solution after centrifugation.

10. Centrifuge for 1 min in a microcentrifuge at 1,000 x g.

37. Seal the bottom of all columns and place them in 2 ml
microcentrifuge tubes containing the previous elution
solution(s).

11. Keep the wash solution after centrifugation.
12. Seal the bottom of all columns and place them in 2 ml
microcentrifuge tubes containing the previous wash
solution(s).

38. Repeat steps 32–36 twice. The total volume of elution
solution (E3) is 500 µl.

13. Repeat steps 9–13 twice. The total volume of wash
solution (W) is 500 µl.

Electrophoresis

14. Seal the bottom of all columns and place columns inunused 2 ml microcentrifuge tubes.

Item

Catalog #Gel Doc EZ Imaging System

1708270

Stain Free Sample Tray

1708274

2x and 4x Laemmli Sample Buffer

1610747

2-mercaptoethanol

1610710

16. Vortex three times for 5 sec and remove the top andbottom seals.

4–20% Criterion TGX Stain-Free Protein Gel,
18 well, 30 µl

5678094

Precision Plus Protein Unstained Standards

1610363

17. Centrifuge for 1 min in a microcentrifuge at 1,000 x g.

Criterion Cell (3x)1656001

18. Discard this wash solution after centrifugation.

10x TGS Buffer

1610732

PowerPac Universal Power Supply

1645070

Materials

15. Add 500 µl of binding buffer to wash the resin and apply
the top seal.

1.5 ml microcentrifuge tubes

19. Seal the bottom of all columns and place them in unused
2 ml microcentrifuge tubes.

–

Additional Materials Required

20. Add 167 µl of elution buffer to elute the bound proteins
and apply the top seal.
21. Vortex three times for 5 sec and remove the top andbottom seals.

Heat block5
Centrifuge
Pipets and pipet tips
Deionized water
Laboratory gloves

22. Centrifuge for 1 min in a microcentrifuge at 1,000 x g.

1.

Prepare 2x and 4x Laemmli buffer.

23. Keep the elution solution after centrifugation.

2.

24. Seal the bottom of all columns and place them in 2 ml
microcentrifuge tubes containing the previous elution
solution(s).

Dissolve the samples from the spin column experimentwith 2x or 4x Laemmli buffer in 1.5 ml micro test tubes
according to Appendix 3.

3.

Vortex for 5 sec and heat the sample for 10 min at 70°C.

4.

Centrifuge for 30 sec at 10,000 x g.

25. Repeat steps 19–23 twice. The total volume of the elution
solution (E1) is 500 µl.

65.

Remove the comb and tape from the gel.

26. Seal the bottom of all columns and place them in unused
2 ml microcentrifuge tubes.

6.

Wash the wells with 1x TGS buffer.

27. Add 250 µl of elution buffer to elute the bound proteins
and apply the top seal.

7.

Place the gel in a Criterion Cell.

8.

Fill with 1x TGS buffer to the fill line.

78

28. Vortex three times for 5 sec and remove the top andbottom seals.
29. Centrifuge for 1 min in a microcentrifuge at 1,000 x g.
30. Keep the elution solution after centrifugation.
31. The total volume of elution solution (E2) is 250 µl.

Note: If you need to order one, we recommend the Bio-Rad Digital Dry
Bath (#1660571).
6 Recipe: 2x Laemmli Buffer: Mix 300 µl of 2-mercaptoethanol, 2,700 µl of 4x
Laemmli Buffer, and 3,000 µl of ultrapure water. 4x Laemmli Buffer: Mix 100 µlof 2-mercaptoethanol with 900 µl 4x Laemmli Buffer.
7 Recipe: Tris/glycine/SDS electrophoresis buffer: To prepare 1 L of buffer,mix 100 ml of 10x TGS buffer and 900 ml of ultrapure water.
8 Tip: Ensure that there is sufficient running buffer in the electrophoresis cell
(as indicated by the fill lines, depending on the number of gels), especiallywhen running at high voltages in order to achieve rapid run times.
5

32. Seal the bottom of all columns and place them in unused
2 ml microcentrifuge tubes.
33. Add 167 µl of 2% SDS solution to elute the boundproteins and apply the top seal.
34. Vortex three times for 5 sec and remove the top andbottom seals.
© 2018 Bio-Rad Laboratories, Inc.

4

Bulletin 7128

Using Design of Experiments (DOE) with Nuvia cPrime to Elucidate Capture Conditions of a Recombinant Protein

9.

Appendix 1: Spin Column Sample Buffers12 (100 mM)

Load samples (20 µl) in the following order:n E. coli sample in binding buffer S
n Unbound sample solution U
n Wash solution W
n Elution solution E1
n Elution solution E2
n Elution solution E3

10. Each Criterion Gel (18 well) can run samples for three spincolumn experiments.
11. Run at 50 V for 15 min and 250 V until the dye front
9
reaches the indicator line at the bottom of the gel.
12. Switch off the power supply and disconnect theelectrical leads.
13. Remove the gel from the Criterion Cell.
10

14. Open the gel cassette using the aluminum lever.

15. Pour a few milliliters of water on the Blot/UV/Stain-Free
11
Tray and place the gel on the tray. Avoid trapping air
bubbles under the gel.

#

pH

NaCl,
mM

1

4.75

0

Citrate-Na

2

4.75

0

Citrate-Na

3

4.75

400

Citrate-Na

4

4.75

800

Citrate-Na

5

4.75

800

Citrate-Na

6

5.75

0

MES-Na7

5.75

0MES-Na

8

5.75

400

MES-Na

9

5.75

400

MES-Na

10

5.75

400

MES-Na

11

5.75

400

MES-Na

12

5.75

800

MES-Na

13

6.75

0

MOPS-Na

14

6.75

0

MOPS-Na

15

6.75

236

MOPS-Na

16

6.75

800

MOPS-Na

17

6.75

800

MOPS-Na

18

6.75

800

MOPS-Na

BufferAppendix 2: Spin Column Binding and Elution Buffers (50 mM)

16. Choose the following settings to activate and image thestain-free gel:
Application: Protein Gels, Stain-Free Gel
Gel Activation: 2.5 min■■ Manually set the Image Exposure time to 0.5–1.0 sec.
		Note: the exposure time is dependent on the gel and		
imager and will vary■■ Make sure that gel activation time and image
		
exposure time are identical for all gels
■■
■■

Spin
columnBinding
Binding NaCl,
pH
mM
BufferElutionpH

Elution
NaCl,mM

Buffer1

4.75

0

Citrate-Na

7.38

1,000

MOPS-Na

2

4.75

0

Citrate-Na

8.75

0

Tricine-Na

3

4.75

200

Citrate-Na

6.00

500

MES-Na

4

4.75

400

Citrate-Na

7.38

0

MOPS-Na

5

4.75

400

Citrate-Na

8.75

500

Tricine-Na

6

5.75

0

MES-Na

6.00

0

MES-Na7

5.75

0MES-Na7.38

500

MOPS-Na

8

5.75

200

MES-Na7.38

500

MOPS-Na

9

5.75

200

MES-Na7.38

500

MOPS-Na

10

5.75

200

MES-Na7.38

500

MOPS-Na

11

5.75

200

MES-Na

8.75

1,000

Tricine-Na

12

5.75

400

MES-Na

6.00

1,000

MES-Na

13

6.75

0

MOPS-Na

6.00

1,000

MES-Na

14

6.75

0

MOPS-Na

8.75

500

Tricine-Na

15

6.75

118

MOPS-Na

7.38

0

MOPS-Na

16

6.75

400

MOPS-Na

6.00

0

MES-Na

17

6.75

400

MOPS-Na

7.38

1,000

MOPS-Na

18

6.75

400

MOPS-Na

8.75

0Tricine-Na

17. Click Run Protocol to begin the activation and exposure.
18. Start data analysis using Image Lab Software(see Appendix 4).

Note: Typically 30 min.
Note: No longer supplied with boxes of gels; order separately (#4560000).
11 Tip: Keep gloves wet when handling the gels.
12
Note: Spin column sample buffers are 2x concentrated binding buffers.
910© 2018 Bio-Rad Laboratories, Inc.

5

Bulletin 7128

Using Design of Experiments (DOE) with Nuvia cPrime to Elucidate Capture Conditions of a Recombinant Protein

Appendix 3: Sample Preparation for Protein Electrophoresis
The following samples are available for analysis after the spincolumn experiments.
Dilute the samples as indicated below and heat for 10 min
at 70°C.
■■

100 µl E. coli sample (~1 mg/ml) in different sample
buffers (S: 1–18)■■

500 µl of unbound material in binding buffers (U: 1–18)■■

500 µl of wash solution in binding buffers (W: 1–18)■■

500 µl of eluted material in elution buffers (E1: 1–18)■■

250 µl of eluted material in elution buffers (E2: 1–18)■■

500 µl of eluted material in 2% SDS (E3: 1–18)
S

UW

Sample, µl

50

50

60

2x Laemmli, µl

5050

4x Laemmli, µl

–

–
–

20

E1

E2

E3

50

50

50

50

50
–

4.

Save this dataset indicating the spin columns as well asthat it is the target band. An example name would be
Spin columns 1–3 SF gel data target analysis.

5.

Manually adjust all 18 target bands so that they become a“total lane” band.

6.

Save this dataset indicating the spin columns as well asthat it is the total lane data. An example name would be
Spin columns 1–3 SF gel data total lane analysis.

7.

Repeat the procedure for the remaining gels and save thedata accordingly (see example images in Appendix 5).

8.

Prior to data export, adjust display data options as
indicated.

50
–

–

Appendix 4: Gel Analysis with Image Lab Software
1. Use the cropping tool to crop all images to the same size.
2.

Use the lane tool to define the lanes of the gel.
1

3.

2

3

4

5

6

7

8

9

10 11

12 13

14 15

16

17

18

Detect target bands manually in each lane. Use the lane
profile tool to make sure the target band is correctly
detected.
1

2

3

4

5

6

7

8

© 2018 Bio-Rad Laboratories, Inc.

9

10 11

12 13

14 15

16

17

18

6

Bulletin 7128

Using Design of Experiments (DOE) with Nuvia cPrime to Elucidate Capture Conditions of a Recombinant Protein

9.

Appendix 5: Stain-Free Imaging Technology Example Images
Spin column samples are run in sequential order:

Compile the intensity values acquired for both the target
protein and total lane intensity into a master Excel (orsimilar software) spreadsheet. Target band intensities
and total lane intensities should be used where indicated.
Each experiment should be normalized to its own input or
supernatant lane. The following calculations can then be
made using these normalized intensity values:

Sample in various binding buffers (S)Unbound (U)■■ Wash (W)■■ Elution 1 (E1)■■ Elution 2 (E2)■■ Elution 3 (E3)■■■■

Target unbound = sum of percentage target unbound and percentage
target wash*[(UTarget/STarget) + (W Target/STarget)]Gel representing experimental data from samples 10–12.

Target bound = 1 – Target unbound#10

Target elution = sum of percentage target in elution 1, elution 2, andelution 3[(E1Target /STarget) + (E2Target/STarget) + (E3Target/STarget)]

S

U

W

E1

#11
E2

E3

S

U

W

#12

E1

E2

E3

S

U

W

E1

E2

E3

Target yield = sum of percentage target in elution 1 and elution 2[(E1Target /S Target) + (E2Target /S Target)]Target purity = Target yield divided by total lane intensity for elution
1 and elution 2[(E1Target/STarget + E2Target/STarget)/(E1Total Lane/STotal Lane + E2Total Lane/STotal Lane)]* Wash intensity for both target and total lane must be divided by 4 toaccount for the increased loading amount.

10. Check that data analysis is within the acceptable range bylooking at center points as well as that U+W+E1+E2+E3= S (± 5%). This should be true for both target intensities
and total lane intensities.
11. If data are within the acceptable range, then input
calculated target bound, target yield, and target purity
into JMP Software to analyze the data. The predictor
profiler and contour plots should be the first visualizationof the data.

© 2018 Bio-Rad Laboratories, Inc.

#

Binding
Binding NaCl,
BindingpH
mM
Target, %ElutionElution NaCl,
Yield
pH
mM
Target, %PurityTarget, %

10

5.75

200

98

7.38

500

78

67

11

5.75

200

99

8.75

1,000

89

63

12

5.75

400

89

6.00

1,000

33

60

Appendix 6: Results

7

Spin
ColumnBinding Binding
Binding NaCl,
Target, Elution
pH
mM
%
pH

Elution
NaCl,
mM

Yield
PurityTarget, Target,
%
%

1

4.75

0

81

7.38

1,000

67

32

2

4.75

0

85

8.75

0

80

37

3

4.75

200

100

6.00

500

15

17

4

4.75

400

100

7.38

0

31

32

5

4.75

400

99

8.75

500

91

45

6

5.75

0

97

6.00

0

2

10

7

5.75

0

98

7.38

500

67

55

8

5.75

200

99

7.38

500

78

58

9

5.75

200

99

7.38

500

60

62

10

5.75

200

98

7.38

500

78

67

11

5.75

200

99

8.75

1,000

89

63

12

5.75

400

89

6.00

1,000

33

60

13

6.75

0

65

6.00

1,000

5

27

14

6.75

0

47

8.75

500

4

27

15

6.75

118

66

7.38

0

26

53

16

6.75

400

28

6.00

0

0

1

17

6.75

400

18

7.38

1,000

1

5

18

6.75

400

22

8.75

0

1

0

Bulletin 7128

Bio-Rad and Bio-Spin are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions.
Precision Plus Protein Standards are sold under license from Life Technologies Corporation, Carlsbad, CA for use
only by the buyer of the product. The buyer is not authorized to sell or resell this product or its components.
All trademarks used herein are the property of their respective owner.

Bio-RadLaboratories, Inc.
Web site bio-rad.com USA 1 800 424 6723 Australia 61 2 9914 2800 Austria 43 01 877 89019 Belgium 32 03 710 53 00 Brazil 55 11 3065 7550
Canada 1 905 364 3435 China 86 21 6169 8500 Czech Republic 36 01 459 6192 Denmark 45 04 452 10 00 Finland 35 08 980 422 00
France 33 01 479 593 00 Germany 49 089 3188 4393 Hong Kong 852 2789 3300 Hungary 36 01 459 6190 India 91 124 4029300
Israel 972 03 963 6050 Italy 39 02 49486600 Japan 81 3 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 31 0 318 540 666
New Zealand 64 9 415 2280 Norway 47 0 233 841 30 Poland 36 01 459 6191 Portugal 351 21 4727717 Russia 7 495 721 14 04
Singapore 65 6415 3188 South Africa 36 01 459 6193 Spain 34 091 49 06 580 Sweden 46 08 555 127 00 Switzerland 41 0617 17 9555
Taiwan 886 2 2578 7189 Thailand 66 2 651 8311 United Arab Emirates 971 4 8187300 United Kingdom 44 01923 47 1301

Life ScienceGroup

Bulletin 7128 Ver A

US/EG

18-05561118

Sig 0118