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Optimization and Scale-Up of HCICBased MAB Purification Processes,

Part 1

onoclonal antibodies (MAbs)

serve important medical
needs in cancer treatment as
well as that of autoimmune
and infectious diseases (1). Antibodies
are also widely used in clinical
diagnostic assays. They can be coated
on solid surfaces to bind specific
analytes, conjugated to reporter
molecules (either as whole antibodies or
fragments) for analyte detection, used in
sensitivity panels for lot-release testing,
and supplied as positive controls in
diagnostic kits (2). Our study evaluates
the use of hydrophobic chargeinduction chromatography (HCIC) for
purification of MAbs that are of
interest for diagnostic applications. Our
objective was to obtain 90% purity
with 90% IgG recovery from a single
chromatographic step a challenging
objective with most experiments
conducted using feedstock derived from
protein-supplemented growth media.
To assess the versatility of the HCIC
approach, we used both concentrated
and unconcentrated dilute feedstocks as
Product Focus: Monoclonal
antibodies (therapeutics, diagnostics)
Process Focus: Downstream
processing

Who Should Read: Process engineers

Keywords: Optimization, scale-up,
mixed-mode chromatography, SDSPAGE, purification platforms, dilute
feedstocks, murine and ovine MAbs
Level: Intermediate
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PALL LIFE SCIENCES (WWW.PALL.COM)

Stphanie Chaudoreille, Natalie Bohrer, Patrick Santambien,

Ren Gantier, Warren Schwartz, and Steven P. Allen

well as a number of different antibody

isotypes and source species. We also
isolated MAbs from different growth
media formulations.
Purification platforms for diagnostic
antibodies can involve a single
chromatographic step, as in the work
reported here. But our findings also
should interest process engineers
developing multistep purification
schemes. HCIC has been evaluated in
development of multistep schemes for
purification of therapeutic antibodies
using strategies based on protein A
affinity capture and those that do not
rely on it (35).
In diagnostic and research
applications, MAbs of different
immunoglobulin classes and isotypes
are derived from a broad range of
species sources. Veterinary therapeutic
applications can require antibodies from
a number of species sources. Such
antibodies are often poorly retained on
protein A sorbents (6). Purification of
murine IgG1 on protein A sorbents, for
example, typically requires binding at
pH 8.5 and then augmenting
feedstock with concentrations of

binding-promoting salt that approach

the antibodies precipitation point (6).
By contrast, no enhanced binding
conditions are required when using the
HCIC sorbent we chose (7). Murine
IgG1 and IgG2a will bind to it directly
from clarified cell-culture supernatant
or from dilute buffer at pH 8.0 (7, 8).
Studies have shown similar binding
capacity values for murine IgG1 and
IgG2a during HCIC: 37 and 34 mg/
mL, respectively (9, 10).
Immunoglobulin E, which is poorly
retained on protein A, also has been
purified by HCIC (11, 12). Brief reports
have also described purification of IgM
with HCIC (10, 13). To explore its broad
selectivity further, we include
purification of murine IgG1, murine
IgG2a, and ovine (sheep) IgG1.
Broadly speaking, process developers
are increasingly focused on strategies to
support enhanced process economics
and robustness. In that context,
sorbents carrying synthetic chemical
ligands are inherently less costly than
those based on protein ligands.
Moreover, the former are typically
compatible with stringent cleaning

Table 1: Objectives, chromatographic conditions, monoclonal antibodies involved (all murine except for Experiment 12), and feedstock compositions; all
murine experiments except 9 and 10 used the same clone of murine IgG1, designated murine IgG1(1); experiment 9 used another clone, murine IgG1(2).
Experiments 110 used supernatant from cell culture growth medium A (a hybridoma serum-free medium (HSFM) supplemented with transferrin
(10 g/mL), insulin (10 g/mL) and Albumax lipid-rich bovine serum albumin at a concentration proprietary to Abbott); experiment 11 came from
medium B (HSFM supplemented by 0.05% F-68 pluronic acid), and experiment 12 came from medium C (Iscoves modified Dulbeccos medium
supplemented by 10% fetal bovine serum). Figures 26 appear in Part 2 of this report.
Experiment, Figure,
and Antibody
Exp. 1, Fig. 2, IgG1(1)

Column
Dimensions
(cm)
0.46 ID 5.0

IgG Load
Concent.
(mg/mL)
1.5*

pH step elution
sequence

Exp. 2, Fig. 3, IgG1(1)

0.46 ID 5.0

1.5a

10

NaCl in binding, wash,

and elution buffers

Exp. 3, Fig. 4, IgG1(1)

0.46 ID 5.0

0.75c

Exp. 4, Fig. 5, IgG1(1)

1.0 ID 9.7

1.5a

Residence time (RT)

with unconcentrated
feedstock

Exp. 5, NA, IgG1(1)

0.66 ID 15

0.043b

500

4.2 mg

Exp. 6, NA, IgG1(1)

0.66 ID 15

0.043b

1,200

10.1 mg

Exp. 7, NA, IgG1(1)

0.66 ID 10.5

0.043b

840

10.1 mg

Increased scale with

unconcentrated
feedstock

Exp. 8, Fig. 6, IgG1(1)

5.0 ID 17.5

0.043b

28.1
103

Antibody type,
feedstock composition
and concentration, and
residence time

Exp. 9, NA, IgG1(2)

1.0 ID 10.0

2.6a

Exp. 10, NA, IgG2a

0.46 ID 5.0

1.2a
a

23

0.8a

81

Influence Evaluated
Preelution wash

Exp. 11, NA, IgG1(1)

1.1 ID 9.0

Exp. 12, NA, ovine IgG1 1.1 ID 9.5

2.8

Load
Vol.
Column
(mL) Loading*
20
36.1 mg

Linear
Veloc.
(cm/h)
60

RT
(min.)
5

Postload
Wash
PBS pH 7.4,
water, 25 mM
Na caprylate

18.1 mg

40

7.5

PBS pH 7.4

5.5, 5.2, 4.8,

4.0, 3.0

20

18.1 mg

40

7.5

PBS pH 7.4 +
1 M NaCl

5.5, 5.2, 4.8,

4.0 (1M NaCl
in all)

45

8.9 mg

80

7.5

PBS pH 7.4

5.5 + 0.5 M
NaCl, 5.0,
4.0, 3.0

180

5.0

PBS pH 7.4

as in Exp. 4

300

3.0

PBS pH 7.4

as in Exp. 4

300

2.1

PBS pH 7.4

as in Expt. 4

3.5 mg

230 L,
300 E

4.6 L,
3.5 E

PBS pH 7.4

as in Exp. 4

38

12.4 mg

80

7.5

PBS pH 7.4

as in Exp. 4

6.5

10.4 mg

60

5.0

PBS pH 7.4

as in Exp. 4

7.6 mg

126

4.3

PBS pH 7.4

as in Exp. 4

7.0 mg

189

3.0

PBS pH 7.4

as in Exp. 4

ID = internal diameter; PBS = phosphate buffered saline; RT = residence time; L = load; E = elute

StepElution pH
Sequence
4.5, 4.0, 3.0

* mg IgG per each mg of solvent

b
30-fold concentrated cell culture supernatant
nonconcentrated cell culture supernatant
c
30-fold concentrated cell culture supernatant, diluted 1:1 with phosphate-buffered saline (PBS augmented with a 1M concentration of NaCl) + 1M NaCl

agents (e.g., 1 M sodium hydroxide)

than the latter. These considerations
suggest a potentially expanded role for
HCIC in purification platform
development.
Affinity chromatography on protein
A sorbents remains the most widely
used strategy for MAb capture and
initial purification (1417). But a number
of researchers have investigated
strategies that do not rely on protein A
(3, 18), or that make use of sorbents
carrying immunoglobulin-selective,
mimetic ligands (19, 20). Development of
platforms without protein A has been
facilitated by a considerable body of
literature describing use of other
chromatographic modes of in
conjunction with protein A affinity
chromatography. Some such techniques
also have been evaluated as alternatives
to protein A for capture
chromatography: cation-exchange
chromatography (CIEX) (21),
hydrophobic-interaction
chromatography (HIC) (22), affinity
chromatography with mimetic ligands
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(19, 20), hydroxyapatite chromatography

(23, 24), and other modes used in
combination (25).
HCIC was introduced by Burton et al.
in 1998 (26). Its mechanism is based on
the pH-dependent behavior of dualmode, ionizable, hydrophobic ligands.
Details concerning chromatographic
mechanism and optimization strategies
are presented below and in Part 2 of this
two-part report (BPI June 2015, in press).
HCIC has been evaluated in schemes for
purification of therapeutic antibodies that
involve two or three chromatographic
steps (35). One study describes its use in
conjunction with CIEX, with evaluation
of both techniques for capture
chromatography (3).
In another study, HCIC was
evaluated as a polishing step directly
following capture on a protein A
sorbent, with anion-exchange
chromatography (AIEX) used as the
third chromatographic step (4). The
authors report that replacing
conventional HIC with HCIC
significantly enhanced process flow.

HCIC requires no addition of lyotropic

salt. Moreover, the need for diafiltration
in advance of AIEX was eliminated
because the product pool from HCIC
was recovered in dilute buffer.
Significantly, the authors report more
general and straightforward HCIC
optimization for antibody purification
than with conventional HIC, thereby
facilitating process development.
In multistep schemes, HCIC has
been shown to contribute effectively to
clearance of CHO host-cell proteins
(CHOPs), DNA, and viruses (3). When
used for capture chromatography,
HCIC can provide better aggregate
clearance than protein A sorbents can
(4). In a related study, clearance of
aggregates, CHOPs, and productrelated impurities was enhanced by
controlling HCIC based on both pH
and the presence of binding-promoting
salt in the wash and elution buffers used
(5). Taken together with our findings
presented below, these results suggest
that HCIC could play a broader role in
MAb platform development.

Figure 1: HCIC mechanism; binding of IgG is

supported by a combination of hydrophobic
interaction and molecular recognition.
Desorption is prompted by electrostatic
charge repulsion through reduction of the
mobile-phase pH.

H
in ydr
te o
ra ph
ct o
i o bi
n c

Adsorption at near-neutral pH
(physiological conditions)
S

pKa = 4.8

ON

E
re lect
pu ro
lsi sta
on ti
c

Desorption at pH 4.05.8
S
O

N+
H

Materials and Methods

Chemicals, Biologicals, and Equipment:

Abbotts diagnostics division provided

cell-culture supernatants (CCSs) for all
experiments. They came from cell
cultures performed in batch spinners,
with hybridoma serum-free media
supplemented by AlbuMAX lipid-rich
bovine serum albumin, porcine
transferrin, and insulin (all from Life
Technologies). Before purification, in
some cases, we concentrated the CCSs
30-fold using tangential-flow filtration
(TFF) with a 50-kD Pellicon cassette
from EMD Millipore to a final
antibody concentration of ~1.5 mg/
mL, then 0.2 m-filtered the resulting
feed before loading it onto a
chromatography column.
MEP HyperCel sorbent from Pall
Life Sciences was initially packed in
columns of 0.46-cm internal diameter
(ID) and 5-cm height from Upchurch
Scientific (IDEX Health and Science).
We optimized the process for a 1.0-cm
ID glass column from Omnifit Ltd.
and scaled up to a 5.0-cm ID 30-cm
column from GE Healthcare. Except
for the scale-up run using a PKL6
chromatography system from Pall, we
ran all tests on an KTA Explorer
chromatography system from GE
Healthcare. All chemicals used in this
study came from Sigma Aldrich and
were rated analytical grade.
We determined total protein
content using a bicinchoninic acid
(BCA) assay kit from Pierce
Biotechnology. Precast polyacrylamide
gels and Mini Protean II
electrophoresis equipment came from
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M ay 2015

Bio-Rad Laboratories. Gels were

stained by SimplyBlue SafeStain
reagent from Life Technologies. We
performed size-exclusion
chromatographic (SEC) analysis using
a TSKgel G4000SWXL column from
Tosoh Bioscience on a highperformance liquid chromatography
(HPLC) system from Waters equipped
with an autosampler and a photodiode
array detector.
Column Packing: The HCIC
sorbent came in slurry form with 1 M
NaCl-containing 20% (v/v) ethanol
~7075% (v/v) concentration. After a
desired volume of slurry was
transferred to a graduated container,
we performed two decantations in 10
column volumes (CV) deionized water
followed by two decantations in 5 CV
packing buffer, phosphate-buffered
saline (PBS) at pH 7.4 as follows:
Sorbent was mixed with buffer or
water and left to settle, then the
supernatant was removed.
A 5075% slurry was finally
prepared through addition of PBS at
pH 7.4.
A few milliliters of packing buffer
were introduced into the column and
drained through its lower plunger,
leaving 1 cm of buffer above the
bottom frit. The outlet column was
closed and the upper plunger primed
to eliminate air bubbles.
Slurry was continuously poured
into the column and left to settle so
that a layer (12 cm) of clear
supernatant became visible at the top
of the column. The upper plunger was
then adjusted so that no air bubbles
were trapped.
The outlet column was opened,
and the pump was operated at a
packing flow rate of 1,000 cm/h until
a stable packed bed was obtained.
The pump was then stopped and
the upper plunger carefully adjusted to
the surface of the bed (with excess
buffer left to drain through the column
inlet).
The column inlet was reconnected
to the pump without introducing air
bubbles, the outlet was opened, and a
flow rate of 1,000 cm/h was applied
for a few minutes. If necessary, the
upper plunger would be finally
readjusted to the bed surface.

Chromatography: Table 1
summarizes the chromatographic
conditions for all experiments. Columns
were equilibrated with a minimum of 5
CV of PBS at pH 7.4, continuing until
influent and eluent showed equivalent
pH and ionic strength. Concentrated
CCSs and unconcentrated material
were loaded onto columns without
preliminary adjustment. After loading
was complete, columns were washed
with PBS at pH 7.4 until absorbance
reached baseline.
For Experiment 1 only, we
performed two additional preelution
wash steps to prompt selective
desorption of bound albumin. We used
deionized water for the first wash and
25 mM sodium caprylate in PBS at pH
7.4 for the second (8). Elution involved a
sequence of 100 mM or 50mM sodium
acetate buffers at different pH levels.
During all the chromatography runs,
we measured absorbance of effluents at
280 nm and collected fractions. For
sorbent regeneration, we used a
minimum of 5CV of 1 N NaOH, and
for reequilibration we used PBS at pH
7.4 or (after a longer storage period) a
1M NaCl, 20% ethanol (v/v) solution.
Assays: To evaluate total protein
content in chromatography fractions,
we used a BCA assay kit following
supplier instructions (27). We ran
sodium-dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE)
analyses using 15-well precast 12%
(w/v) gels (28). Samples were diluted
threefold in Laemmli sample buffer
containing 5% (v/v) -mercaptoethanol.
We loaded 12 L of diluted sample into
each well of the gel, then applied 50 V
for 10 minutes and 200 V for 42
additional minutes. SimplyBlue
SafeStain gel staining was a microwave
procedure, whereas destaining used
deionized water. For SEC analyses, we
injected 30 L of sample diluted
twofold in mobile phase (0.05 M
sodium phosphate and 0.1 M Na2SO4
at pH 6.8) onto a TSKgel
G4000SWXL column, then ran it at
0.5 mL/min for 45 minutes.

Results and Discussion

Our findings are best understood in

connection with the mechanism of
HCIC on MEP HyperCel sorbent

(Figure 1). At near-neutral pH, the

MEP ligand is uncharged and supports
binding by hydrophobic interaction. In
addition to its hydrophobic properties,
the ligand is immunoglobulin selective
(8, 13). A combination of affinity and
hydrophobic interactions typically
allows immunoglobulin binding at
near-neutral pH and physiological ionic
strength. Generally, no addition of
lyotropic or other binding-promoting
salt is required (8), although adding salt
could be evaluated as a means of
providing increased binding, with
binding capacities <20 mg/mL observed
at physiological ionic strength.
As Figure 1 illustrates, desorption is
prompted by electrostatic charge
repulsion, which is induced when
mobile-phase pH is reduced
(establishing a net-positive charge on
both ligand and bound protein). HCIC
optimization typically begins with
determining the optimum pH for
selective elution of a target antibody. To
eliminate potential ambiguities
associated with pH control or
interpretation of findings from
pH-gradient experiments, a pH-step
elution sequence is often used to
determine that value. But some
investigators have successfully used pH
gradients during their optimization
studies (3).
Control of HCIC chromatography
also can be established through addition
of a binding-promoting salt to elution or
preelution wash buffers. Here, HCIC is
controlled similarly to that conventional
HIC. For example, if an impurity is
found to coelute with the target
antibody even at an optimum elution
pH, then further optimization can be
achieved by adding a binding-promoting
salt to the elution or preelution wash
buffer. That will prompt selective
retention of either the impurity or the
target antibody. We combined both
control mechanisms, as described in
Part 2 next month.
Finally, we investigated the influence
of residence time and feedstock
concentration. In addition, we compared
isolation results from murine IgG1 from
albumin-supplemented and albuminfree growth media. We also purified
antibodies representing additional
isotypes and species sources. Table 1

COMBINING

affinity and
hydrophobic
interactions typically
provides for
immunoglobulin
binding at near-neutral
pH and physiological
ionic strength.
summarizes the chromatographic
conditions, antibodies, and feedstock
compositions for all experiments. It
includes a summary statement of
objectives for each experiment or group
of experiments. In Part 2 of this twopart report, experiments are numbered
according to their Table 1 listings.

References, Part 1

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Continued on page 46
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Continued from page 46

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At the time this work was done, Stphanie

Chaudoreille was an R&D scientist, and
Patrick Santambien was a senior R&D
manager at Pall Life Sciences, 48, Avenue des
Genottes, 95800 Cergy St. Christophe, France.
Natalie Bohrer is a scientist in global protein
sciences at Abbvie, 1 North Waukegan Road,
North Chicago, IL, 60064. Ren Gantier is
R&D director of biopharm applications, and
Warren Schwartz is senior technical director
of chromatography at Pall Life Sciences, 20
Walkup Drive, Westborough, MA 01581.

34 BioProcess International

13(5)

M ay 2015

Corresponding author Steven P. Allen is

manager of biologics process design Abbott
Diagnostics Division, 100 Abbott Park Road,
Abbott Park, IL 60064; 1-224-668-1006;
steven.allen@abbott.com.
For electronic or printed reprints, contact
Rhonda Brown of Foster Printing Service,
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x194. Download personal-useonly PDFs
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