A Nuvia™ IMAC Resin–Based Purification Workflow for
the Production of Highly Pure Recombinant Proteins
TechNote

Louisa Vang and Xuemei He
Bio-Rad Laboratories, 6000 James Watson Drive, Hercules, CA 94547

Protein Purification

Bulletin 6964

Abstract
A three-column workflow has been developed for the purification of a histidine-tagged recombinant protein from E. coli
extract. The macroporous Ni2+-charged Nuvia IMAC Resin provides efficient capture of the target protein moleculeswith excellent selectivity, binding capacity, and recovery. The subsequent mixed-mode chromatography steps, performedwith Nuvia™ cPrime™ Resin followed by CHT™ Ceramic Hydroxyapatite Media, eliminate leached Ni2+ ions and offer
further clearance of residual impurities from host cells.
Introduction
Immobilized metal affinity chromatography (IMAC) is one of themost popular tools for the preparation of recombinant proteinsin a single step. Proteins naturally rich in histidine or cysteine
residues, as well as those with histidine residue clusters
engineered to their N- or C-terminus via recombinant DNA
technology, form strong chelation complexes with transition
metals, such as Ni2+, Cu2+, Co2+, or Zn2+, immobilized on
chromatography matrices. After impurities are washed off, thebound target protein molecules are selectively eluted from theIMAC column with buffers containing metal chelator imidazole.
As with other affinity chromatography techniques, purification
with IMAC is robust and requires minimal optimization ofconditions. It is typically performed at physiological pH andshows good tolerance for a wide variety of chemicals (Bio-Radbulletin 6859), which is crucial to maintaining the structural
integrity and biological activity of target protein molecules.
Proteins prepared with IMAC are often pure enough forstructural and functional characterization studies.
In recent years, the use of IMAC has been extended to themanufacture of protein therapeutics (Bhattacharyya et al. 2004),
vaccines (Angov et al. 2003, Darko et al. 2005, Ockenhouse etal. 2006, Stoute et al. 2007), and adeno-associated virus (AAV)(Koerber et al. 2007). However, protein pharmaceuticals areheld to extremely high purity standards to ensure drug efficacy
and the safety of patients. In addition to initial target extraction
from crude bioprocess mixtures, further polish purificationis necessary for sufficient clearance of contaminants, suchas host cell proteins (HCPs) and DNA, and leachables fromchromatography media. In this study, we demonstrate a threecolumn worfklow for the purification of a histidine-tagged protein(Figure 1). Nuvia IMAC, a rigid macroporous high-capacity resin
designed for process production at high flow rates, was used
to capture histidine-tagged protein from E. coli lysate. Furtherpurification was facilitated by chromatography with two mixedmode media, Nuvia cPrime and CHT Ceramic Hydroxyapatite.
This purification sequence resulted in effective removal ofhazardous contaminants such as endotoxins and leached Ni2+and the significant reduction of host cell proteins and DNA.
E. coli lysate

Nuvia IMAC Resin

Bind-Elute Mode

Nuvia cPrime Resin

Flow-Through Mode

CHT Type I Media

Bind-Elute Mode

Fig. 1. Purification workflow for a histidine-tagged target protein fromE. coli lysate.

Material and Methods
GeneralE. coli cell lysate containing the target histidine-tagged protein
was prepared by the Center for Biocatalysis and Bioprocess,
University of Iowa. All chromatography was conducted on a
BioLogic DuoFlow QuadTec™ 10 System (Bio-Rad Laboratories).
Chromatography media Nuvia IMAC, Nuvia cPrime, and CHT
Type I, 40 μm (Bio-Rad) were packed and regenerated following
manufacturer’s instructions (bulletins 10044307, 10023853,
and 6086, respectively). Protein fractions were analyzed bySDS-PAGE using Criterion™ Precast Gels (Tris-HCl 4–20%
linear gradient), Bio-Safe™ Coomassie Stain, and Gel Doc™
EZ Gel Documentation System (all Bio-Rad). The clearanceof HCPs and double-stranded DNA (dsDNA) was determinedby E. coli HCP ELISA Kit (Cygnus Technologies) and ddPCR™
E. coli Residual DNA Quantification Kit (Bio-Rad), respectively.
Endotoxin contamination in protein samples was detected withthe Endpoint Chromogenic LAL Assays (Lonza). Leached Ni2+

A Nuvia™ IMAC Resin–Based Purification Platform for the Production of Highly Pure Recombinant Proteins

was quantified by Exova using inductively coupled plasma mass
spectrometry (ICP-MS). Protein concentration was quantified
with Bradford Protein Assay (Bio-Rad).

Table 1. Design of experiment (DoE) setup.

Experiment

Elution Buffer
Sodium Phosphate
Concentration, mM

Elution Buffer
Sodium Chloride
Concentration, M

Elution Buffer pH

115026.5

250

26.5

35007.5

4150

06.5

550

0

6.5

6100

177150

27.5

8100

17950

27.5

10150

0

7.5

Capture purification of histidine-tagged protein by immobilized metal affinity
chromatography using Nuvia IMAC Resin

Intermediate polish purification by hydrophobic cation exchange
chromatography using Nuvia cPrime Resin

The eluate from the Nuvia IMAC column was loaded at 300 cm/hrdirectly onto a 5 ml Nuvia cPrime Column (1.25 x 4 cm) preequilibrated with 10 CV of buffer B. The target histidine-tagged
protein was recovered in the flow-through fractions. This column
was then regenerated with 5 CV of 50 mM Tris-HCl, 5 mM
sodium chloride, pH 7.5 (buffer D), 5 CV of 50 mM sodiumphosphate, 2 M sodium chloride, pH 7.5 (buffer E), followedby 5 CV of 1 N sodium hydroxide.
Further purification by ceramic hydroxyapatite media chromatography

CHT Ceramic Hydroxyapatite Type I, 40 µm Media wasemployed for further purification of target protein. A design ofexperiment (DoE) study was performed to screen for the optimal
elution condition. The effects of sodium phosphate and sodium
chloride concentrations and buffer pH were evaluated using a
two-level full factorial screening design as suggested by JMP
Software (jmp.com) (Table 1). Mini Bio-Spin® Columns, eachcontaining 100 μl of CHT Media pre-equilibrated with buffer B,
were used in this screening study. Pre-equilibrated CHT Mediawas mixed at room temperature for 3 min with agitation in a spincolumn with 500 µl of load, which contained ~0.4 mg histidinetagged protein purified using the above described Nuvia cPrime
chromatography step. Unbound material was removed at theend of the incubation by centrifugation at 1,000 x g for 1 min.
Elution of target protein was achieved by incubation with each
elution buffer to be tested for 10 min with agitation, followedby centrifugation at 1,000 x g for 1 min. These eluates wereanalyzed by SDS-PAGE. The target protein yield, as reflected by
the target protein band density, and target protein purity werequantified using Image Lab™ Software (Bio-Rad). A standard
least squares model was employed to obtain the response
surfaces using JMP Software.

Note: Experiments 6 and 8 are the center points

For scale-up purification, a 1 ml CHT Type I, 40 µm Column(0.56 x 4.0 cm) was equilibrated with 10 CV of buffer B. The
partially purified histidine-tagged target protein from theprevious two steps was applied onto this CHT Column. The
loaded column was washed with 10 CV of 50 mM sodium
phosphate, 400 mM sodium chloride, pH 7.5 (buffer F). Target
protein was eluted by 10 CV of 50 mM sodium phosphate,
1.2 M sodium chloride, pH 7.5 (buffer G). The used column
was regenerated by 2 CV of 50 mM Tris-HCl, 5 mM sodiumphosphate, 2 M sodium chloride, pH 7.5 (buffer H) followedby 5 CV of 1 N sodium hydroxide.
Results
The open pores of Nuvia IMAC allow efficient mass transfer underdynamic conditions. Its hydrophilic inert surface results in minimal
nonspecific binding through charge or hydrophobic interactions.
As demonstrated in Figure 2, the histidine-tagged target protein
was efficiently captured from the E. coli lysate by chelation withNuvia IMAC Resin and eluted with over 95% recovery and near
homogeneous purity as visualized by SDS-PAGE. The 10%
breakthrough dynamic binding capacity of target protein wasestimated as 50–60 mg per ml of resin (data not shown).
A1
4.0

2

3

45

Fractions
6789101112

100.0% Buffer C

3.5
3.0

75.0

Elutedtarget

2.5A 280, AU

Nuvia IMAC Resin was packed in a 2.2 x 25 cm column andequilibrated with 10 column volumes (CV) of 50 mM sodiumphosphate, 10 mM imidazole, 300 mM sodium chloride, pH 7.5(buffer A). E. coli cell lysate was centrifuged at 30,000 rpm for
5 min to collect the supernatant, which was then applied onto
the Nuvia IMAC Column at 150 cm/hr. The loaded column waswashed with 5 CV of buffer A followed by 5 CV of 50 mM
Tris-HCl, 5 mM sodium phosphate, pH 7.5 (buffer B). The boundtarget protein was eluted with 10 CV of 50 mM Tris-HCl, 5 mM
sodium phosphate, 125 mM imidazole, pH 7.5 (buffer C).
Manufacturer’s instructions (Bio-Rad bulletin 10044307)
were followed to regenerate the column.

2.0

50.0

1.5
1.0

25.0

0.500

1020Time, minFig. 2A. Capturing histidine-tagged protein from E. coli cell lysate using aNuvia IMAC Column. Chromatogram of histidine-tagged protein purification ona Nucia IMAC Column. A 280 (—); % buffer C (--); AU, absorbance units.
© 2017 Bio-Rad Laboratories, Inc.

Bulletin 6964

A Nuvia™ IMAC Resin–Based Purification Platform for the Production of Highly Pure Recombinant Proteins

B123456789HNOHNOHOFig. 4. Ligand structure of Nuvia cPrime Resin.
Table 2. Nuvia cPrime chromatography in flow-through mode:
Correlation of buffer conductivity and Ni 2+ clearance efficiency.
Fig. 2B. Capturing histidine-tagged protein from E. coli cell lysate usinga Nuvia IMAC Column. SDS-PAGE analysis of column fractions. Lane 1,E. coli cell lysate; lane 2, Precision Plus Protein™ Standards; lanes 3–9, column
fractions 3–9. Target protein containing fractions 7 and 8 were pooled andsubjected to next purification step.

The predicted pI of the target protein, calculated based on its
polypeptide sequence, is ~6. The protein is thus expected tocarry a net negative charge at pH ~7.5. We tested multiple ion
exchange resins for polish purification (data not shown). The
hydrophobic cation exchange resin Nuvia cPrime was eventuallychosen, as it predominantly binds basic host cell proteins whileleaving the acidic target protein in the flow-through fractions(Figure 3). In addition, the Nuvia cPrime Resin ligand contains anelectron-rich carboxylic moiety (Figure 4), which makes this resin
an excellent scavenger of the Ni2+ ions leached from the IMAC
resin at pH 7.5. The conductivity of preequilibration buffer used
for the Nuvia cPrime Column has a significant effect on the Ni2+clearance efficiency (Table 2). Buffer B is preferred, as it ensuresmaximum retention of Ni2+ by Nuvia cPrime Resin.
246Fractions
8 10 12 14 16 18 20 22 24 26 28 30

1.00

10.90
0.80

30.70

A 280, AU

0.60
0.50
0.40
0.30

20.20

40.10
0.00

BufferConductivity,
mS/cma

Ni2+,
µg/gb

50 mM sodium phosphate,
25 mM NaCl, pH 7.5, buffer I

14.560.120

50 mM Tris-HCl, pH 7.5,5 mM sodium phosphate, buffer B

7.02

0.014

Buffer Composition

abDetermined at 23°C on a BioLogic DuoFlow QuadTec 10 System.
ICP-MS analysis, per gram of solution. Detection limit is 0.002 μg/g.

For the second polish step we used CHT Media, a naturally
occurring mixed-mode chromatography media that provides
unmatched clearance of process-related impurities (Figure 5).
CHT has been widely used in the downstream processing oftherapeutic biologics (Gagnon et al. 2006). In the purification
workflow we present here, CHT offers additional clearance ofendotoxins, proteins, and nucleic acids from expression hostcells (Table 3). The residual Ni2+ ions in the Nuvia cPrime
Column flow-through pool were effectively eliminated by CHT,
most likely through their strong interaction with the phosphoryl
groups on this chromatography media (Figure 6). The eluate
from the CHT Media contained less than 151 endotoxin units
per mg of purified protein, or less than 8 EU/50 μg dose ofvaccine, which was significantly lower than the acceptable
criteria of 350 EU per 50 µg dose for 70 kg human bodyweight (Angov et al. 2003). Additionally, the low conductivity
buffer employed for the flow-through operation of theNuvia cPrime Column allowed direct loading of the target
protein-containing material onto the CHT Column. Therefore,
feedstream manipulation was minimized, which greatlyreduced the overall time and cost associated with target
protein production.
Table 3. Summary of process impurity clearance.

01020Time, min30Fig. 3. Intermediate polishing purification of histidine-tagged protein
in flow-through mode using a Nuvia cPrime Column. Peak 1, histidinetagged target protein; peak 2, impurities eluted by buffer D; peak 3, impurities
eluted by buffer E; peak 4, impurities stripped by 1 N NaOH. A 280 (—);
AU, absorbance units.

Samples from
Purification Steps

Endotoxin,
EU/mga

Ni2+,
µg/gb

dsDNA,ng/mgc

E. coli lysate

1.05 x 105—

1.34 x 104

2.87 x 105

Nuvia IMAC Column

5.13 x 10

3

1.400

4.63 x 10

2

1.11 x 104

Nuvia cPrime Column<3.60 x 103

0.014

2.42 x 102

6.03 x 102

CHT Column<1.51 x 10NotdetectedNot
detected

1.46 x 102

2HCP,ng/mgd

LAL assay, per milligram of protein.
ICP-MS analysis, per gram of solution. Detection limit is 0.002 μg/g.
c ddPCR analysis, per milligram of target protein. Detection limit is 1.2 pg/ml.
d ELISA, detection limit is 1.6 ng/ml.
ab© 2017 Bio-Rad Laboratories, Inc.

Bulletin 6964

A Nuvia™ IMAC Resin–Based Purification Platform for the Production of Highly Pure Recombinant Proteins

246Angov E et al. (2003). Development and pre-clinical analysis of a Plasmodium
falciparum merozoite surface protein-1(42) malaria vaccine. Mol Biochem
Parasitol 128, 195–204.

300

0.6250Target protein
eluted by buffer G0.4

2000.31500.21000.1

5000Buffer 1 N NaOHHPre-elution wash
buffer F0102030Time, minConductivity, mS/cm

0.5A 280, AUReferences

Fractions
8 10 12 14 16 18 20 22 24 26 28 304050Fig. 5. Further purification of target protein using a CHT Column.
Target protein eluted from the CHT Column was collected in fractions 18–22.
A 280 (—); conductivity (—); AU, absorbance units.

Bhattacharyya MS et al. (2004). Recombinant factor VIII for haemophilia. An
overview of production technologies. CRIPIS 4, 2–8.
Darko CA et al. (2005). The clinical-grade 42-kilodalton fragment of merozoite
surface protein 1 of Plasmodium falciparum strain FVO expressed in
Escherichia coli protects Aotus nancymai against challenge with homologous
erythrocytic-stage parasites. Infect Immun 73, 287–297.
Gagnon P et al. (2006). A ceramic hydroxyapatite-based purification platform.
Simultaneous removal of leached Protein A, aggregates, DNA, and endotoxinsfrom mAbs. BioProcess International 4, 50–60.
Koerber JT et al. (2007). Engineering adeno-associated virus for one-steppurification via immobilized metal affinity chromatography. Hum Gene Ther 18,367–378.
Ockenhouse CF et al. (2006). Phase I safety and immunogenicity trial of FMP1/
AS02A, a Plasmodium falciparum MSP-1 asexual blood stage vaccine. Vaccine
24, 3,009–3,017.
Stoute JA et al. (2007). Phase 1 randomized double-blind safety andimmunogenicity trial of Plasmodium falciparum malaria merozoite surface
protein FMP1 vaccine, adjuvanted with AS02A, in adults in western Kenya.
Vaccine 25, 176–184.

Visit bio-rad.com/NuviaIMAC for more information.
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Precision Plus Protein Standards are sold under license from Life Technologies
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is not authorized to sell or resell this product or its components.

Fig. 6. Schematic representation of functionalities of CHT Media.

Conclusions
We employed three chromatography columns in sequence
to obtain highly purified histidine-tagged recombinant proteinfrom E. coli. This workflow requires minimum feed conditioning
during transitions between chromatography steps, which
greatly minimizes labor and the consumption of materials.
This robust purification process provides the productquality, productivity, and economics demanded by today’s
downstream manufacturing.

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