From Optimization to Automation: Multidimensional (Multi-D)
Histidine-Tag Protein Purification
Jeff Habel, Bio-Rad Laboratories, Inc., 6000 James Watson Drive, Hercules, CA 94547

The purification of proteins requires multiple iterations of individual column purification,
fractionation, visualization, and pooling of fractions for the next column. Significant
time is spent optimizing the first round of purification and using the information gainedon subsequent batches of protein purification. A well-designed Multi-D method
incorporates the optimizations of the individual column purifications steps into asingle automated method, allowing hands-free reproducible high-fidelity purifications
every time.

IMAC Multi-D Method Generation

To determine IMAC conditions for an automated Multi-D method, optimal %B was determinedby using the scouting feature of ChromLab™ Software to scout %B (Figure 2A) and byanalyzing the effect of pre-elution wash %B on the subsequent AEX step (Figure 2B).

An automated IMAC purification workflow Multi-D method was generated in ChromLab Software
using the optimal conditions determined for each of the individual columns. Method phases(colored blocks in Figure 4) from existing tandem and Multi-D templates were used to assemble
a custom IMAC purification method.

A. IMAC %B column wash scouting
8,000

Typical Single-Column
Method%B

2-D Affinity Desalting
Template

IMAC Purification Multi-D Method
Loop 1 Wash
Loop 2 Wash

7,000

25%

Loop Wash

Loop 3 Wash
IMAC Equilibration

6,000

P6 Equilibration
Loop 1 Wash

20%
5,000
λ280, mAU

Abstract

IMAC Optimization

Here, a typical capture (immobilized metal affinity chromatography, IMAC), intermediate(anion exchange, AEX), and polish (size exclusion, SEC) purification workflow with an
N-terminal small ubiquitin-like modifier tagged and C-terminal 6x histidine-tagged
superfolder green fluorescent protein (SUMO-6xHis GFPsf referred to as just GFPsf
going forward) on the NGC™ Chromatography System is used to show the developmentof an optimized automated 4-D Multi-D method highlighting the recovery andreproducibility of the final protein product.

15%

4,000

DS 5 Equilibration

IEX Equilibration

Equilibration

AC-1 Equilibration

IMAC Buffer Change

Wait for Sample Load

Wait for Sample LoadSample Application

AC-1 Sample Application

IMAC Sample Application

Column Wash

AC-1 Column Wash

IMAC Column Wash

AC-1 Elution

IMAC Elution

DS Buffer Change

P6 Buffer Change

Elution

3,000

SEC Equilibration

10%DS-5-1 Sample App/Elution

2,000

P6 Sample Application
P6 Elution
IEX Buffer Change

5%

IEX Sample Application
IEX Column Wash

1,000

IEX Elution

0%0

SEC Buffer Change
SEC Sample Application
SEC Elution

01234567Column volume8910Wait to Depressurize

11AC-1 Clean

AC-1 Clean

End RunEnd RunColumn
Equilibration

IMACPurification
Column 1Desalting
Column 2IEX
Purification
Column 3
SEC
Purification
Column 4
SystemReconditioning

Fig. 4. Graphical representation of theconversion of a single-column method
into a ChromLab 2-D template and IMAC
purification workflow Multi-D method. The
backbone of a single column method is theequilibration, sample application, column wash,and elution phases. These phases are modifiedand recycled to make the second column in theChromLab 2-D templates. Additional system
wash phases are generated to equilibrate theloops at the beginning of the method, clean
the IMAC column, and flush the system at theend of the method. The template is further
expanded to generate the IMAC purification
workflow Multi-D method. The expansion
occurs by recycling the same phases fromthe 2-D template to account for the additional
columns in the final Multi-D method. 96% of theMulti-D method phases shown here (24 of 25)
are recycled directly from the 2-D template withIEX elution being the only phase generated
by hand.

B. Effect on downstream IEX elution
10%B Wash

1,600

1,600

1,400

1,400800

11,20060040040020020000IMAC Purification Workflow
Traditional Sequential
Workflow
12341IMAC

2Dialysis or Dilution

3AEX

4SEC

IMAC

Dialysis or Dilution

AEX

SEC

Automated Multi-D Workflow

1Desalting

AEX

SEC

8,000

IMAC

Desalting

AEX

SEC

7,000

λ280, mAU

λ280, mAU

8.5

4,000
8.0

3,000

IMAC Column
Sample volume, %B elution window,
elution volume

IEX Column
pH, %B gradient elution profile,elution volume

Desalting Column
Sample volume, elution volume

SEC Column
Sample volume

2,000
1,000

7.5

002468 10 12 14 16 18 20 22 24 26 28 30
Column volume

10,000
9,000
8,000
7,000

2,000
1,600
1,200

17,00016,00015,00014,00013,00012,00011,00010,000
9,000
8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0%B

00		 40		 80		120		160		200		240		280Volume, ml

IMAC
4,800
4,400
4,000
3,600
3,200
2,800
2,400
2,000
1,600
1,200
800
400
0

Desalting1320 360

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76
Column volume

030%
20%

0246810 12 14 16 18 20 22 24 26 28 30
Column volume

Fig. 3. ENrich Q AEX Column pH and %B scouting. A buffer blending module was inserted into the NGC Chromatography
System and the Tris buffer system was used for the ENrich Q scouting. A, pH scouting runs using Tris pH 7.5, 8.0, and 8.5
generated by the buffer blending module for the duration of the entire method. Tighter GFPsf binding is demonstrated by increasing
amounts of salt required to elute the protein as pH increases further from the theoretical protein pI of 6.8. B, using pH 8.0 from theprevious scouting, the linear gradient %B endpoint was scouted in increments of 10%B. A GFPsf peak shift to the linear gradient
begins at 50% and reaches completion by 70%B. 495 nm wavelength was used to account only for the GFPsf signal and notcontaminants or buffer effects (for example, imidazole absorption at 280 nm). A 280 (—); A495 (—); conductivity (—).

D. Percentage recovery
IEX

SEC

23Column

Peak Area (495 nm)% Recovery*

IMAC

5,753.51

100.0

IEX

5,523.5

96.0

SEC

5,169.46

89.8* Defined by the area of the peak of interest divided by the area of theIMAC peak, multiplied by 100.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420

Volume, ml

50%
40%

4,000

1,000

060%

5,000

2,000

400

70%

6,000

3,000800

B. Linear gradient %B scouting

5,000SEC

C. Multi-D method recovery

pH

6,000

IEX

2,400

The ChromLab Software Anion Exchange template was used to load desalted GFPsf onto
an Enrich Q Column. Optimal pH (Figure 3A) and %B (Figure 3B) were determined using thesoftware’s scouting feature.

1IMAC

Desalting

B. Multi-D method reproducibility

A/1–A/19 A/20–A/40 A/41–A/61 A/62–A/82

IEX Optimization

9,000

Variables for Multi-D method creation:IMACFig. 2. IMAC %B wash scouting and its effects on downstream IEX elution. A, six scouting runs of column wash %B
from 0–25%B in 5%B increments were run on a Bio-Scale™ Mini Nuvia™ IMAC Column using the ChromLab Software scouting
feature. With increasing %B the leading shoulder of the 280 nM elution peak decreases without significant decreases in the GFPsf
495 nm trace up to 10%B. At 15%B wash we start to see significant GFPsf elution during the wash step and at 20%B all of the GFPsf
has been eluted during the wash. B, ENrich™ Q Anion Exchange Column linear gradient elutions of IMAC purified GFPsf. The left
represents the elution from a sample experiencing a default 3%B wash and the right from a sample that underwent a 10%B wash
while bound to the IMAC column. The contaminant peaks seen with 3%B can cause complications when using threshold fraction
collection. The lack of small impurity peaks on the leading shoulder of the 10%B sample allows for a low threshold value to be usedfor collection of the eluate to the loop resulting in maximum recovery and purity. Based on these data 10%B IMAC pre-elution wash
was determined to be optimal and used to generate the Multi-D method. A 280 (—); A495 (—); conductivity (—).

A. pH scouting

Optimization Parameters

A. Multi-D IMAC purification workflow

38 39 40 41 42 43 44 45 46 47 48 49 50
Column volume

37 38 39 40 41 42 43 44 45 46 47 48
Column volume

Fig. 1. Schematic of the 4-D Multi-D configuration of the NGC System. This schematic represents an NGC Quest™ 10 Plus System
populated with a third tier, a sample pump, two buffer inlet valves, two column switching valves, and an outlet valve. One of the column switchingvalves is plumbed as a loop valve replacing the static loop on the inject valve. A fluidic line from outlet valve port 2 is plumbed into the injection
port of the inject valve. All of the chromatography columns used are located on a single column switching valve. All of the static loops used in theMulti-D method are plumbed on the loop.

800600λ495, mAU

1The IMAC Multi-D method created in ChromLab Software (Figure 4) was used in a validationrun (Figure 5A) as well as in three additional runs to assess method reproducibility (Figure 5B).
ChromLab Software peak integration was used to determine percentage recovery of GFPsf at
the IMAC, IEX, and SEC steps (Figure 5C and D).

1,000

1,000

1Final Automated IMAC Purification

l280, mAU

1,200l280, mAU

l280, mAU

2

1,800l280, mAU

3%B Wash
1,800Fig. 5. The IMAC purification Multi-D method. A, 2 ml of clarified GFPsf lysate was initially loaded onto a 5 ml Nuvia IMAC Columnusing the sample pump. The protein was washed with 10%B, step eluted from the column, and stored in a 10 ml static loop on
the loop valve position 1. The protein was loaded from the loop onto the 50 ml Bio-Scale™ Mini Bio-Gel® P-6 desalting column andeluted isocratically with the protein collected into a 20 ml static loop at position 2 of the loop valve. The 20 ml protein sample wasloaded onto a 1 ml ENrich Q Ion Exchange Column and eluted with a linear gradient. Using threshold collection the protein wasshuttled into a 5 ml static loop on position 3 of the loop valve. The protein was then loaded onto a 120 ml Sephacryl S300-HR column
in a single injection and eluted isocratically collecting 1.5 ml fractions. B, offset chromatograms of three separate 11.5 hr IMAC
purification Multi-D method runs show high purification reproducibility. C and D, percentage recovery from a single run calculatedusing ChromLab Software generated peak area values. 495 nm wavelength was used to account only for the GFPsf signal and notcontaminants or buffer effects (for example, imidazole absorption at 280 nm). A 280 (—); A495 (—).

Conclusion■■

Automated Multi-D methods enable consistent and reproducible hands-free protein purification■■

Optimized methods for each step are vital to the construction of Multi-D methods to maximize 		
reproducibility and recovery■■

Multi-D methods can be constructed using the Tandem/2-D templates in ChromLab Software following thestandard equilibration, sample application, column wash, and elution process of a single-column method
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