Mixed-Mode Chromatography for mAb S Aggregate Removal:
Comparison of CHT ™ Ceramic Hydroxyapatite, Capto adhere,
and Capto adhere ImpRes
Xuemei He
Bio-Rad Laboratories, Inc., 6000 James Watson Drive, Hercules, CA 94547

Process Separations

Bulletin 6749

Abstract

In the purification of monoclonal antibodies, the separation of monomers from product-related
impurities is a major challenge. Here, we highlight the importance of screening various media
to determine the optimal one for an efficient purification process and illustrate that CHT
Ceramic Hydroxyapatite is an effective mixed-mode chromatography media.
Introduction
Monoclonal antibodies (mAbs) remain a predominant classof therapeutic protein products on the market because of theirwide range of applications in disease treatment and diagnosis.
Over the years, upstream technology advancements havehelped improve the titer of target antibodies, from merely
1 g/L two decades ago to 10–13 g/L in fed-batch processes
and up to 25 g/L in perfusion cultures today (Gronemeyer et
al. 2014). However, these advancements have often adversely
affected impurity composition and concentration upon harvest.
This has had a significant impact on downstream processing.
Elevated levels of antibody aggregates are often associated withthe overproduction of mAbs due to the mispairing of disulfide
bonds and the unfolding or denaturation of drug molecules atcell growth temperatures (25°C or above) (Rathore et al. 2013).
The strong acidic elution conditions needed for Protein A affinity
chromatography, commonly used for the capture of mAbs
from clarified tissue culture fluid, can trigger structural changes
and promote the oligomerization of pH-sensitive molecules.
Additional column chromatography steps are necessary for theclearance of such product-related impurities. The separation ofmAb monomers and aggregates has been a major challenge
in downstream manufacturing as these molecules exhibit
very similar physical and chemical properties and sometimes
identical chromatographic behavior. Baseline separation ofthese mAb species is often difficult to achieve, and extensive
process development is required to balance the purity and yieldof the monomeric mAbs.

The molecule of interest in this present study, mAb S, washarvested from Chinese hamster ovary (CHO) cell culture.
Typically, a Protein A affinity chromatography eluate contains
a mixture of mAb S monomers (~75%) and aggregates(dimers and other oligomers, ~25%). We separated themAb S monomers from the aggregates using three mixedmode chromatography media, CHT, Capto adhere, andCapto adhere ImpRes, which have all been extensively
researched for their applications in mAb aggregate clearance(Gagnon 2009). We compared the purity and recovery ofmonomeric mAb S purified with these chromatography media.
Our results underscore the importance of screening multiple
media to develop an efficient downstream production processwith the highest possible target purity and recovery.
Materials and Methods
Chromatography Media and Columns

Bio-Scale™ Mini Cartridges (0.56 x 4 cm) were packed
with CHT Ceramic Hydroxyapatite Type I Media, 40 µm(Bio-Rad Laboratories), Capto adhere Media (GE Healthcare
Life Sciences), or Capto adhere ImpRes Media (GE Healthcare
Life Sciences) to give a final bed volume of 1 ml. The monomer
content in eluate fractions or pools was determined using
an ENrich™ SEC 650 Column (Bio-Rad Laboratories) with
1x phosphate buffered saline (PBS) as the mobile phase.

Mixed-Mode Chromatography for mAb S Aggregate Removal: Comparison of CHT™ Ceramic Hydroxyapatite, Capto adhere, and Capto adhere ImpRes

Chromatography SystemA

mAb S eluted from the mixed-mode columns was quantified
using the Peak Integration feature within the NGC System’s
ChromLab™ Software. The peak area of each individual elution
fraction was measured in ml * AU. Total mAb S loaded onto thecolumn was determined by injecting 1 ml of purified mAb S into
the NGC System, in column bypass mode, and measuring thepeak area of the flow-through mAb S.

n

S mA
i=1
i

Monomer recovery (%) = 100 xi

Mixture of monomer
and dimer

60
40

100

20
0

0
0

2 4 6

8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Volume, ml

Column:	1 ml CHT packed in a Bio-Scale Mini Cartridge (0.56 x 4 cm)
Sample:	2.8 mg affinity-purified mAb S in 1 ml 10 mM sodium phosphate, pH 7.0
Flow rate:
300 cm/hr
Equilibration:
10 mM sodium phosphate, pH 7.0, 10 column volumes (CVs)
Post-loading wash: 10 mM sodium phosphate, pH 7.0, 5 CVs
Elution:	Buffer A
10 mM sodium phosphate, pH 7.0
	Buffer B
10 mM sodium phosphate, 1 M sodium chloride, pH 7.0
A to B linear gradient, 0–100%B, 20 CVs

B
100

100

90
80

99

70
60

98

50
40

97

30
20

96

10
0

95
A8

A9

A10

A11

A12
A13
A14
Elution fraction

A15

A16

A17

Fig. 1. Purification of mAb S on a CHT Column. A, removal of mAb S
aggregates using a 0–1,000 mM sodium chloride gradient. OD 280 (—);
conductivity (—); %B (—). The blue vertical bands represent where fractions
were collected. B, mAb S monomer content in individual elution fractions (– –)and accumulated monomer recovery in pooled elution fractions (– –).
A1–2 A3–4 A5–6 A7–8

A9–11

A12–14 A15

200

600

© 2015 Bio-Rad Laboratories, Inc.

160
400

l280, mAU

Results
The mAb S aggregates were well separated from themonomer using the CHT Column with a 0–1,000 mM sodium
chloride gradient. The bulk of the mAb S monomer was
eluted at ~50%B, which is equivalent to ~500 mM sodium
chloride (Figure 1A). Elution of the monomer was complete
at ~75%B or 750 mM sodium chloride, and increasing the salt
concentration resulted in leaching of the undesired mAb S
oligomers from the column (Figure 1B). Step elution of mAb
S was then performed using a sodium phosphate buffer
containing 550 mM sodium chloride (Figure 2). Fractions A5
to A9 were pooled, and the monomer content in this eluate
was determined to be 99.5% with an overall mAb S monomer
recovery of 82.7%.

%B80
Monomer

mt A tmi: Monomer content in an elution fraction; determinedby SEC analysis with an ENrich SEC 650 Column
mt: Monomer content in the starting material; determined
to be 0.7412 in this study
Ai: Integration area of individual elution fraction (ml * AU)
At: Integration area of total mAb S loaded (ml * AU)

A10–12 A13–15 A16–18 A19–21 A22–24 A25–26

Fraction monomer content, %Calculation of Monomer Recovery

A7–9

120
80

200

40

Conductivity, mS/cm

mAb S was purified from a CHO cell culture harvest byProtein A affinity chromatography and used immediately. Prior
to loading onto the columns, the mAb S solution was adjusted
to the desired pH with 1 M Tris HCl and filtered through a0.22 µm membrane. The total mAb S concentration in thetest solution was 2.8 mg/ml with monomer content of 74.12%.

A4–6

100

200

l280, mAU

Test Protein Solution

A1–3

300

Accumulated monomer recovery, %Chromatographic separations and analyses were performedon an NGC™ 10 Chromatography System (Bio-Rad
Laboratories), which provides simultaneous monitoring ofabsorbance at 280 nm, conductivity, and pH.

0

0
0

2

4

6

8

10 12 14 16 18 20 22 24 26 28 30 32 34 36
Volume, ml

Column:1 ml CHT packed in a Bio-Scale Mini Cartridge (0.56 x 4 cm)
Sample:
2.8 mg affinity-purified mAb S in 1 ml 10 mM sodium phosphate, pH 7.0
Flow rate:
300 cm/hr
Equilibration:
10 mM sodium phosphate, pH 7.0, 10 CVs
Post-loading wash: 10 mM sodium phosphate, pH 7.0, 5 CVs
Elution:
10 mM sodium phosphate, 550 mM sodium chloride, pH 7.0, 15 CVs

Fig. 2. Elution of mAb S monomers from a CHT Column. Elution was carriedout using 10 mM sodium phosphate and 550 mM sodium chloride, pH 7.0.
Fractions A5 to A9 were pooled. OD 280 (—); conductivity (—). The blue verticalbands represent where fractions were collected.

Bulletin 6749

Mixed-Mode Chromatography for mAb S Aggregate Removal: Comparison of CHT™ Ceramic Hydroxyapatite, Capto adhere, and Capto adhere ImpRes

A
A34–36A37–39A40–42

A43–45

A46–48

A49–51

A52–54 A55–56

12

8

120

pH

80

4

40
0

0
0

2

4

6

8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38
Volume, ml

Column:1 ml Capto adhere packed in a Bio-Scale Mini Cartridge (0.56 x 4 cm)
Sample:
2.8 mg affinity-purified mAb S in 1 ml 10 mM sodium phosphate, pH 7.0
Flow rate:
300 cm/hr
Equilibration:
50 mM sodium phosphate, pH 8.0, 10 CVs
Post-loading wash: 50 mM sodium phosphate, pH 8.0, 5 CVs
Elution:Buffer A
50 mM sodium phosphate, pH 8.0
Buffer B
50 mM sodium acetate, pH 5.0
A to B linear gradient, 0–100%B, 20 CVs

B
5.6

5.4

98

5.3
97
5.2

5.4

30

5.3

20

5.2

10

5.1

Fraction pH

5.5

40

5.0

0
A49		 A50		

A51		A52		 A53

Elution fraction

Fig. 3. Purification of mAb S on a Capto adhere Column. A, removal ofmAb S aggregates using a pH 8–5 gradient. OD 280 (—); pH (—); %B (—).
The blue vertical bands represent where fractions were collected. B, mAb S
monomer content in individual elution fractions (– –) and its correlation withfraction pH (– –). C, accumulated mAb S monomer recovery in pooled elution
fractions (– –) and its correlation with fraction pH (– –).

To further assess the effect of the elution pH on final mAb S
monomer purity and recovery, step elutions under various
pH conditions were performed. The results are summarizedin Table 1. The chromatography of mAb S elution at pH 5.5
is shown in Figure 4. The purity of the product was found tobe very sensitive to the pH of the elution buffer. A minor pH
drop, from 5.5 to 5.4, resulted in a tripling of the aggregate
content in the eluate. The pooled product from a pH 5.4 step
elution failed the quality control (QC) specification of ≥99.5%
monomer content. Furthermore, despite the fact that 14 CVsof eluate were collected, monomer recovery was only ~53%.
Table 1. Pooled elution fractions from the Capto adhere Column
using 50 mM sodium acetate buffer at different pH.

Elution pH

mAb S
MonomerContent, %mAb S
Monomer
Recovery, %EluateVolume, CV

5.2

96.9

76.9

14

5.3

98.1

64.9

14

5.4

98.8

52.5

14

5.5

99.7

48.8

14

A31–33

5.5

99

A34–36 A37–39

A40–42 A43–45 A46–48 A49–51

12

100
Fraction pH

Fraction monomer content, %

100

5.6

50

A48		

80
l280, mAU

l280, mAU

160

60

8

60

pH

A31–33200

C
Accumulated monomer recovery, %Preliminary design of experiments (DoE) studies were
performed to screen for mAb S purification conditions with
Capto adhere Media. Binding was maximal with buffers
containing ≤50 mM sodium chloride at pH above 8.0. mAb
S was only partially eluted from the column under a modest
acidic condition (pH 5.0). At pH 4.0, all mAb S species were
eluted from the Capto adhere Column without differentiation ofmonomer and higher molecular weight species. The additionof sodium chloride to the elution buffer further hampered
target protein recovery, which indicates a strong hydrophobic
interaction between target molecules and this hydrophobic
anion exchange media (data not shown). A gradient of pH
8–5 was therefore employed in an effort to elute mAb S
monomers selectively from the Capto adhere Column (Figure
3A). Dependency of monomer content and elution pH wasobserved, with the later fractions containing significantly morehigh molecular weight species (Figure 3B) and exhibiting aplateau of mAb S monomer recovery (Figure 3C).

40

4

20

96

5.1

95

5.0
A48		

A49		 A50		 A51		
Elution fraction

A52		A53

0

0
0

2

4

6

8

10 12 14 16 18 20 22 24 26 28 30 32 34 36
Volume, ml

Column:1 ml Capto adhere packed in a Bio-Scale Mini Cartridge (0.56 x 4 cm)
Sample:
2.8 mg affinity-purified mAb S in 1 ml 50 mM sodium phosphate, pH 8.0
Flow rate:
300 cm/hr
Equilibration:
50 mM sodium phosphate, pH 8.0, 10 CVs
Post-loading wash: 50 mM sodium phosphate, pH 8.0, 5 CVs
Elution:
50 mM sodium acetate, pH 5.5, 15 CVs

Fig. 4. Elution of mAb S monomers from a Capto adhere Column. Elution
was carried out using 50 mM sodium acetate, pH 5.5. Fractions A35 to A48
were pooled. OD 280 (—); pH (—); %B (—). The blue vertical bands represent
where fractions were collected.
© 2015 Bio-Rad Laboratories, Inc.

Bulletin 6749

Mixed-Mode Chromatography for mAb S Aggregate Removal: Comparison of CHT™ Ceramic Hydroxyapatite, Capto adhere, and Capto adhere ImpRes

Compared with Capto adhere, the binding of mAb S
by Capto adhere ImpRes was more sensitive to theconcentration of sodium chloride in the buffer. However,
the elution of mAb S from this mixed-mode media seemed
to be solely dependent on the pH of the buffer, as revealed
by DoE screening (data not shown). Again, a pH gradient wasused to resolve mAb S monomers from aggregates(Figure 5A). The pH of the elution fractions, monomer
content, and monomer recovery were analyzed and theresults are shown in Figures 5B and 5C.

Buffers at different pH were used to elute mAb S fromthe Capto adhere ImpRes Column in step mode (Table 2).
Figure 6 depicts the chromatogram resulting from the elutionof mAb S with 50 mM sodium acetate (pH 5.4). Overall,
Capto adhere ImpRes offered better recovery of mAb S
monomer than Capto adhere. The purity of mAb S monomerin the eluate met the QC specification of ≥99.5% monomer
content and was consistent at pH 5.4 and higher; however,monomer recovery was only 50–62% after 14 CVs of eluate
were collected under these conditions.

A

Table 2. Pooled elution fractions from the Capto adhere ImpRes
Column with 50 mM sodium acetate buffer at different pH.

A61–63 A64–66 A67–69 A70–72 A73–75 A76–78 A79–81 A82–84 A85–86

200

8

100

4

pH

12

0

0
2

4 6

EluateVolume, CV

5.3

98.6

68.3

14

5.4

99.6

61.7

14

5.5

99.6

50.5

14

8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Volume, ml

A1–2 A3–4 A5–6 A7–8

Column:	1 ml Capto adhere ImpRes packed in a Bio-Scale Mini Cartridge(0.56 x 4 cm)
Sample:
2.8 mg affinity-purified mAb S in 1 ml 50 mM sodium phosphate, pH 8.0
Flow rate:
300 cm/hr
Equilibration:
50 mM sodium phosphate, pH 8.0, 10 CVs
Post-loading wash: 50 mM sodium phosphate, pH 8.0, 5 CVs
Elution:Buffer A
50 mM sodium phosphate, pH 8.0
Buffer B
50 mM sodium acetate, pH 5.0
A to B linear gradient, 0–100%B, 20 CVs

5.6

99

5.5
5.4

98

5.3
97

5.2

96

Fraction pH

100

5.0
A78		

A79

A80
A81
Elution fraction

A82

A12–14A15–17

A18–20 A21

12

160
8

120
80

4

40
0

0
2

4

6

8

10 12 14 16 18 20 22 24 26 28 30 32 34 36
Volume, ml

Column:	1 ml Capto adhere ImpRes packed in a Bio-Scale Mini Cartridge(0.56 x 4 cm)
Sample:
2.8 mg affinity-purified mAb S in 1 ml 50 mM sodium phosphate, pH 8.0
Flow rate:
300 cm/hr
Equilibration:
50 mM sodium phosphate, pH 8.0, 10 CVs
Post-loading wash: 50 mM sodium phosphate, pH 8.0, 5 CVs
Elution:
50 mM sodium acetate, pH 5.4, 15 CVs

Fig. 6. Elution of mAb S monomers from a Capto adhere ImpRes Column.
Elution was carried out using 50 mM sodium acetate, pH 5.4. Fractions A4
to A18 were pooled. OD 280 (—); pH (—); %B (—). The blue vertical bands
represent where fractions were collected.

5.1

95

A9–11

200

0

B

Fration monomer content, %mAb S
Monomer
Recovery, %pH

0Elution pH

mAb S
MonomerContent, %l280, mAU

l280, mAU

300

A83

80

5.6

70

5.5

60

5.4

50
40

5.3

30

5.2

Fraction pH

Accumulated monomer recovery, %

C

20
5.1

10

5.0

0
A78		

A79

A80
A81
Elution fraction

A82

A83

Fig. 5. Purification of mAb S on a Capto adhere ImpRes Column.
A, removal of mAb S aggregates using a pH 8–5 gradient. OD 280 (—); pH (—);%B (—). The blue vertical bands represent where fractions were collected.
B, mAb S monomer content in individual elution fractions (– –) and its
correlation with fraction pH (– –). C, accumulated mAb S monomer recovery
in pooled elution fractions (– –) and its correlation with fraction pH (– –).
© 2015 Bio-Rad Laboratories, Inc.

Bulletin 6749

Mixed-Mode Chromatography for mAb S Aggregate Removal: Comparison of CHT™ Ceramic Hydroxyapatite, Capto adhere, and Capto adhere ImpRes

Discussion
Aggregates of therapeutic mAbs often demonstrate different
bioactivity/potency profiles, storage stability, immunogenicity,and pharmacodynamic/pharmacokinetic properties thantheir monomeric counterparts (Vázquez-Rey and Lang 2011).
These product-related impurities must be effectively removed
during the manufacturing process to ensure the safety andefficacy of the final drug formulation. A high level of aggregates
was present in the mAb S harvest, which could have been
an indication of the intrinsic hydrophobicity of mAb S or the
exposure of its hydrophobic amino acid residues due to
misfolding, unfolding, or denaturation. The mixture of monomers
and aggregates of mAb S could not be separated by traditional
cation exchangers (data not shown). Three mixed-mode media,
CHT, Capto adhere, and Capto adhere ImpRes, were tested
for their efficiency in clearing mAb S aggregates; their optimal
performances are summarized in Table 3.
Table 3. Comparison of CHT, Capto adhere, and Capto adhere ImpRes
in the clearance of mAb S aggregates.

Media

mAb S
MonomerContent, %mAb S
Monomer
Recovery, %EluateVolume, CV

CHT*

99.5

82.7

5

Capto adhere**

99.5

48.8

14

Capto adhere
ImpRes***

99.5

61.7

14

*

 tep elution with 10 mM sodium phosphate and 550 mM sodium chloride,S
pH 7.0** Step elution with 50 mM sodium acetate, pH 5.5*** Step elution with 50 mM sodium acetate, pH 5.4

The contamination level of mAb S aggregates in therecovered mAb S monomer fractions was effectively reduced
to below 0.5% by all three media. However, CHT offered
the best monomer recovery in the smallest eluate volume(Table 3). Since the isoelectric point (pI) of mAb S wasestimated at 6.9 (data not shown), it should bind to Capto
adhere–based media through strong electrostatic interaction.
The difficult elution of mAb S from these two hydrophobic
anion exchangers suggests that a significant hydrophobic
interaction was also involved in mAb S binding to thesecolumns. A more stringent acidic condition was necessary
for protonating mAb S molecules in order to promote theirdissociation from these chromatography media; however,this was carried out at the expense of selectivity between
monomers and aggregates. To satisfy the QC specificationof ≥99.5% monomer in the final product, elution had tobe terminated at pH 5.5 and pH 5.4 for Capto adhere andCapto adhere ImpRes, respectively. As a result, the recovery
of mAb S monomer from these chromatography media
was compromised. Tailing was observed and elution wasincomplete even after 15 CVs of elution buffer were applied.
Conclusions
In the commercial production of therapeutic proteins, both
purity and yield are essential to product quality and process
efficiency and economics. For the hydrophobic and mildly
acidic mAb S molecules, our data show that CHT is thechromatography media of choice for a robust production
process with minimal loss of monomeric target molecules.
References
Gagnon P (2009). IgG aggregate removal by charged-hydrophobic mixed mode
chromatography. Curr Pharm Biotechnol 10, 434–439.
Gronemeyer P et al. (2014). Trends in upstream and downstream processdevelopment for antibody manufacturing. Bioengineering 1, 188–212.
Rathore AS et al. (2013). Aggregation of monoclonal antibody products:
Formation and removal. Biopharm International 26, 40–45.
Vázquez-Rey M and Lang DA (2011). Aggregates in monoclonal antibody
manufacturing processes. Biotechnol Bioeng 108, 1,494–1,508.

© 2015 Bio-Rad Laboratories, Inc.

Bulletin 6749

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