Minimizing On-Column Monoclonal Antibody
Aggregate Formation
Giorgio Carta,1 Payal Khandelwal, 2 Mark Snyder 2
1
2

Department of Chemical Engineering, University of Virginia, Charlottesville, VA
Bio-Rad Laboratories, Inc., 6000 James Watson Drive, Hercules, CA

Antibody Purification

Bulletin 7000

Abstract

The formation of aggregates is a common occurrence during monoclonal antibody (mAb)
purification and can happen for several reasons. Aggregates are undesirable due to thepossibility of altered pharmacodynamics, pharmacokinetics, and/or increased immunogenicity.
Generally, the formation of aggregates is shown to be product or process related. Here weshow how on-column aggregate formation is dependent on the resin type and on the resin’sinteractions with the mAb loaded. Among the various resins tested, Nuvia™ HR-S Resin,a high-resolution cation exchanger, demonstrated the lowest proportion of on-column
aggregate formation with two mAbs. On-column aggregate formation can therefore be
minimized by proper resin screening and subsequent optimization of resin type used for
purification process development.
Introduction
Aggregate formation can happen due to a variety of reasons,such as self-association of monomers or protein nucleation(Lebendiker and Danieli 2014). Aggregates are highly
undesirable in final purified products, especially in therapeuticmAbs. Their presence can lead to different bioactivity andpotency profiles, storage stability, and pharmacodynamic andpharmacokinetic properties relative to the monomeric versions(Lang et al. 2011).
Because many current-day mAbs have a relatively high pI,
cation exchangers (CEX) are often chosen for removal of
aggregates, charge variants, and other impurities in the
purification workflow (Shukla et al. 2007, Stein andKiesewetter 2007). CEX purification platforms offer mild
processing conditions, which have been thought to induce
neither conformational changes nor unfolding during protein
binding (Staby et al. 2005).
However, multiple recent studies have reported two-peak
behavior during mAb elution from CEX resins, due either toconformational changes in the protein during the binding step
(Gillespie et al. 2012) or to reversible self-association of themAbs at high salt and protein concentrations during the elution
step (Luo et al. 2014). Both conformational changes and mAb
self-association lead to on-column aggregates. However, thedegree of on-column aggregate formation may be resin- andmAb-specific.

To explore this possibility, a series of different CEX resins
were analyzed to determine the effects of their individual
characteristics on on-column aggregate formation. Results
showed that the proportion of on-column aggregate formation
depends in part on the different surface extenders, poresizes, and pore size distributions of the resins used and inpart on the interactions between the mAbs and the selected
resin’s structures (Guo and Carta 2015, Guo et al. 2016, Recket al. 2017). This article discusses the results obtained withresins that can be used for process-scale manufacturing ofmAbs and the importance of selecting the ideal resin for such
purifications. Based on the results obtained, Nuvia HR-S, astrong cation exchanger, emerges as an ideal process resin
to minimize the levels of on-column aggregate formation under
the tested conditions.
Materials and Methods
Chromatography resins

Fractogel EMD SO3– (M) and Eshmuno S Resins were obtained
from EMD Millipore. According to the manufacturer’s literature,both have a polyacrylate backbone grafted with charged
polymers or “tentacles” to facilitate binding of proteins.
POROS 50 HS and POROS XS Resins were obtained from
Thermo Fisher Scientific. They are based on a cross-linkedpoly(styrene-divinylbenzene) backbone with a polyhydroxyl
surface coating functionalized with sulfopropyl groups.

Minimizing On-Column Monoclonal Antibody Aggregate Formation

Table 1. Mean particle diameter and charge densities of all resins used.

ab

FractogelSO3 – (M)Eshmuno S

POROS 50 HS

POROS XS

Capto S
ImpActCapto S
ImpResNuvia S

Nuvia HR-S

Mean particle
diameter, µm

74

85

50

50

50

36–44

b

85 ± 15b

50 ± 10bCharge density,µeq/mlb

70–110

130–160

90–150

100–180aa50–100b

135b88-120b

37–63

Guo and Carta 2015.
Values obtained from manufacturer literature.

Capto S ImpAct and Capto S ImpRes Resins were obtained
from GE Healthcare. Both are based on a rigid, high-flow
agarose matrix modified with dextran surface extenders.
Nuvia HR-S and Nuvia S Resins were obtained from
Bio-Rad Laboratories. They are based on an acrylamide
polymeric backbone functionalized with sulfopropyl groups.
Nuvia HR-S does not contain grafted polymers whereas
Nuvia S is grafted with polymeric surface extenders. The
physical properties of the resins are summarized in Table 1.
Comparison of on-column aggregate formation on seven resins after different
retention times

Resins were packed into 0.5 cm diameter × 5 cm long columns
with an actual packed bed volume of 1.00 ± 0.04 ml. Eachcolumn was equilibrated with five column volumes (CV) of
acetate buffer containing 40 mM Na+ and then loaded with
0.5 ml of 2 mg/ml mAb A in the acetate buffer. After loading,the flow was stopped and the column was held idle for different
hold times ranging from 0 to 1,000 min (data shown only for
the 0 and 1,000 min hold time). Two CVs of load buffer werethen applied followed by a 20 CV gradient to 1 M NaCl in load
buffer. All chromatograms were normalized to a total peakarea proportional to the amount of protein injected per unit ofcolumn volume. This was done in order to account for small
differences in actual protein feed concentration and column
volumes, thus facilitating comparisons between runs. The
default load and elution flow rate was 0.5 ml/min, but flow
rates of 0.25 and 1 ml/min were also used.
Calculation of hydrodynamic radii of the eluted species on POROS XS
and Nuvia HR-S

All calculations were done as described in Guo et al. 2016.
Monoclonal antibodies

Monoclonal antibody A was a glycosylated IgG2 antibody witha molecular mass of 150 kDa and a calculated pI of 8.7 basedon its amino acid sequence. Monoclonal antibody B had amolecular mass of 150 kDa and a calculated pI of 7.
Results and Discussion
On-column aggregate formation is dependent on the resin type

Monoclonal antibody A was loaded onto columns packed
with seven different resins. The load buffers used in these runs
were an acetate buffer containing 40 mM Na+ at pH 5.0 for the
Fractogel and acetate buffer containing 20 mM Na+ at pH 4.8
for all other resins. An elution flow rate of 0.3 ml/min was usedwith Eshmuno S and Nuvia S whereas all other columns wereoperated at an elution flow rate of 1 ml/min. These conditions
were selected to provide the best resolution between peaks forthe different resins. Antibody was held on the various resins fortimes ranging from 0 to 1,000 minutes.
© 2017 Bio-Rad Laboratories, Inc.

As shown in Figure 1, for the conditions chosen, the protein
eluted in a single peak on all resins when elution was started
immediately after loading and washing the column (0 minutehold time). However, different patterns of elution were seen foreach of the resins when employing a 1,000 minute hold time.
All resins showed a two-peak elution behavior with the firstpeaks appearing at the same retention time as the 0 minutehold time runs. The second elution peaks, however, showed
different retention times and percentage of total eluted protein
for all seven resins. Such late-eluting peaks were previously
found to be a mixture of monomers and higher molecular mass
species, aggregates, that appeared to be stable over long
periods of time (Guo et al. 2014, Guo and Carta 2014). Further
studies showed that such two-peak behavior was a result of
resin-induced protein conformational changes that occurred
gradually and involved the fragment crystallizable (Fc) regionof the mAbs (Guo et al. 2015).
Nuvia HR-S yielded the least amount of eluted protein in thesecond peak (~10%) (Table 2). This result is in agreement withpreviously reported data showing that the “tentacle type”
Fractogel displayed strong two-peak elution behavior (Guo et al.
2014, Guo and Carta 2014), whereas only one peak eluted froma macroporous CEX resin without “tentacles” under otherwise
identical conditions (Guo and Carta 2015).
The predominant one-peak elution profile of mAb A on
Nuvia HR-S appears to be related to the resin’s internal structure.
The tested resins (excluding Nuvia HR-S and POROS 50 HS)
contain grafted polymers designed to enhance binding capacity.
In such cases, binding is not restricted to monolayer coverage,
but potentially occurs throughout the grafted polymer layer(Almodóvar et al. 2011, Almodóvar et al. 2012). Therefore, theseresins demonstrated a distinct two-peak elution behavior witha relatively large fraction of late-eluting protein.
In contrast, POROS 50 HS does not have grafted polymeric
extenders. Despite this, it showed a distinct two-peak separation
with a pronounced late-eluting peak. But POROS 50 HS has
a bimodal pore size distribution, which includes large “throughpores,” about 500 nm in diameter, as well as small pores, about
22 nm in diameter. It is possible that proteins bind in thesesmall pores, similar to the way they bind to a grafted polymer
layer, yielding a large second elution peak.
Nuvia HR-S, on the other hand, has an open pore structure
without bimodal pores or grafted polymers and shows the least
percentage of protein elution in the second peak (Table 2).

Bulletin 7000

Minimizing On-Column Monoclonal Antibody Aggregate Formation

80

0.6

70

0.5

60

0.4

50

0.3

40

0.2

30

0.1

20

0

8

10

12

14

16

18

20

10

B. Eshmuno S - 0.3 ml/min, pH 4.8
1

70

0.8

50
40

0.4

20

0
8

10

12

Normalized absorbance

0.8
0.6
0.4
0.2

14

60
0.6

16

18

20

16

18

20

30
0.2
0
8

10

12

14

18

20
0

10

20

10
8

10

12

14

70

0.7

70

60

0.6

60

0.4

50

40

0.3

40

30

0.2

30

20

0.1

20

0.1

10

0

10

0

16

18

20

0.5

50

0.4

40

0.3

30

0.2

20
8

10

12

14

16

18

20

10

CV

CV

16

18

20

CV

80

0.6

CV

16

0.8

0.5

14

30

0.2

G. Nuvia HR-S - 1 ml/min, pH 4.8

60

12

40

20

70

10

50
0.4

CV

F. Nuvia S - 0.3 ml/min, pH 4.8

8

60

0.6

40

0.4

10

70

50

80

50

12

0.8

0.7

80

1

10

14

80
0.8

70

30
0.2

D. Capto S ImpAct - 1 ml/min, pH 4.8
80

CV

E. Capto S ImpPres - 1 ml/min, pH 4.8

8

1

60
0.6

CV

0

C. POROS 50 HS - 1 ml/min, pH 4.8
80

Conductivity, mS/cm

0.7

Conductivity, mS/cm

Normalized absorbance

A. Fractogel SO3 – - 1 ml/min, pH 5.0

Fig 1. Nuvia HR-S caused theleast amount of on-column
aggregate formation of mAb A.
The load buffer for all resins from
B to G was acetate buffer containing
20 mM Na+ at pH 4.8 and for
A was acetate buffer containing
40 mM Na+ at pH 5.0. The elution
flow rate was 1 ml/min on all resins
except B and F, which had a flowrate of 0.3 ml/min. 0 min hold time,(– – –); 1,000 min hold time, (——);
conductivity, (——). Data in A-C andF courtesy of Dr. Giorgio Carta.

Table 2. Percentage of on-column aggregate formed on different resins (protein eluting in 2nd peak after 1,000 min hold time).
Eshmuno S

POROS 50 HS

Capto S
ImpActCapto S
ImpResNuvia S

Nuvia HR-S

87

44

51

65

50

73

10

Two-peak elution behavior is also dependent on the inherent
stability of the mAb to be purified. Due to differences in stability,
different mAbs may show dissimilar behaviors on the same
resin under the same conditions. To demonstrate this principle,
a second monoclonal antibody, mAb B, with an approximate
pI of 7 and MW of 150 kDa, was studied. This antibody issimilar to the mAb A in size, but differs in its pI. Size exclusion
chromatography (SEC) analysis on fresh mAb B confirmedthat it contained only the monomeric species (Guo et al. 2016).
Monoclonal antibody B was loaded onto Nuvia HR-S and
POROS XS, and its elution profiles after zero and 30 min
hold times were studied. This antibody exhibited a two-peakelution behavior for zero hold times (Guo et al. 2016) andthree-peak elution behavior for longer hold times (30 min)on the POROS XS (Figure 2A). In contrast, a single peak
was observed even after a 30 minute hold time on Nuvia
HR-S (Figure 2B). In-line hydrodynamic radius measurements
confirmed that the single elution peak obtained for Nuvia HR-S
consisted exclusively of monomeric species with radii of about
5.3 nm. However, with POROS XS, the hydrodynamic radii ofthe species increased from 5.3 nm (corresponding to the firsttwo elution peaks) to about 7.5 nm, indicating the formationof aggregates.

© 2017 Bio-Rad Laboratories, Inc.

Both Nuvia HR-S and POROS XS have an average particle sizeof 50 µm and, hence, similar resolution. Results also indicatedsimilar elution peak widths from both resins. Therefore, thevariances in the elution profile at different hold times are notdue to any differences between the resolving powers of thetwo resins. Instead, these elution behaviors are most likely due
to the distinct way in which mAb B interacted with the resins’surfaces and structures.
A. POROS XS

B. Nuvia HR-S

0.5

9

0.6

9

0.4

8

0.5

8

7

0.3

6

0.2

5

0.1

4
0

0.4

7

0.3
6

0.2

5

0.1

4

0
4

5

6

7

CV

8

9

10

Hydrodynamic radius, nm

On-column aggregate formation depends on unique mAb-resin structureinteractions

Normalized absorbance

Percentage of on-column
aggregation, %FractogelSO3 – (M)

4

5

6

7

8

9

10

CV

Fig. 2. Comparison of elution behavior and on-column aggregate formationof mAb B. Columns packed with POROS XS (A) and Nuvia HR-S (B) with a
30 min hold time followed by a 20 CV 0–1 M NaCl gradient in 40 mM sodium
acetate at pH 5. For POROS XS, the first two peaks contain species withhydrodynamic radii of 5.3 nm, consistent with monomeric mAb B, while the third
peak shows hydrodynamic radii increasing to about 7.5 nm, corresponding to a
dimeric species. For Nuvia HR-S, a single peak with hydrodynamic radii of 5.3 nm
indicates the presence of only monomeric species. Hydrodynamic radii ( ).
Data courtesy of Dr. Giorgio Carta.

Bulletin 7000

Minimizing On-Column Monoclonal Antibody Aggregate Formation

Retention time of mAb B monomers depends on resin internal structure

In contrast to the results seen from mAb A, mAb B eluted asa monomer in two peaks at the zero time point on POROS XS(Figure 1, Guo et al. 2016). Yet mAb B eluted in a single peak on
Nuvia HR-S. To explore this behavior further, the two monomer
peak fractions from the POROS XS Column were separately
dialyzed against the original load buffer and then individually
re-injected into either the POROS XS Column or the Nuvia HR-S
Column. Re-injecting the fractions from the two peaks into
the POROS XS Column resulted in the same two-peak elution
behavior as the fresh mAb sample (Figure 3A). In contrast,
reloading on the Nuvia HR-S Column led to the same single
peak regardless of which POROS XS Resin peak-produced
fraction was re-injected (Figure 3B). This confirms that thetwo primary mAb B elution peaks from POROS XS contained
monomers. The POROS XS contains two different pore sizes
that bind mAb B with different strengths and at different rates,
explaining the two-peak behavior. Nuvia HR-S, which has auniform pore size, produced a single peak.
B. Nuvia HR-S

1.2

1.2

1

1

Normalized absorbanceNormalized absorbance

A. POROS XS

0.8
0.6
0.4
0.2
0

0.8
0.6
0.4
0.2
0

4

5

6

7

8

4

CV

5

6
CV

7

8

Fig. 3. Comparison of elution profiles on POROS XS and Nuvia HR-S.
Elution profiles of fresh mAb B sample were overlaid with the results obtainedby re-injecting fractions from the POROS XS with 0 min hold time followed by
a 20 CV 0–1 M NaCl gradient in 40 mM sodium acetate at pH 5. A, Resultsfrom POROS XS. B, Results from Nuvia HR-S. Conditions were identical for
all runs. Fresh mAb sample (––––); first peak fraction (– – –); second peakfraction (- – - – ). Data courtesy of Dr. Giorgio Carta.

Conclusion
Of all the resins tested under the mentioned conditions,
Nuvia HR-S, a high-resolution CEX resin, shows minimal
formation of on-column aggregates, irrespective of mAb
type. Nuvia HR-S also demonstrated single-peak elution
behavior after mAb B had been held on the resin for up to
30 minutes. We know this behavior is not due to its particle
size because POROS XS, which has the same particle size
as Nuvia HR-S, demonstrated a different elution behavior
and aggregate formation. Prior studies have also shown that,when compared with another high-resolution agarose-based
resin, Nuvia HR-S demonstrated comparable total aggregate
removal, but at significantly higher recovery rates (Ng et al. 2014).
These results underscore the importance of screening multiple
resins during mAb purification process development, especiallyfor aggregation-prone mAbs, not only to minimize the potentialof on-column aggregate formation, but also to optimize recovery.
Without this screening, it is difficult to predict the interaction andelution behavior of different mAbs on any given resin.
Acknowledgements
The authors express their appreciation to Jing Guo, Shaojie
Zhang, Arch Creasy, and Jason Reck from the University ofVirginia for generating the data included in this article.
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Capto S ImpAct and Capto S ImpRes are trademarks of General Electric Company.
DynaPro NanoStar and MiniDAWN TREOS are trademarks of Wyatt Technology
Corporation. Fractogel EMD SO3– (M) and Eshmuno S are trademarks of Merck KGaA,
Darmstadt, Germany. POROS XS and 50 HS are trademarks of Thermo Fisher Scientific.

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