Protein Purification

Reference Methods for Packing Columns with Nuvia IMAC Resin:
From Lab-Scale to Process-Scale
ProtocolImmobilized metal affinity chromatography (IMAC) is an effective
chromatography tool for preparing recombinant proteins naturallyrich in histidine/cysteine residues, as well as those with a histidine
residue cluster engineered to their N- or C-terminus via recombinant
DNA technology. While a small quantity of protein prepared on alaboratory bench is often sufficient for structural and functional
characterization studies, process-scale columns are needed in themanufacturing of recombinant proteins for therapeutic applications.
We have developed effective protocols for packing Nuvia IMAC
Resin in various column sizes to fulfill the needs of lab-scale tocommercial production.
Product Characteristics
Nuvia IMAC is a rigid macroporous high-capacity metal affinity
resin with good tolerance to a wide variety of chemicals employedfor protein purification (bulletins 6859 and 6964). It is built on therobust UNOsphere base beads with nitrilotriacetic acid (NTA)
as the chelating ligand for di- or trivalent metal ions. Its liganddensity and particle size distribution are optimized to facilitatehigh dynamic binding capacity (DBC) while maintaining excellent
pressure flow properties. Nuvia IMAC Resin is chemically stable
under conditions commonly employed for protein preparation andcolumn regeneration (Table 1).

Table 1. Properties of Nuvia IMAC Resin.
Property

Description

LigandNitrilotriacetic acid (NTA)

Particle size

38–53 µm

Total ligand density≥18 µmol/ml

Dynamic binding capacity*>40 mg/ml at 300 cm/hr

Compression factor

1.20–1.30

Recommended linear flow rate

50–300 cm/hrPressure vs. flow performance

Under 2 bar at flow rate of 300 cm/hrin DI water (20 x 20 cm packed bed,
1.2 compression factor)

pH stability

2–14

Shipping solution

2% benzyl alcohol or 20% ethanol

Regeneration

50 mM EDTA, pH 8.0 (stripping)
1 N NaOH (CIP/SIP)
0.1 M Ni2SO4 (recharging)

CIP solution

1 N NaOH

Sanitization

1 N NaOH

Storage conditions

20% ethanol

Chemical compatibility**

Reducing agents (β-ME, TCEP, DTT)
Denaturing agents (GnHCl)
Detergents (Triton X-100, NP-40, CHAPS,CHAPSO)
Additives (glycerol)

Chemical stability***

48 hr at 1 N NaOH
1 week at 0.01 N HCl

Shelf life

5 years* 	10% breakthrough capacity determined with 1.2 mg/ml of a 40 kD
histidine-tagged protein in 50 mM sodium phosphate, 5 mM imidazole,
300 mM NaCl (pH 7.5).
** No recharge required. Refer to the instruction manual for a complete list.
*** Recharge required. Refer to the instruction manual for a complete list.

Bulletin 7028

Reference Methods for Packing Columns with Nuvia IMAC Resin: From Lab-Scale to Process-ScaleAbbreviations
a — front section of peak width at 10% of peak height bisectedby line denoting Ve

2.		A s — asymmetry factorb — latter section of peak width at 10% of peak height bisectedby line denoting Ve

3.		

Cs — resin slurry concentration

Determination of resin slurry in packing solution (Vr) needed to packa column to desired bed height (Hp)
Eq. 3

Vr = 3.14 x R2 × Hp × CF/(Cs/100)

Eq. 4

Vt = Vr × Cs

Preparation of resin in packing solution
a. Transfer appropriate amount of resin slurry in storage
solution into a column and drain to remove the storage
solution

CF — compression factor
CV — column volumes

b. Wash the resin with three column volumes (CV) of packing
solution to fully remove the storage solution

HETP — height equivalent to a theoretical plate
Hp — bed height/height of packed columnc. A dd packing solution to resuspend the resin to make slurrywith desired concentration

L — bed height, cm
N — number of theoretical plates
4.		Ve — peak elution volume or time

Evaluation of column efficiency
The packing quality of a column should be subjected toefficiency tests, which typically include determination of theheight equivalent to a theoretical plate (HETP) or reducedheight equivalent to a theoretical plate (rHETP), as well as theasymmetry factor (A s). These tests should be repeated asnecessary during the working life of a column to ensure that itmeets the performance requirements for specific purification.

Vr — resin slurry in packing solution
Vs — settled bed volume
Vt — volume of resin transferred into a column
Vp — volume of packed column
R — radius of column
rHETP — reduced height equivalent to a theoretical plate
W½h — peak width at peak’s half height in volume or time

VeCalculations
Determination of resin slurry concentration (Cs)

Absorbance/Conductivity

1.		

Method 1

a. Transfer 10 ml of resin slurry [Nuvia IMAC Resin isprovided as a 50% (v/v) slurry in 20% ethanol or 2%
benzyl alcohol] into an Econo-Pac™ Chromatography
Column (catalog #7321010)

W1/2h width at half heightab. Apply gentle suction at the column outlet to remove
solution (Note: Avoid drying the resin)

b10% of peakc. A dd water to remove resin off the column wall. Stop thesuction when a stable bed is formed

Injectiond. Wait 5 to 10 min and record the settled bed volumeFig. 1. A simulated chromatogram illustrating the calculation of HETP, rHETP,
and A s values.

Time/Volumee. Calculate Cs by dividing the settled bed volume (Vs)by 10 ml:
Eq. 15.		Calculation of the number of theoretical plates

Cs (%) = 100 × Vs/10 = 10 × Vs

Method 2a. Transfer 10 ml of resin slurry into a graduated cylinder

Eq. 5

N = 5.54 × (Ve/W½h )2

Eq. 6

HETP = L/Nb. Allow resin to settle overnightEq. 7 	rHETP = 10,000 × HETP/average beaddiameter, µm)

c. Record the Vs

Eq. 8

A s = b/ad. Calculate slurry concentration using Eq. 1
Definition of compression factor (CF)Eq. 2

CF = Vt /Vp© 2023 Bio-Rad Laboratories, Inc.

2

Bulletin 7028

Reference Methods for Packing Columns with Nuvia IMAC Resin: From Lab-Scale to Process-Scale

Performing Column Qualification Tests

7.

 onnect the column top adaptor to a chromatography system.
COpen the bottom outlet and consolidate the column bed bypumping packing solution through the column at 60 cm/hr.

8.

 hen a stable bed is formed, stop the pump and close theWbottom outlet.

9.

 lose the column top outlet and push the adaptor downC
slowly to compress the column to the desired H p.

Method 1
Efficiency test with acetone as the probe

Probe solution composition:	2% v/v acetone in
nanopurified water
Probe solution volume:		

1% of Vp

Test velocity:			

100 cm/hrEluent:				nanopurified water

10. Connect the column to a chromatography system againand condition the column with 3 CV of packing solution
at 600 cm/hr downflow.

Method 2
Efficiency test with sodium chloride (NaCl) as the probe

Probe solution composition:	0.8 M NaCl in
nanopurified water
Probe solution volume:		

1% of Vp

Test velocity:			

100 cm/hr

11. Perform column qualification test.
Nuvia IMAC Resin can be packed in a Vantage L Column (MilliporeSigma) with excellent consistency, and post-packing conditioning
improves column efficiency (Table 2). Packing at a CF higher than
1.2 may result in slight column fronting; however, this has little
effect on the rHETP or the pressure flow properties of the packedcolumns (Table 3, Figures 2 and 3). For example, the integrity of acolumn packed with Nuvia IMAC Resin at a CF of 1.28 is wellmaintained even at a linear flow rate of 800 cm/hr (Figure 3).

Eluent:				0.4 M NaCl in
nanopurified water
A packed column is equilibrated with 1.5 CV of eluent
at test velocity before the probe solution is injected.
Reference Column Packing Protocols
Please review the column manufacturer’s instruction manual
thoroughly before performing any of the following lab-scale orprocess-scale procedures.

Table 2. Reproducibility of Nuvia IMAC Resin packed in a Vantage L Column(1.6 x 20 cm) using nanopurified water as the packing mobile phase.
Column efficiency
before conditioning

Lab-Scale Column Packing
Packing Nuvia IMAC Resin in a Vantage L Column (1.6 x 20 cm)

1.

2.
3.

4.

Calculate Vr (typically at a Cs of 45–65%) requiredfor packing a column of desired Hp at a specific CF
using Eq. 3.
 et the bottom frit of the column with packing solution andW
remove air; then close the bottom outlet of the column.

6.

Insert the top adaptor into the column and tighten the seal.
Avoid trapping air between the adaptor and the supernatant.

© 2023 Bio-Rad Laboratories, Inc.

AsrHETP

As

1

1.12

2.67

1.11

rHETP
2.41

2

1.12

2.53

1.05

2.43

3

1.00

3.29

0.92

2.24

4

1.29

2.52

1.00

1.90

5

1.41

3.27

1.35

2.38

Table 3. Effect of CF on the qualification of Nuvia IMAC Resin packed in aVantage L Column (1.6 x 20 cm) using nanopurified water as the packing
mobile phase.

 inse the interior wall of the column to wash down resin
Rparticles.
 llow the resin to settle in the column for 30 min or until a clearA
supernatant of 2–3 cm has developed above the resin bed.

ColumnColumn efficiency was evaluated using acetone as the probe (Method 1).

 eave 1–2 cm of packing solution at the bottom of the column.
L
Transfer an appropriate amount of resin (Vr ) into the column.
Use an extension tube or a packing reservoir, if needed, toensure the entire resin slurry is transferred in one operation.

5.

Column efficiency
after conditioning

CFAsrHETP

1.26

0.78

2.29

1.24

0.84

2.43

1.22

0.92

2.52

1.20

1.11

2.41

Column efficiency was evaluated using acetone as the probe (Method 1).

3

Bulletin 7028

Reference Methods for Packing Columns with Nuvia IMAC Resin: From Lab-Scale to Process-Scale80

8.

 onnect the top valve to a pump and immediately open theCbottom valve.

9.

 onsolidate the column bed by pumping packing solution
Cthrough the column at 60 cm/hr.

Pressure, psi

7060
5040

10. When a stable bed is formed, stop the pump and close thebottom valve.

302011. L oosen the seal slightly and lower the top adaptor toapproximately 1 cm above the consolidated bed surface,and then tighten the seal again.

100100200

300

400

500
600
Flow rate, cm/hr

700

800

900

12. C
 lose the top valve and open the bottom valve. Slowly screw
down the top adaptor to compress the bed to the desired H p.

Fig. 2. Pressure flow curve of Nuvia IMAC Resin packed in a Vantage L Column(1.6 x 20 cm) at a CF of 1.2.

13. Condition the column with 2 CV of packing solution at
320 cm/hr downflow, or at 43–54 psi, as hardware allows.

120Pressure, psi

100

14. Perform column qualification test.

80

CF is crucial for packing Nuvia IMAC Resin in a BPG 100/500
Column (Cytiva) (Table 4). It appears that a CF of 1.28 provides thebest packing quality among all compression factors tested. Similarto packing resin in a Vantage L Column, flow conditioning a packed
column can further improve its efficiency (Table 5). The pressure
flow property of Nuvia IMAC Resin packed in a BPG 100/500
Column (10 x 20 cm) is shown in Figure 4. The recommended
maximum flow rate for performing protein purification with thiscolumn is 320 cm/hr.

6040200100200300400500600
Flow rate, cm/hr

700800900Fig. 3. Pressure flow curve of Nuvia IMAC Resin packed in a Vantage L Column(1.6 x 20 cm) column at a CF of 1.28.

Table 4. Effect of CF on packing Nuvia IMAC Resin in a BPG 100/500 Column(10 x 20 cm) using nanopurified water as the packing mobile phase.

Pilot/Process-Scale Column Packing
Packing Nuvia IMAC Resin in a BPG 100/500 Column (10 x 20 cm)

1.

2.

As

1.25

1.35

rHETP
4.72

1.28

1.16

3.91

1.3

1.21

4.86

1.34

0.99

4.77

 et the bottom frit of the column with packing solution.
W
Remove air and then close the bottom outlet of the column.

Column efficiency was evaluated using sodium chloride as the probe (Method 2).

3.

Leave 1–2 cm of packing solution at the bottom of the column.

4.

 ransfer the appropriate amount of resin slurry (Vr ) into
Tthe column.

5.

 inse the interior wall of the column to wash down resin
Rparticles. Allow resin to settle in the column for 30 min or
until a clear supernatant of 2–3 cm has developed above
the resin bed.

6.

CF

 alculate the Vr (typically at a Cs of 45–65%), required
Cfor packing a column of desired Hp at a specific CF
using Eq. 3.

Table 5. Effect of post-packing conditioning of a column packed with
Nuvia IMAC Resin (10 x 20 cm) at a CF of 1.28 using nanopurified water
as the packing mobile phase.
Column efficiency
before conditioning ower the top adaptor to 1 cm below the liquid surface.
L
Gently shake the adaptor to remove air trapped near theseal or underneath the adaptor.

7. 	Tighten the seal and lower the top adaptor further to remove
any remaining air through the waste line of the top valve.

© 2023 Bio-Rad Laboratories, Inc.

Column efficiency
after conditioning

Column Column conditioning

AsrHETP

As

rHETP

1

Downflow 3 CV
at 200 cm/hr,
23 psi

0.80

2.73

0.94

3.00

2Downflow 2 CV
at 367 cm/hr,
53 psi

0.86

2.40

1.05

2.42

3Downflow 2 CV
at 317 cm/hr, 54 psi

1.32

8.05

0.99

2.94

Column efficiency was evaluated using acetone as the probe (Method 1).

4

Bulletin 7028

Reference Methods for Packing Columns with Nuvia IMAC Resin: From Lab-Scale to Process-Scale50

10. Inflate the seal to six bars. Condition the column with 2 CVof packing solution at 200 cm/hr downflow.

40

11. Perform column qualification test.

30

While the CF is important for packing Nuvia IMAC Resin in anInPlace Column (Table 6), the resin Cs has no significant effect oncolumn efficiency (Table 7). The pressure flow property of Nuvia
IMAC Resin packed in an InPlace Column (20 x 20 cm) is shownin Figure 5. The recommended maximum flow rate for performing
protein purification with this column is 300 cm/hr.

Pressure, psi

6020100100150200250300
Flow rate, cm/hr

350400

Table 6. Effect of CF on packing Nuvia IMAC Resin in an InPlace Column(20 x 20 cm) by axial compression.

Fig. 4. Pressure flow curve of Nuvia IMAC Resin packed in a BPG 100/500 Column(10 x 20 cm) at a CF of 1.29.

CFAsrHETP

Packing Nuvia IMAC Resin in an InPlace Column (Verdot Ips2) (20 x 20 cm) by axialcompression1.2

1.03

2.84

1.25

1.19

1.80

1.

 alculate the Vr (typically at a Cs of 45–65%) required forCpacking a column of desired Hp at the specific CF using Eq. 3.
Program the control console to set the desired H p.

1.3

0.77

4.97

Nanopurified water is used as the packing mobile phase and sodium chloride is theprobe for column efficiency evaluation (Method 2).

2.

 et the bottom frit of the column with packing solution andW
remove air, and then close the bottom outlet of the column.

Table 7. Effect of C s on packing Nuvia IMAC Resin in an InPlace Column(20 x 20 cm) by axial compression.
C s, %

As

 eave 1–2 cm of packing solution at the bottom
Lof the column.

45

0.94

2.1155

1.19

1.80

65

1.11

2.21

4.

Transfer appropriate amount of resin slurry (Vr ) to the column.

Nanopurified water is used as the packing mobile phase and sodium chloride is theprobe for column efficiency evaluation (Method 2).

5.

 inse the interior wall of the column to wash down resinRparticles. Allow resin to settle in the column for 30 min or
until a clear supernatant of 2–3 cm has developed above
the resin bed.

6.

7.

50
45
40
35
Pressure, psi

3.

 ower the top adaptor to 1 cm below the liquid surface.
L
Gently shake the adaptor to remove air trapped near theseal or underneath the adaptor.

Close the column top valve and open the bottom valve.

9.

 et the piston speed to 200 cm/hr to compress the resin toS
the desired H p. Close the bottom valve when compression
is completed. Note: Using a higher piston speed may cause
column fronting.

© 2023 Bio-Rad Laboratories, Inc.

30
252015105Inflate the seal to four bars and lower the piston at300 cm/hr to remove any remaining air trapped inside
the piston through the waste line of the top valve.

8.

rHETP

0100

150

200

250
300
Flow rate, cm/hr350

400

Fig. 5. Pressure flow curve of Nuvia IMAC Resin packed in an InPlace Column(20 x 20 cm) at a CF of 1.2.

5Bulletin 7028

Reference Methods for Packing Columns with Nuvia IMAC Resin: From Lab-Scale to Process-Scale

Troubleshooting Tips
Observation

Possible Cause

High rHETP■Underpacked column■

Compress column further■

Clogged column screen/frit■

Clean screen/frit■ robe volume too large/unoptimized efficiency
Ptest condition■

 odify injection loop to reduce probe volume or use anMalternative test probe■

Unevenly packed column■

Compress column further or repack■

Channels in column bed■

Repack■Overcompressed column■

Repack using a lower CF■

Packing pressure/flow rate too high■Use a lower packing pressure/flow rate■

 robe volume too large/unoptimized efficiency
Ptest condition■

 odify injection loop to reduce probe volume or use anM
alternative test probe■

Interaction between test probe and resin■

Use an alternative test probe■

Underpacked column■

Compress column further■

Air trapped under column adaptor/piston■

Eliminate air■

Space between column adaptor/piston and bed■

Adjust adaptor/piston■

Clogged column screen/frit■

Clean column screen/frit■

Presence of fine particles■

Decant to remove fines■

Contaminated resin■

Clean or replace resin■

Overcompressed column■

Repack using a lower CF■

Channels in column bed■

Compress column further or repack■Interaction between test probe and resin■

Use an alternative test probe■

Plugged or contaminated resin■

Clean or replace resin■

Hardware configuration■

 se slower flow rate to consolidate the bed and thenUlower the adaptor to the desired bed height followed byconditioning with high flow rate

Peak fronting

Peak tailingHigh column pressure

Split peakShoulder peak

Channeling when packing small columns

Correction

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