Cation exchange frontal chromatography for the removal of monoclonal antibody aggregates

Journal of Chromatography A, 1599 (2019) 152–160

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Cation exchange frontal chromatography for the removal of
monoclonal antibody aggregates
Matthew T. Stone ∗ , Kristen A. Cotoni, Jayson L. Stoner
EMD Millipore Corporation, United States

a r t i c l e

i n f o

Article history:
Received 26 November 2018
Received in revised form 7 April 2019
Accepted 8 April 2019
Available online 9 April 2019
Keywords:
Cation exchange chromatography
Frontal chromatography
Continuous loading chromatography
Overloaded chromatography
Monoclonal antibody aggregates

a b s t r a c t
®

A low ligand density cation exchange (CEX) chromatography resin, Eshmuno CP-FT resin, was investigated for the removal of aggregates from monoclonal antibody (mAb) feeds using a continuous loading
process. Removing mAb aggregates with a CEX resin using continuous loading is advantageous relative
to a bind/elute loading process, because the resin can use nearly its full capacity to bind the aggregates
®
enabling much higher loadings. The removal of mAb aggregates with Eshmuno CP-FT resin using a continuous loading process was found to be consistent with a frontal chromatography mechanism where the
mAb monomer initially binds to the column and is subsequently displaced by dimers and higher molec®
ular weight aggregates. The removal of mAb aggregates with Eshmuno CP-FT resin using a continuous
loading process was compared with six other commercially available strong CEX chromatography resins
and found to correlate with their ionic densities, but not their mAb static binding capacities. The influence
®
of pH, conductivity, residence time, and mAb concentration on the removal of aggregates with Eshmuno
CP-FT resin using a continuous loading process was also investigated. Finally, the percentage of aggre®
gates in a mAb feed was varied to examine the effect on the removal of aggregates with Eshmuno CP-FT
resin using a continuous loading process.
© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license ( />
1. Introduction
It is critical to remove aggregates during the downstream purification of monoclonal antibodies (mAbs) as they can increase the
risk of an immunogenic response in patients [1–4]. Unlike other
impurities, such as host cell proteins and DNA, mAb aggregates contain an Fc binding domain and are not typically separated from the
monomeric product during a protein A chromatography capture
step [5,6]. The chromatographic separation of mAb aggregates from
the monomer is particularly difficult, as they have nearly identical
isoelectric points and hydrophobicities. Aggregates are most commonly removed with bind/elute chromatography processes using
ion exchange, mixed-mode, hydrophobic interaction, or hydroxyapatite media [5,7,8]. However, there is interest in the development
of CEX chromatography processes that use continuous loading for
the removal of mAb aggregates rather than bind/elute loading
[9–12]. CEX chromatography using continuous loading allows the
resin to be loaded with the mAb feed until it is completely occupied by the aggregates, which is significantly higher than bind/elute

∗ Corresponding author.
E-mail address: (M.T. Stone).

processes where the resin must bind both the monomer and aggregates. For instance, if a mAb feed containing 10% aggregates can
be loaded up to 50 g/L with a bind/elute loading process using a
CEX resin, then it has the potential to be loaded up to 500 g/L by
a continuous loading process assuming the CEX resin has a similar capacity for both the mAb monomer and aggregates. Higher
loadings of the CEX resin are advantageous because they require
significantly smaller volumes of both resin and buffer shrinking the
footprint of the mAb downstream purification process. Continuous
loading processes using CEX media to purify mAb feeds have been
previously reported as overloaded chromatography [9,10,13]. We
suggest the more precisely defined term frontal chromatography as
has been described by Rachinskii [14], Jonsson [15], Hill et al. [16],
and Ahuja [17] to describe the mechanism of separation observed
in these processes.
Frontal chromatography is characterized by the continuous
loading of the column under conditions where all the components
of a mixture will bind with the resin [17]. This mechanism separates the components of a mixture into fronts based on their relative
strength of interaction with the resin [14,15]. The weakest interacting component will elute from the column first in a pure form. The
next front eluted from the column will be composed of the weakest
interacting component plus the next strongest interacting compo-

/>0021-9673/© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( />0/).


M.T. Stone et al. / J. Chromatogr. A 1599 (2019) 152–160

nent. Successive fronts will exit the column as mixtures including
all components of the previously eluted fronts. This process will

continue until the column has reached its full capacity for the most
strongly interacting component.
Frontal chromatography is not commonly used for preparative
scale purification of proteins, however Hill et al. demonstrated that
it could be applied for the preparative purification of the weakest interacting component within a mixture [16]. The removal of
mAb aggregates with a CEX resin is an opportunity for the preparative scale application of frontal chromatography because the mAb
monomer interacts less strongly with the CEX resin relative to the
aggregates. Thus, the monomer will elute from the CEX column
first as a single component and the column can continue to be
loaded with the mAb feed until it is completely occupied by aggregates. In practice, some monomer is likely to still be retained by
the column after the aggregates begin to elute. However, a good
monomer recovery can still be achieved by this method, because
at higher loadings the amount of monomer retained is only a small
percentage of the total monomer processed.
Herein, we report our investigation into a low ionic density CEX
®
resin, Eshmuno CP-FT resin, for the removal of mAb aggregates
using a continuous loading process. To understand if the removal
®
of aggregates with Eshmuno CP-FT resin using continuous loading was consistent with a frontal chromatography mechanism we
measured the composition of both the eluted and retained components as the loading of the resin was varied. The removal of
®
aggregates from a mAb feed with Eshmuno CP-FT resin using
a continuous loading process was then compared with six other
commercially available strong CEX chromatography resins and the
percentage of aggregates in the elution pool were compared to the
ionic density and mAb static binding capacity of the resins. In addition, the influence of several different process conditions including
pH, conductivity, residence time, and mAb feed concentration were
examined to understand how each of these factors influenced the
removal of aggregates with Eshmuno® CP-FT resin using a continuous loading process. Finally, we examined how the removal of mAb

®
aggregates with Eshmuno CP-FT resin using a continuous loading
process was influenced by the percentage of aggregates in the mAb
feed.

2. Experimental
2.1. Materials
2.1.1. Enrichment of mAb05 and mAb02 feeds with aggregates by
high pH hold
A high pH hold process was used to induce aggregates for both
the mAb05 and mAb02 feeds. The process was modified from the
procedure reported by Potty and Xenopoulos [18]. First, internally
generated mAb-containing Chinese hamster ovary cell cultures
were clarified and subjected to protein A capture chromatography.
The resulting protein A elution pool was then adjusted to a neutral
pH. The mAb concentration of the elution pool ranged from 10 g/L
to 20 g/L. The neutralized mAb solutions were gently stirred and
5 M sodium hydroxide was added dropwise until the solution pH
reached 11.0. Care was taken to avoid increasing the solution pH
above 11.0, which could cause significant degradation of the mAb
protein. The mAb solution was held at pH 11.0 for 30 min and then
1.0 M hydrochloric acid was added dropwise until the solution was
reduced to pH 5.0. The percentage of aggregates in the resulting
solution was determined by analytical size-exclusion chromatography. The pH cycling procedure was repeated up to four times until
the desired percentage of aggregates was obtained. The resulting
mAb solution was then dialyzed into the desired buffer. This process
was found to generate variable percentages of aggregates. The per-

153

centage of aggregates in an overly enriched mAb feed was lowered
to the desired level by combining with an untreated mAb feed.

2.1.2. Resins
The following CEX chromatography resins were used in this
®
investigation including Eshmuno CP-FT resin (500 mL, catalog
number: 1200930500, EMD Millipore Corporation, Burlington,
®
MA, 01803, USA), Eshmuno CPX resin (500 mL, catalog number:
1200830500, EMD Millipore Corporation, Burlington, MA, 01803,
®
USA), Poros XS Strong Cation Exchange Resin (50 mL, catalog number: 4404338, Thermo Fisher Scientific Inc., Waltham, MA, 02451,
®
USA), Poros 50 HS Strong Cation Exchange Resin (50 mL, catalog number: 1335906, Thermo Fisher Scientific Inc., Waltham, MA,
®
02451, USA), Toyopearl Gigacap S-650 M (100 mL, part number:
0021833, Tosoh Corporation, Minato-Ku, Tokyo, 105–8623, Japan),
®
Capto S ImpAct (100 mL, product number: 17371702, GE Healthcare Bio-Sciences AB, Uppsala, Sweden), and SP SepharoseTM Fast
Flow (300 mL, product number: 17072901, GE, Healthcare BioSciences AB, Uppsala, Sweden).

2.2. Methods
2.2.1. Standard procedure for removal of aggregates using frontal
chromatography
For all experiments, a glass chromatography column (Omnifit
Benchmark Column 6.6 mm/100 mm, 6.6 mm diameter, 100 mm
length, SKU: 006BCC-06-10-AF, Diba Industries, Danbury, CT
06810, US) was packed to a height of 3 cm with 1.0 mL of the CEX
chromatography resin. Continuous loading chromatography experiments were performed using a GE Healthcare Life Sciences ÄKTA

avant 25. Before each experiment, the columns were equilibrated
with the same buffer as that of the mAb feed solution for 10 CV.
Before a column was reused for additional experiments it was first
washed with the loading buffer for 10 CV, stripped with the loading
buffer also containing 1.0 M sodium chloride for 15 CV, cleaned with
0.5 M sodium hydroxide for 5 CV, and equilibrated with loading
buffer for 15 CV.

2.2.2. Analytical size exclusion chromatography
Analytical size-exclusion chromatography of proteins was performed using a Waters 2695 Separation Module, a Waters Dual ␭
Absorbance Detector, and a Tosoh Biosciences TSKgel G3000SWxl
column (part number: 08541, column size: 300 × 7.8 mm, Tosoh
Bioscience LLC, King of Prussia, PA, USA). The isocratic mobile phase
was a solution of 50 mM sodium phosphate and 150 mM sodium
chloride at pH 7.0. The column was run at a flow rate of 1.00 mL/min
for 20 min and the UV detector was set to a wavelength of 280 nm.
The percentage of aggregates was calculated based on the areas of
the HPLC peaks.

2.2.3. UV spectroscopic analysis of protein concentration
UV spectroscopic analysis of protein solution concentration
was performed with a Thermo Scientific GENESYS 10S UV–vis
Spectrophotometer. The concentration of the mAb05 and mAb02
fractions was determined by measuring their absorbance at
280 nm in a disposable plastic cell having a 1.0 cm path length.
The absorbance was divided by the extinction coefficient of the
mAbs (mAb05 = 1.419 mL·g−1 ·cm−1 , mAb02 = 1.467 mL·g−1 ·cm−1 )
and the pathlength to calculate the protein concentration. The
monomer recovery was calculated based on the concentration of
the mAb in a fraction as determined by UV spectroscopic analysis and the percentage of monomer in a fraction as determined by

analytical size-exclusion chromatography.


154

M.T. Stone et al. / J. Chromatogr. A 1599 (2019) 152–160

®

Fig. 1. SEC chromatograms of individual fractions of a mAb05 feed processed through a 1.0 mL packed column of Eshmuno CP-FT resin at a residence time of 3 min at various
loadings (left). The mAb05 feed for this experiment examining the composition of proteins in the elution fractions had a concentration of 15 g/L with 10% total aggregates
(5% dimers) and was dialyzed into a buffer composed of 100 mM sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm. SEC chromatograms of high salt elutions from a
®
1.0 mL packed column of Eshmuno CP-FT resin loaded with various amounts of the mAb05 feed at a residence time of 3 min (right). The mAb05 feed used for this experiment
examining the composition of proteins that were retained by the column had a concentration of 16 g/L with 11% total aggregates (5% dimers) and was dialyzed into a buffer
composed of 100 mM sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm.

2.2.4. Determination of CEX resin ionic density
A 1 mL portion of gravity settled CEX resin in 20% ethanol was
measured in a small plastic column. To remove the ethanol from the
CEX resin it was three times suspended in 5 mL of water and then
the water was removed by suction from the column. The sulfonate
group on the CEX resin was converted to a sulfonic acid group by
three times suspending the resin in 5 mL of 1.0 M hydrochloric acid
for 5 min and then removing the hydrochloric acid by suction. To
remove the remaining hydrochloric acid the resins were suspended
in 5 mL of water that was then removed by suction and this process
was repeated until the pH of the water removed was neutral. The
CEX resin was then transferred into a 200 mL glass beaker. To the
glass beaker was also added 80 mL of 1.0 M sodium chloride and

a 1.0 mL solution of phenolphthalein at a concentration of 1% by
weight in ethanol. The solution was titrated with a 0.01 M solution
of sodium hydroxide and the end of the titration was indicated
when the solution changed to a pink color. The ionic density of the
CEX resin was calculated by the following formula:
ionic denisity =

VNaOH × CNaOH
Vresin

In this equation VNaOH is the volume of sodium hydroxide
titrated into the suspension of CEX resin, CNaOH is the concentration
of sodium hydroxide, and Vresin is the volume of the resin that was
titrated. The ionic density of the CEX resins is the average of two
separate measurements.

to centrifuge and a portion of the supernatant mAb05 solution was
diluted 20-fold. The UV absorbance of the 20-fold diluted solution
at 280 nm was determined as described in Section 2.2.3. The process
of preparing a 20-fold dilution and measuring the UV absorbance
of the solution at 280 nm was performed in triplicate for each tube
and the average of the three measurements was used to calculate
the mAb05 concentration. The static binding capacity of the CEX
resin for mAb05 was then determined by the following formula:
static binding capacity
=

(V control × Ccontrol ) − (V resin treated × Cresin treated )
Vresin

In this equation Vcontrol and Vresin treated are the total volume of
the mAb05 control solutions and the total volume of the mAb05
solutions treated with the resin, respectively. The Ccontrol and the
Cresin treated are the concentration of mAb05 in the control tubes
after they were rotated for 4 h and the concentration of mAb05 in
the tubes treated with the resin after they were rotated for 4 h,
respectively. Vresin is the volume of the resin added to the resin
treated solutions. The static binding capacity of the CEX resins for
mAb05 is the average of two separate preparations of the resin
slurry that were each measured in triplicate.
3. Results
3.1. Mechanism for the removal of aggregates

2.2.5. Determination of CEX resin static binding capacity for
mAb05
A 1 mL portion of gravity settled CEX resin in 20% ethanol was
measured in a small plastic column. To remove the ethanol from
the CEX resin it was three times suspended in an acetate buffer
composed of 100 mM sodium acetate at pH 5.0 and 5.0 mS/cm and
then the acetate buffer was removed by suction. The CEX resin was
added to a 50 mL centrifuge tube. To the centrifuge tube was also
added 9 mL of the acetate buffer to give a 10% resin slurry. A 1.0 mL
portion of the 10% resin slurry was added to a 15 mL centrifuge tube.
To the 15 mL tube was also added 1.5 mL of the acetate buffer and
2.5 mL of a mAB05 solution at a concentration of 10 g/L with 0.4%
aggregates that was dialyzed into the acetate buffer. The resulting
slurry contained 0.1 mL of CEX resin and mAb05 at a concentration
of 5 g/L. Controls omitting the resin were prepared by adding 2.5 mL
of the acetate buffer and 2.5 mL of the dialyzed mAB05 solution to
15 mL tubes. The 15 mL tubes containing CEX resin and the control

tubes were rotated for 4 h. The 15 mL tubes were then subjected

®

We investigated the removal of aggregates with Eshmuno CPFT resin using a continuous loading process to determine if the
separation of the mAb monomer from the aggregates was consistent with a frontal chromatography mechanism. A mAb05 feed
containing 10% aggregates was dialyzed into a 100 mM sodium
acetate buffer at pH 5.0 and 5 mS/cm then loaded onto a column of
®
the Eshmuno CP-FT resin. The composition of the fractions eluted
from the column were analyzed by size exclusion chromatography
(Fig. 1, left). Initially, only the monomer was observed to elute from
the column. At a loading of 600 g/L, dimers were also detected in
the elution. However, no higher molecular weight aggregates were
observed up to a loading of 1000 g/L where the experiment was
ended. These results are consistent with a frontal chromatography
mechanism in which pure monomer eluted in the first front and
then the dimers coeluted with the monomer in a second front. The
®
Eshmuno CP-FT resin was not loaded with a sufficient amount
of the mAb05 feed to observe a third front that would be com-


M.T. Stone et al. / J. Chromatogr. A 1599 (2019) 152–160

155

Fig. 2. The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (left) and the cumulative percentage of aggregates in the elution pool as a
function of the cumulative mAb05 monomer recovery in the elution pool (right). The mAb05 feed was processed through a 1.0 mL packed column of a CEX chromatography
resin at a residence time of 3 min. The mAb05 feed had a concentration of 15 g/L with 11% total aggregates (5% dimers) and was dialyzed into a buffer composed of 100 mM

®
®
®
®
sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm. CEX media legend: C1 - Eshmuno CPX resin, C2 – Poros XS, C3 – Poros 50 HS, C4 - Toyopearl Gigacap S-650 M,
®
C5 – Capto S ImpAct, C6 – SP SepharoseTM Fast Flow. The percentages included on the plots in black font indicate the cumulative monomer recovery at the last fraction
before the cumulative percentage of aggregates exceeded 1%.

posed of the higher molecular weight aggregates along the with
the monomer and dimers.
Next, we examined the components that were retained by the
®
Eshmuno CP-FT resin after it was loaded with various amounts
of a mAb05 feed containing 11% aggregates that was dialyzed into
a 100 mM sodium acetate buffer at pH 5.0 and 5 mS/cm. After a
specific loading of the column was completed the components of
the mAb05 feed retained by the column were eluted in a subsequent step using a high salt buffer. We found that as the loading of
the mAb05 feed was increased, the amount of monomer retained
by the column decreased and the amount of aggregates increased
(Fig. 1, right). For example, at a loading of 200 g/L, the composition of mAb05 feed that was retained by the column consisted of
71% monomer while at a loading of 1000 g/L, the percentage of
monomer retained by the column decreased to 9%. The composition of the aggregates that were retained by the column included
dimers and higher molecular weight aggregates. These results are
consistent with a frontal chromatography mechanism, in which the
mAb monomer is initially retained by the column and subsequently
displaced by aggregates.

Table 1
Ionic density and static binding capacity for mAb05 determined for the CEX resins.

Note that the percentage of aggregates in the elution pool corresponds to a resin
loading of approximately 1000 g/L. However, there were variations in the final loading of the CEX resins which ranged from 967 g/L to 1000 g/L.

3.2. Removal of mAb aggregates by continuous loading of various
CEX chromatography resins

tion may indicate an influence of the base bead because Capto
S ImpAct and SP SepharoseTM Fast Flow have agarose resin base
beads while the other CEX resins are composed of polymeric base
beads.
Next, we measured the ionic density and the mAb05 static binding capacities of these seven strong CEX resins to determine if either
of these attributes correlated with the efficient removal of aggregates using a continuous loading process (Table 1). The ionic density
of a CEX resin was determined by converting the sulfonate groups
to sulfonic acids and then titrating the acidified resin with sodium
hydroxide in the presence of a phenolphthalein indicator. The static
binding capacity of the CEX resins was measured using a mAb05
feed dialyzed into 100 mM sodium acetate at pH 5.0 and 5 mS/cm.
We plotted the cumulative percentage of aggregates in the elution
pool at a loading of approximately 1000 g/L as a function of both
variables (Fig. 3).
We found that there was a rough correlation between the ionic
density and the percentage of aggregates in their elution pool. For
®
instance, Eshmuno CP-FT resin had the lowest ionic density at 37
␮eq/mL and had the lowest percentage of aggregates in the cumu®
®
lative elution pool at 1.9%. Eshmuno CPX resin, Poros XS, and
®
Poros 50 HS had intermediate ionic densities of 70–81 ␮eq/mL
and very similar percentages of aggregates in the elution pool of

®
7.5–7.8%. Toyopearl Gigacap S-650 M and SP SepharoseTM Fast
Flow had the highest ionic densities of 188–210 ␮eq/mL and the
highest percentages of aggregates in the elution pool of 8.8–9.7%.

®

The ability of Eshmuno CP-FT resin to remove aggregates using
a continuous loading process was compared with six commerciallyavailable strong CEX chromatography resins having sulfonate
ligands. We selected a solution pH of 5.0 and a conductivity of
5 mS/cm because a sulfonate CEX resin will typically have a high
capacity for the mAb monomer and aggregates under these solution conditions. A mAb05 feed containing 11% aggregates was
dialyzed into 100 mM sodium acetate at pH 5.0 and 5 mS/cm, loaded
onto the CEX column, and the compositions of the elution fractions were determined. Under these solution conditions five of
®
®
the CEX resins including Eshmuno CP-FT resin, Eshmuno CPX
®
®
®
resin, Poros XS, Poros 50 HS, and Toyopearl Gigacap S-650 M
showed a gradual breakthrough of the aggregates as is consistent
®
with a frontal chromatography mechanism (Fig. 2). Eshmuno CPFT resin is an outlier of these five as it removed significantly more
aggregates with a better monomer recovery. The elution pool for
®
Eshmuno CP-FT resin had 0.8% aggregates with a 92% monomer
recovery at a loading of 741 g/L. By contrast, the elution pool for
the other CEX chromatography resins all exceeded 1% aggregates
®

before their monomer recoveries reached 50%. Capto S ImpAct
TM
Fast Flow did not show a gradual increase in
and SP Sepharose
the cumulative percentage of aggregates in the elution pool as is
expected for a frontal chromatography mechanism. This observa-

CEX resin

®

Eshmuno
CP-FT resin
®
Capto S
ImpAct
®
Eshmuno CPX
resin
®
Poros 50 HS
®
Poros XS
®
Toyopearl
Gigacap
S-650M
SP SepharoseTM
Fast Flow

ionic density
(␮eq/mL)

static binding
capacity for
mAb05 (g/L)

aggregates in
elution at
loading of
˜
1000
g/L

37

69

1.9%

64

89

8.9%

70

71

7.5%

78
81
188

50
68
100

7.8%
7.8%
9.7%

210

66

8.9%

®


156

M.T. Stone et al. / J. Chromatogr. A 1599 (2019) 152–160

Fig. 3. The cumulative percentage of aggregates in the elution pool at a loading of approximately 1000 g/L as a function of the ionic density of the CEX resin (left) and the
cumulative percentage of aggregates in the elution pool at a loading of approximately 1000 g/L as a function of the static binding capacity of the CEX resin for mAb05 (right).
The CEX static binding capacity was measured with a mAb05 feed at a concentration of 5 g/L with 0.4% aggregates that was dialyzed into a 100 mM sodium acetate buffer at

pH 5.0 and a conductivity of 5.0 mS/cm.

Fig. 4. The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (top left) or mAb02 loading (bottom left) and the cumulative percentage
of aggregates in the elution pool as a function of the cumulative mAb05 monomer recovery in the elution pool (top right) or the cumulative mAb02 monomer recovery in
the elution pool (bottom right). The feeds were processed through a 1.0 mL packed column of Eshmuno® CP-FT resin at a residence time of 3 min. The mAb05 feed had a
concentration of 12 g/L with 10% total aggregates (6% dimers) and was dialyzed into a buffer composed of 50 mM sodium acetate at pH 4.5, pH 5.0, pH 5.5, or pH 6.0. The
mAb02 feed had a concentration of 13 g/L with 6% total aggregates (4% dimers) and was dialyzed into 50 mM sodium acetate at pH 4.0, pH 4.5, or pH 5.0. The conductivities
of the acetate buffers for the mAb05 and mAb02 feeds were adjusted to 5.0 mS/cm and 2.5 mS/cm respectively by the addition of sodium chloride. The percentages included
on the plots in black font indicate the cumulative monomer recovery at the last fraction before the cumulative percentage of aggregates exceeded 1%.

Capto® S ImpAct does not follow this trend as it has an intermediate ionic density of 64 ␮eq/mL, but still has a highest percentages
of aggregates in the elution pool of 8.9–9.7%. However, it might not
®
be appropriate to compare the dependence of Capto S ImpAct and
SP SepharoseTM Fast Flow if they are not operating according to
a frontal chromatography mechanism as is suggested by shape of
their curves in Fig. 2.
We did not find a correlation between the mAb05 static binding capacity of the CEX resins and the percentage of aggregates in
®
their elution pool. For instance, SP SepharoseTM Fast Flow, Poros
®
®
XS, Eshmuno CP-FT resin, and Eshmuno CPX resin all had very
similar mAb05 static binding capacities of 66 g/L, 68 g/L, 69 g/L,
and 71 g/L respectively, but varied significantly in the percent®
age of aggregates in their elutions. Neither Poros 50 HS that
®
had the lowest static binding capacity of 50 g/L or Toyopearl
Gigacap S-650 M that had the highest static binding capacity of

100 g/L showed the lowest percentage of aggregates in their elution
pools.
3.3. Influence of solution pH
The influence of solution pH on removal of aggregates with
®
Eshmuno CP-FT resin using a continuous loading process was
investigated with both a mAb05 feed and a mAb02 feed. A mAb05
feed containing 10% aggregates and a mAb02 feed containing 6%
aggregates were dialyzed into acetate buffers that varied in pH. We
observed for both the mAb05 and mAb02 feeds that as the solution pH was lowered more aggregates were removed with a higher
monomer recovery (Fig. 4). Based on their isoelectric points, both
mAb05 (pI = 8.1) and mAb02 (pI = 8.24) are more strongly charged
at a lower solution pH. Thus the removal of aggregates was most
efficient at a lower solution pH where the electrostatic interactions


M.T. Stone et al. / J. Chromatogr. A 1599 (2019) 152–160

157

Fig. 5. The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (top left) or mAb02 loading (bottom left) and the cumulative percentage
of aggregates in the elution pool as a function of the cumulative mAb05 monomer recovery in the elution pool (top right) or the cumulative mAb02 monomer recovery in
®
the elution pool (bottom right). The feeds were processed through a 1.0 mL packed column of Eshmuno CP-FT resin at a residence time of 3 min. The mAb05 feed had a
concentration of 12 g/L with 11% total aggregates (6% dimers) and was dialyzed into a buffer composed of 50 mM, 100 mM, 150 mM, or 200 mM sodium acetate at pH 5.0
having a conductivity of 2.8 mS/cm, 4.7 mS/cm, 6.2 mS/cm, or 8.2 mS/cm respectively. The mAb02 feeds had a concentration of 13 g/L with 6% total aggregates (4% dimers) and
was dialyzed into a 50 mM sodium acetate buffer at pH 4.0 having a conductivity of 2.5 mS/cm, 5.0 mS/cm, 7.0 mS/cm, or 9.0 mS/cm. The conductivity of the buffers used for
dialysis of the mAb02 feed were adjusted by the addition of sodium chloride. The percentages included on the plots in black font indicate the cumulative monomer recovery
at the last fraction before the cumulative percentage of aggregates exceeded 1%.

between the positively charged mAb monomer/aggregates and the
negatively charged resin are strongest.
3.4. Influence of solution conductivity
The influence of solution conductivity on the removal of aggre®
gates with Eshmuno CP-FT resin using a continuous loading
process was investigated with both a mAb05 feed and a mAb02
feed. A mAb05 feed containing 10% aggregates and a mAb02 feed
containing 6% aggregates were dialyzed into acetate buffers that
varied in conductivity. We found that as the solution conductivity was decreased, more aggregates were removed from both the
mAb05 feed and the mAb02 feed with a higher monomer recovery (Fig. 5). However, there was a significant departure from this
trend for the mAb05 feed at 2.8 mS/cm in which less aggregates
were removed with a lower monomer recovery than was observed
for the three other mAb05 feeds having higher solution conductivities. One potential explanation for this outlier could be that the
mAb05 monomer is too strongly bound to the resin at this low
solution conductivity inhibiting displacement by the aggregates as
is required for an efficient separation with a frontal chromatography mechanism. No such exception was observed for the removal of
aggregates from the mAb02 feeds where the most efficient solution
condition was at the lowest conductivity of 2.5 mS/cm.
3.5. Influence of residence time
The influence of residence time on the removal of aggregates
®
with Eshmuno CP-FT resin using a continuous loading process
was investigated using a mAb05 feed containing 11% aggregates.
We observed that as the residence time was increased more aggregates were removed from the mAb05 feed with a higher monomer

recovery (Fig. 6). Longer residence times are to likely result in the
more efficient removal of aggregates with a frontal chromatography mechanism because mass transfer of the aggregates into the
resin limits displacement of the bound monomers. However, longer
residence times are not desirable as they will require longer loading times. For instance, increasing the residence time from 3 min
to 6 min increased loading time for the mAb05 feed from 3.3 h to

6.7 h.
3.6. Influence of mAb feed concentration
The influence of the mAb feed concentration on the removal of
®
aggregates with Eshmuno CP-FT resin using a continuous loading
process was investigated with a mAb05 feed containing 10% aggregates. We observed that as the concentration of the mAb05 feed was
decreased more aggregates were removed with a higher monomer
recovery (Fig. 7). However, lowering the concentration of the mAb
feed is not desirable, because longer loading times are required to
process the larger volumes of the mAb feed. For instance, decreasing
the concentration of the mAb05 feed from 15 g/L to 5 g/L increased
the loading time from 3.3 h to 10 h.
3.7. Influence of the percentage of aggregates in the mAb feed
To examine the influence of the percentage of aggregates in the
mAb feed on the removal of aggregates with Eshmuno® CP-FT resin
using a continuous loading process, six mAb05 feeds were prepared with varying percentages of aggregates ranging from 1.9%
to 14.6%. We observed for all six mAb05 feeds that a specific loading of the feed could be selected where the level of aggregates in
the elution pool was reduced below 1% with a monomer recovery
greater than 85% (Fig. 8, top left and right). We also observed that


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M.T. Stone et al. / J. Chromatogr. A 1599 (2019) 152–160

Fig. 6. The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (left) and the cumulative percentage of aggregates in the elution pool
as a function of the cumulative mAb05 monomer recovery in the elution pool (right). The feed was processed through a 1.0 mL packed column of Eshmuno® CP-FT resin at
a residence time of 1 min (180 cm/h), 2 min (90 cm/h), 3 min (60 cm/h), or 6 min (30 cm/h). The mAb05 feed had a concentration of 15 g/L with 11% total aggregates (5%
dimers) and was dialyzed into a buffer composed of 100 mM sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm. The percentages included on the plots in black font
indicate the cumulative monomer recovery at the last fraction before the cumulative percentage of aggregates exceeded 1%.

Fig. 7. The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (left) and the cumulative percentage of aggregates in the elution pool as a
®
function of the cumulative mAb05 monomer recovery in the elution pool (right). The mAb05 feed was processed through a 1.0 mL packed column of Eshmuno CP-FT resin
at a residence time of 3 min. The mAb05 feed had an initial concentration of 15 g/L with 10% total aggregates (6% dimers) and was dialyzed into a buffer composed of 100 mM
sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm. Portions of the mAb05 feed were diluted to 5 g/L and 10 g/L with the dialysis buffer. The percentages included on
the plots in black font indicate the cumulative monomer recovery at the last fraction before the cumulative percentage of aggregates exceeded 1%.

Fig. 8. The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (top left) and the cumulative percentage of aggregates in the elution
pool as a function of the cumulative mAb05 monomer recovery in the elution pool (top right). The cumulative percentage of aggregates in the elution pool as a function of
®
the loading of mAb05 aggregates (bottom). The mAb05 feeds were processed through a 1.0 mL packed column of Eshmuno CP-FT resin at a residence time of 3 min. The
mAb05 feeds had a concentration of 15 g/L with 1.9%, 3.7%, 7.3%, 10.4% 12.2%, or 14.6% total aggregates and were dialyzed into a 100 mM sodium acetate buffer at pH 5.0
and a conductivity of 5.0 mS/cm. The percentages included on the plots in black font indicate the cumulative monomer recovery at the last fraction before the cumulative
percentage of aggregates exceeded 1%.


M.T. Stone et al. / J. Chromatogr. A 1599 (2019) 152–160

159

Table 2
The effective loading range for the removal of aggregates from mAb05 feeds with varying percentages of aggregates.
percentage of aggregates in mAb05
feed

minimum loading at which the
monomer recovery in the elution
pool was ≥85% (g/L)

maximum loading at which the
aggregates in the elution pool was
≤1% (g/L)

observed effective
loading range (g/L)

1.9%
3.7%
7.3%
10.4%
12.2%
14.6%

700
681
596
583
511
446

>1000
>1000
>1000
827
660
535

>300
>319

>404
245
149
89

as the percentage of aggregates in the mAb05 feed was increased,
the aggregates eluted from the column at lower loadings. The percentages of the aggregates in the elution pool were also plotted as a
function of the loading of aggregates (Fig. 8, bottom). The resulting
plot shows that all six feeds had a similar shape that is consistent
with a frontal chromatography mechanism where the aggregates
should not begin to elute until they have exceeded the capacity of
®
the Eshmuno CP-FT resin.
We also noted that as the percentage of aggregates in the mAb05
feed was increased, the effective loading range for the removal of
aggregates became narrower (Table 2). For this experiment, we
defined the effective loading range as starting when the cumulative monomer recovery in the elution pool exceeded 85% and
ending when the cumulative percentage of aggregates exceeded
1%. The effective loading range for the removal of aggregates was
found to increase as the percentage of aggregates in the feed was
decreased. The effective loading ranges for the 1.9%, 3.7%, and 7.3%
feeds were not fully determined, as the percentage of aggregates
in the elution pool remained below 1% at a loading of 1000 g/L.
The effective loading range for the 1.9% and 3.7% feeds are likely
to extend significantly beyond 1000 g/L as the percentage of aggregates in the elution pool was only 0.2% and 0.3% respectively when
the experiment ended.
4. Discussion
®

First, we sought to confirm that Eshmuno CP-FT resin was

removing aggregates from a mAb05 feed according to a frontal
chromatography mechanism when using a continuous loading process. The composition of the mAb05 feed that eluted from the
®
Eshmuno CP-FT resin as well as the composition of the mAb05
feed that was retained by the column was determined as the loading
was varied (Fig. 1). We observed that the mAb05 monomer eluted
from the column in the earliest fractions and the dimers did not
begin to elute until 600 g/L. At lower loadings the composition of
mAb05 feed that was retained by the column consisted primarily of
monomer, but as the loading was increased the retained monomer
was displaced by dimers and higher molecular weight aggregates
[14,16]. We also noted that dimers were the only types of aggregates observed in the elution fractions while higher molecular
weight aggregates were completely retained by the column. This
suggests that the higher molecular weight aggregates are forming
a third front that has yet to elute from the column at the end of
the experiment. The results of both experiments indicate the sep®
aration of the mAb monomer from the aggregates with Eshmuno
CP-FT resin using a continuous loading process is consistent with a
frontal chromatography mechanism.
®
Next, we compared the removal of aggregates with Eshmuno
CP-FT resin using a continuous loading process from a mAb05
feed with six commercially available strong CEX chromatography
resins having sulfonate ligands (Fig. 2). We chose to compare the
strong CEX resins at a solution pH of 5.0 and a conductivity of
5 mS/cm where they should have a high capacity for the mAb
aggregates. If a CEX resin efficiently removes aggregates from a

mAb feed using a continuous loading process under these solution conditions, then very high loadings of the resin should be
®

®
®
possible. Eshmuno CP-FT resin, Eshmuno CPX resin, Poros XS,
®
®
Poros 50 HS, and Toyopearl Gigacap S-650 M showed a gradual
increase in the percentage of the aggregates in the elution pool as
®
is expected with a frontal chromatography mechanism. Eshmuno
CP-FT resin removed significantly more aggregates with a higher
monomer recovery than the other CEX chromatography resins.
®
Capto S ImpAct and SP SepharoseTM Fast Flow showed an immediate breakthrough of aggregates in the elution pool indicating that
they are not removing aggregates according to a frontal chromatography mechanism under these solution conditions. These two CEX
resins are both composed of an agarose base bead and we speculate that this factor may be responsible for inhibiting a frontal
chromatography mechanism under these solution conditions as all
the other CEX resins are composed of polymer base beads. The percentage of aggregates in the elution pool for all seven CEX resins
was plotted as a function of their ionic density and their mAb05
static binding capacity (Table 1, Fig. 3). A low percentage of aggregates in the elution pool was found to correlate with a low ionic
density while no correlation was observed with the static binding
capacities of the CEX resins for mAb05. A lower ionic density CEX
resin may facilitate efficient removal of mAb aggregates by frontal
chromatography because it likely has fewer electrostatic interactions with the monomer allowing it to be more easily displaced by
the aggregates.
It is important to note that we compared the removal of aggregates with CEX resins using a continuous loading process at a single
solution condition where CEX resins typically have high capacities
for the mAb monomer and aggregates. CEX resins with higher ionic
densities may remove aggregates more efficiently at a higher solution pH and/or conductivity where the strength of the electrostatic
interaction between positively charged mAb monomer/aggregates
and the negatively charged resin will be weaker. However, operating a CEX resin at a higher pH/conductivity will reduce its capacity

for mAb aggregates and thus limit the amount of the mAb monomer
that can be purified by continuous loading before elution of aggregates will occur.
Then we examined the influence of solution pH and conductivity
®
on the removal of aggregates with Eshmuno CP-FT resin using a
continuous loading process from both a mAb05 feed and a mAb02
feed. We found that more aggregates were removed with higher
monomer recoveries at lower solution pHs (Fig. 4) and conductivities (Fig. 5), which favor strong electrostatic interactions between
the positively charged mAb monomer/aggregates and the negatively charged resin. One exception to this trend was observed with
®
mAb05 at 2.8 mS/cm, where Eshmuno CP-FT resin was significantly less efficient for the removal of aggregates than the other
higher conductivities mAb05 feeds investigated. One explanation
is that at a solution conductivity of 2.8 mS/cm the mAb05 monomer
®
is too strongly bound to the Eshmuno CP-FT resin thus preventing
displacement by aggregates and inhibiting separation by a frontal
chromatography mechanism [12]. Liu, et al. also observed that the
®
removal of aggregates from a mAb feed using Poros 50 HS CEX


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M.T. Stone et al. / J. Chromatogr. A 1599 (2019) 152–160

resin with a continuous loading process became increasingly efficient as the solution conductivity was decreased from 18 mS/cm to
5 mS/cm, however at 3 mS/cm the removal of aggregates was significantly worse [10]. However, no such exception was observed for
the mAb02 feed where the removal of aggregates was most efficient
at the lowest conductivity of 2.5 mS/cm.
We also investigated the influence of the flow-rate and the mAb

feed concentration on the removal of aggregates from a mAb05 feed
®
with Eshmuno CP-FT resin using a continuous loading process. The
removal of aggregates was found to be most efficient at longer residence times (Fig. 6) and lower mAb feed concentrations (Fig. 7).
®
Liu, et al. also reported that the removal of aggregates with Poros
50 HS resin using a continuous loading process was most efficient
at longer residence times [10]. Longer residents times are likely
to be advantageous for the removal of aggregates using a frontal
chromatography mechanism because they give more time for mass
transfer of the aggregates into the resin. However, using longer residence times and lower mAb concentrations will also increase the
resin loading time and thus could decrease the productivity of the
resin to an unreasonably low level.
Finally, we investigated how the percentage of aggregates in
the mAb feed influenced the removal of mAb aggregates with
®
Eshmuno CP-FT resin using a continuous loading process. We
tested six different mAb05 feeds with levels of aggregates that
varied from 1.9% to 14.6%. We found that as the percentage of
aggregates in the mAb05 feed was increased, the aggregates began
eluting from the resin at lower loadings. The level of aggregates in
all six mAb05 feeds could be reduced to less than 1% with monomer
recoveries greater than 85% at a particular loading (Fig. 8). However,
purifying mAb05 feeds containing higher percentages of aggregates
using a continuous loading process is more challenging, because the
effective operating range was found to decrease as the percentage
of aggregates in the feed was increased (Table 2). A mAb05 feed
with a higher percentage of aggregates must be processed at lower
loadings and over narrower ranges to remove a sufficient amount
of aggregates with a good monomer recovery. While a mAb05 feed

a with lower percentage of aggregates can be processed at higher
loadings with a significantly wider effective loading range.
5. Conclusions
®

A low ionic density CEX chromatography resin, Eshmuno CPFT resin, was investigated for the removal of aggregates from
mAb feeds using a continuous loading process. The removal of
®
mAb aggregates with Eshmuno CP-FT resin using a continuous
loading process was found to be consistent with a frontal chromatography mechanism, whereby the mAb monomers are initially
retained by the column and are subsequently displaced by aggre®
gates. Eshmuno CP-FT resin was found to be significantly more
effective for the removal of mAb aggregates using a continuous
loading process compared to six commercially available strong CEX
chromatography resins under solution conditions where CEX resins
typically have high capacities for mAb monomer and aggregates.
We found that the efficient removal of aggregates using a continuous loading process correlated with CEX resins having lower ionic
densities while no correlation was observed with their mAb static
binding capacities. Optimization studies found that the removal of
®
aggregates with Eshmuno CP-FT resin using a continuous loading process was more efficient at lower solution pHs and lower
solution conductivities, which favor strong electrostatic interactions between the positively charged mAb monomer/aggregates
®
and the negatively charged Eshmuno CP-FT resin. An important
exception to this trend in the solution conditions was observed at

the lowest conductivity for the mAb05 feed. Optimization studies
®
also found that Eshmuno CP-FT resin removes more aggregates
with higher monomer recoveries at longer residence times and

®
lower mAb feed concentrations. Eshmuno CP-FT resin efficiently
removed aggregates from mAb feeds containing between 1.9% and
14.6% aggregates using a continuous loading process, however the
mAb feeds with lower percentages of aggregates had much wider
effective loading ranges.
Conflict of interest disclosure
The authors are employees of EMD Millipore Corporation which
®
sells Eshmuno CP-FT resin.
Acknowledgements
The authors thank James Hamzik, Lars Peeck, Dominic Zorn,
Romas Skudas, Paul Turiano, Lloyd Gottlieb, Michael Schulte, David
Beattie, and Matthias Jöhnck for their support and encouragement.
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