Purification of an Acidic IgM with a Strong Anion Exchange
Resin Tailored for Large Biomolecule Purification
Jamie C Greenwood II, Payal Khandelwal, William H Rushton, and Carsten Voss
Bio-Rad Laboratories, Inc., 6000 Alfred Nobel Drive, Hercules, CA 94547

Antibody Purification

Bulletin 7127

Abstract

IgM antibodies are one of the fastest growing groups of diagnostic and therapeutic candidates.
The large size of IgMs makes their purification very challenging. Bio-Rad has developed anew strong anion exchange resin (AEX), Nuvia HP-Q, to overcome the multiple issues facedwhen purifying large biomolecules. Purification of an acidic IgM was performed using Nuvia
HP-Q Resin, which resulted in good binding and purification. Further polishing of the Nuvia
HP-Q eluate pool on the mixed-mode CHT Ceramic Hydroxyapatite Media led to highly
pure IgM, with minimal product- and process-related impurities. The workflow resulted in>99% IgM purity, as measured by size exclusion chromatography (SEC) and SDS-PAGE
analysis. This AEX-based purification platform helps overcome the limitations of traditional ion
exchange resins for IgM purification and provides a successful and scalable platform strategy.
The purified IgM is suitable for diagnostic applications and potentially for pharmaceuticalapplications.

Introduction
IgM molecules are penta- and hexameric protein multimers
with a size of 900–1,000 kD. Like other antibodies, they areinvolved in immune responses in mammals. Such recombinant
antibodies are used in diagnostic and some therapeutic
applications. IgM purification presents multiple challenges.
IgM molecules do not have a strong Protein A binding site.
Therefore, affinity purification with Protein A resins is not
an option. Other affinity ligands, like Protein L and variants
thereof, have not shown the same performance as Protein A
resins with respect to dynamic binding capacity (DBC) andligand stability over multiple cycles. Additionally, the large sizeof IgMs has direct impact on several process parameters inchromatographic purification. The dynamic binding capacity ofIgM is significantly reduced in comparison to smaller proteinsbecause of the inability of IgM to penetrate small pores andconsequent insufficient utilization of the pore volume. Theslow diffusion of these large molecules also impacts thechromatography steps that are dependent on mass transport.
To overcome these large biomolecule purification issues,
Bio-Rad developed the Nuvia HP-Q Anion Exchange Resin(bulletin 7078). It is built on the UNOsphere epoxide base beadthat provides fast mass transfer kinetics and low nonspecificbinding. The particle size of Nuvia HP-Q is optimized to offer
high DBC at fast flow rates without excessive backpressure.
Its pore size is optimized for easy accessibility and adsorptionof large biomolecules, like IgMs, and the internal spacer
length and ligand density facilitate optimal binding of large
biomolecules even at high flow rates. Traditional ion exchange
resins have kinetic limitations due to nonoptimized pore sizes
for large biomolecules, which affects purification productivity.
The design and construction of Nuvia HP-Q Resin helps inovercoming this challenge.
In the present study, the purification of an acidic IgM isdemonstrated with a two-step workflow using Nuvia HP-Q
for capture and CHT Type II Ceramic Hydroxyapatite Media,
40 µm (CHT II 40) for polish purification. The capture step
generated ~63% purity and ~83% recovery of the IgM. The
polish step produced an IgM fraction with >99% purity and>77% recovery. In addition, excellent clearance of otherimpurities, such as host cell protein (HCP) and nucleic acids,
was also achieved. These results establish Nuvia HP-Q as agreat capture resin for large biomolecule purification.

Purification of an Acidic IgM with a Strong Anion Exchange Resin Tailored for Large Biomolecule Purification

Materials and MethodsResults and DiscussionGeneralOptimization of Binding and Elution Conditions

The acidic IgM (pI ~6.7) was expressed in HEK cells via
transient expression. Foresight Prepacked Columns
containing Nuvia HP-Q (catalog #12007020) and CHT Type II,
40 μm (#732-4736) were used and column chromatography
was conducted on an NGC Discover 10 Pro Chromatography
System (#7880011) (Bio-Rad Laboratories, Inc.). Protein
fractions were analyzed by Criterion XT Gel (#3450129)
under nonreducing conditions and Criterion TGX Stain-Free
Precast Protein Gel (4–20% linear gradient; #5678093) under
reducing conditions (Bio-Rad). Imaging was performed using
the ChemiDoc MP Imaging System (#17001402; Bio-Rad).
A Quant-iT PicoGreen dsDNA Assay Kit (Thermo FisherScientific Inc.) was used to determine DNA concentration.
HCP analysis was performed using a HEK 293 HCP ELISA Kit(Cygnus Technologies).

Multiple experiments were performed with a range of pH andsalt conditions to determine the optimal binding and elution
conditions of the IgM on Nuvia HP-Q Resin. Eluted proteinfrom the initial trial runs was analyzed for purity and recovery.
Using a linear NaCl gradient at pH 7.5 resulted in best purity
and recovery values for this purification (Figure 1).
A
8.2

Purity, %≤10≤20
≤30
≤40
≤50
≤60
≤70
≤80>80

8.0Elution, pH 7

7.8

Optimization of Binding and Elution Conditions on Nuvia HP-Q Resin

Optimal binding and elution conditions were determined byon-column scouting of different pH conditions using linear
NaCl gradients. For the initial screening, a pH range from
6.5 to 8.5 was tested. All chromatography runs wereperformed on an NGC Discover 10 Pro Chromatography
System using 1 ml Foresight Columns.

7.6
7.4
7.2
7.06.80Capture Purification of the Acidic IgM with Nuvia HP-Q Resin

50100150
Elution [NaCl], mM

200BThe acidic IgM had a calculated pI of 6.7. Approximately
400 ml of the cell culture supernatant with ~2 mg ofIgM antibody was adjusted to pH 7.5 with 800 ml of theequilibration buffer, buffer A (20 mM Na2HPO4/NaH2PO4,pH 7.5), before loading. The Foresight Nuvia HP-Q Column
was equilibrated with 10 column volumes (CV) of equilibrationbuffer. The IgM sample was then applied to the column at120 cm/hr. The column was washed with 20 CV of buffer Aand elution of the bound IgM was done with a linear gradient
to 200 mM NaCl.

8.2
8.0Recovery, %
≤10
≤20
≤30
≤40
≤50
≤60
≤70
≤80>80

10Elution, pH 7

7.8
7.6
60

7.4

50
40

7.2

Polish Purification of the Acidic IgM with CHT II 40 Mixed-Mode Media

30
207.0

CHT Type II Media, 40 µm (CHT II 40) was used for polishing
purification. The pH of the pooled fractions A28 to A37 fromthe capture step was adjusted to 7.0 with 0.1 M H3PO4. Thecolumn was pre-equilibrated with 10 CV of 1 M NaCl,
20 mM Na2HPO4/NaH2PO4, pH 7.0 (buffer C). The pooled
fractions from the capture step were applied to it at 120 cm/hr.
The column was washed with 10 CV of buffer C. The purified
IgM was eluted with a linear phosphate gradient to 400 mM
NaPi with buffer D.

6.8050100
150
Elution [NaCl], mM200Fig. 1. Elution of the acidic IgM from Nuvia HP-Q Resin. A, effect of thebuffer pH and NaCl concentration on the purity of the eluted IgM; B, effect ofthe buffer pH and NaCl concentration on the recovery of the eluted IgM.

Size Exclusion High Performance Liquid Chromatography (SEC-HPLC)

Analysis was performed on a Dionex UltiMate 3000 Systemusing a MAbPac SEC-1 Column (Thermo Fisher Scientific).
A sample volume of 3 µl (Nuvia HP-Q eluate) or 10 µl(CHT II 40 eluate) was injected. Analytical separation wasachieved in isocratic mode using 50 mM Na3PO4, 200 mM
arginine, 300 mM NaCl, pH 6.8 as running buffer at
0.8 ml/min for 12 min.
© 2018 Bio-Rad Laboratories, Inc.

2Bulletin 7127

Purification of an Acidic IgM with a Strong Anion Exchange Resin Tailored for Large Biomolecule Purification

Capture Purification of the Acidic IgM with Nuvia HP-Q Resin

Polish Purification of the Acidic IgM with CHT II 40 Media

Nuvia HP-Q is a high-capacity strong anion exchange resinwith a pore size optimal for easy accessibility and adsorptionof large biomolecules like IgMs. The capture purification wasperformed at pH 7.5. Since the calculated pI of the IgM was6.7, it is expected to carry a negative charge at higher pH,
making it ideal for capture with an AEX resin. The use of Nuvia
HP-Q Resin eliminated the potential need for removal of any
leached Protein A from downstream samples, which is alimitation of using a Protein A affinity resin for capture.

CHT Ceramic Hydroxyapatite is a mixed-mode media
widely used in monoclonal antibody polishing for removal ofaggregates, endotoxins, HCPs, and DNA (Gagnon et al. 2006).
Its applicability for IgM purification has been shown previously(Greenwood et al. 2017). CHT also has the advantages ofoperating well at neutral pH and generally requiring mild saltconcentrations for elution (Gagnon et al. 2014).
The polish step utilized the capability of CHT II 40 to bindstrongly to IgM relative to contaminants and IgG. CHT Type II
has a very low affinity for albumin and is particularly wellsuited for the purification of many species and classes ofimmunoglobulins. Biomolecules can bind to it by calcium
affinity and/or cation exchange interactions (bulletin 6902). The
carboxyl or phosphate groups on biomolecules bind to thecalcium in CHT while amino groups or other positively charged
moieties bind via cation exchange to the phosphate groups.
Since hydroxyapatite binds IgM strongly at physiological pH
and conductivity, mild nondenaturing conditions can be usedfor elution. We used a phosphate gradient for IgM elution. TheIgM peak was seen with the 280 nm probe between fractions
A13 and A20 (Figure 3). These fractions were collected forfurther analytical assays.

A/15–A/17 A/18–A/20 A/21–A/24 A/25–A/27 A/28–A/30 A/31–A/33A/34–A/36 A/37–A/39 A/40–A/42B/1–B/2

30

3,000

20

2,000
1,000

1000Column:

1820222426

28 30 32 34Column volume, ml

36384042320

Foresight Nuvia HP-Q, 1 ml

Equilibration: 10 CV of 20 mM Na 2HPO4/NaH2PO4, pH 7.5 (buffer A)
Sample:

400 ml cell culture supernatant adjusted to pH 7.5 with 800 ml
equilibration buffer

Flow rate:

120 cm/hr

Wash:

20 CV buffer AElution:

10 CV linear gradient from 0 to 20% buffer B

A3 A4 A5 A/6 A/7 A/8 A/9

A/10–A/12

A/13–A/15 A/16–A/17 A/18–A/19 A/20–A/21 A/22–A/23

20028044l 3 (280 nm), mAU

16Conductivity, mS/cml 3 (280 nm), mAU404,000160240200120160120808040400046810121416Column volume, ml

182022

Buffer A:

20 mM Na 2HPO4/NaH2PO4, pH 7.5

Column:Buffer B:

1 M NaCl, 20 mM Na 2HPO4/NaH2PO4, pH 7.5

Equilibration: 10 CV of 1 M NaCl, 20 mM Na 2HPO4/NaH2PO4, pH 7.0

Fig. 2. Capture purification of IgM on Nuvia HP-Q Resin. Chromatography
run of IgM on Nuvia HP-Q with a 0–200 mM NaCl linear gradient. 260 nm (n);280 nm (n); conductivity (n); %B (n).

Conductivity, mS/cmAn IgM peak was seen with the 280 nm probe between
fractions A28 and A37 (Figure 2). The peak is seen somewherein the middle of the salt gradient, coinciding with the 125–150 mM
optimal elution salt concentration. Small aliquots of theeluted samples were kept aside to analyze on gels for purity
determination (data shown later). The remainder of the fractions
was pooled to run on the CHT II 40 Column for subsequent
polishing of the target protein.

Foresight CHT II 40, 1 ml

Sample:Nuvia HP-Q elution pool, supplemented with NaCl, with pHadjusted to 7.0 using diluted H3PO4

Flow rate:

120 cm/hr

Wash:

10 CV buffer CElution:

20 CV linear gradient from 0 to 100% buffer D

Buffer C:

1 M NaCl, 20 mM Na 2HPO4/NaH2PO4, pH 7.0

Buffer D:

1 M NaCl, 400 mM Na2HPO4/NaH2PO4, pH 7.0

Fig. 3. Polish purification of IgM on CHT II 40. Chromatography run of thepreviously eluted IgM on CHT II 40 with a 20–400 mM phosphate gradient in
the presence of 1 M NaCl. 260 nm (n); 280 nm (n); conductivity (n); %B (n).

© 2018 Bio-Rad Laboratories, Inc.

3

Bulletin 7127

Purification of an Acidic IgM with a Strong Anion Exchange Resin Tailored for Large Biomolecule Purification

The resulting IgM after the polish purification was run
on a nonreducing Criterion XT Gel (Figure 4A) and areducing Criterion TGX Gel (Figure 4B) for purity analysis.
The CHT II 40 eluate showed a single band of IgM in thenonreducing gel and two bands for the heavy and the lightchains, respectively, on the reducing gel. Contaminantbands were not observed in these lanes.

A55

4540A 280, mAU35A12345678kDIgM

503025209LMW impurities(serum albumin, transferrin)

15IgM heavy chain

10250

5150

0010075

246Time, min

810126Time, min

81012B
140

Product- and processrelated impurities

5037IgM

120B

100
2 3 4 567 8

9kD
250
A 280, mAU

1150
100
Productandprocessrelated
impurities

75 IgM heavy chain

60405037252080

20IgM light chain

015010Fig. 4. Reduced SDS-PAGE analysis of IgM purified from the Nuvia
HP-Q and CHT II 40 Columns. A, nonreducing Criterion XT Gel; B, reducing
Criterion TGX Gel. A and B, lane 1, protein molecular weight (MW) marker;
lane 2, culture supernatant; lane 3, Nuvia HP-Q flowthrough; lane 4, pooled
Nuvia HP-Q eluate; lane 5, CHT II 40 flowthrough; lane 6, CHT II 40 wash;lane 7, pooled CHT II 40 eluate; lane 8, DNA peak from the CHT II 40 plot;
lane 9, protein MW marker.

24Fig. 5. SEC analysis of the Nuvia HP-Q and CHT II 40 eluates. Analysis wasperformed on a Dionex UltiMate 3000 System using a MAbPac SEC-1 Column.
A sample volume of 3 µl Nuvia HP-Q eluate (A) or 10 µl CHT II 40 eluate (B) wasinjected. Analytical separation was achieved in isocratic mode using 50 mM
Na3PO4, 200 mM arginine, 300 mM NaCl, pH 6.8 as running buffer at 0.8 ml/min
for 12 min. LMW, low molecular weight.

Analysis of the contaminant levels in each of the samples
showed a reduction in the DNA and host cell protein
content after the Nuvia HP-Q purification. Polish purification
with CHT II 40 resulted in minimizing these contaminants
further as shown in Table 1.

The purity was further confirmed by SEC analysis.
Contaminant peaks were seen on the SEC plot with theNuvia HP-Q eluate (Figure 5A) but only a single sharp peakfor IgM was seen on the SEC plot with the CHT II 40 eluate(Figure 5B).

Table 1. Final purification data.
SampleCulturesupernatantPurity, %*Recovery, %* DNA, pg/µl** HCP, ppm***

20—

2,332

>200,000

Nuvia HP-Q EP

63

83

1,187

21,455CHT II 40 EP>9977

Not
detectable

109

EP, elution pool; HCP, host cell protein.
* Determined by SEC-HPLC.
** DNA content determined by Quant-iT PicoGreen dsDNA Assay Kit.
*** HCP content determined by HEK 293 HCP ELISA Kit.

© 2018 Bio-Rad Laboratories, Inc.

4

Bulletin 7127

Purification of an Acidic IgM with a Strong Anion Exchange Resin Tailored for Large Biomolecule Purification

Conclusions
This nonaffinity-based platform approach using Nuvia
HP-Q Anion Exchange Resin produces a >99% pure IgM,
which meets the purification requirements for diagnostic
IgM manufacturing. This approach shows high potential for
pharmaceutical purification as well. The process involvesonly two steps and requires a narrow range of optimized
conditions, making it suitable for scalable purification. Using
this state-of-the-art media with high capacity and selectivity
for large biomolecules also helps overcome the affinitybased challenges of low binding and/or recovery during
IgM purification.
Visit bio-rad.com/AcidicIgM for more information.
Acknowledgements
We deeply appreciate the support and collaboration of the Bio-Rad Antibodies
R&D team in supplying cell culture supernatants and engaging in fruitfuldiscussions.

References
Gagnon P et al. (2006). A ceramic hydroxyapatite–based purification platform.
Simultaneous removal of leached protein A, aggregates, DNA, and endotoxinsfrom mAbs. BioProcess Int 4, 50–60.
Gagnon P et al. (2014). IgM purification with hydroxyapatite. BioProcess Int 12,40–51.
Greenwood JC II et al. (2017). Development of a non-affinity based purification
platform for neutral/basic IgMs. Bio-Rad Bulletin 6966.

© 2018 Bio-Rad Laboratories, Inc.

5Bulletin 7127

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