BioPharm

INTERNATIONAL

mAb Purification

The Science & Business of Biopharmaceuticals

Electronically reprinted from December 2013

Comparing Protein A Resins for

Monoclonal Antibody Purification
Shohei Kobayashi and
Yasufumi Ueda

Photo Credit: SCIEPRO/Getty Images

A prototype
Protein A resin
is evaluated
for purification
performance,
reusability,
and cost
performance.

W
Shohei Kobayashi and
Yasufumi Ueda are in the
API process development department,
Pharmaceutical Technology Division,
Chugai Pharmaceutical, Tokyo, Japan.

it h g re ate r e conom ic
pressure on monoclo n a l a n t i b o d y (m A b)
produc t ion for t herapeutic and research uses, antibody
titers in mammalian cell culture have
increased dramatically over the past
20 years. As a consequence, dow nstream processing must accept and
handle higher titers of mAbs in harvested cell-culture fluid (HCCF), and
vendors of mAb purification technolog ies must develop chromatog raphy
resins with high binding capacity to
meet the demand. In addition, more
cost-efficient cleaning procedures are
necessary to extend the lifetime of
chromatography resins and to reduce
the cost for cleaning and validation.
A team from Chugai Pharmaceutical
( Japan) investigated the mAb purification performance of a new alkali-

BioPharm International www.biopharminternational.com December 2013

tolerant, protot y pe Protein A resin

(Resin 3, MabSelect SuRe LX prototype, GE Healthcare), which has the
potential to address the demand for
a more advanced, cost-effective mAb
purification technology.
The methodolog y for the production of monoclonal antibodies from
a cell line by hybridization of mouse
mye lom a a nd mou s e sple e n c e l l s
from an immunized donor was first
published in 1975 (1). As a technolog y that permits the generation of
monoclonal antibodies against almost
any target molecule, it immediately
gained great interest as a source for
potential drug candidates. Although
it took some time for the first therapeutic mAb to become commercially
available in 1986 (2), the market for
therapeutic m Abs has since g row n
rapidly. MAbs have proved to be suc-

mAb Purification

Table I: Properties of Protein A resins; degree of alkali resistance is indicated by +/-.

Resin

Ligand

Average particle size (m)

Alkali resistance

Matrix

Binding capacity*

rProtein A

90

+/-

Agarose

~27 g/L resin

Resin 2 (MabSelect SuRe)

Alkali-tolerant Protein A

85

+++

Agarose

~35 g/L resin

Resin 3 (MabSelect SuRe

LX prototype)

Alkali-tolerant Protein A

85

+++(+)

Agarose

~60 g/L resin

Resin 1 (rProtein A
Sepharose 4 Fast Flow)

* Typical dynamic binding capacities according to resin manufacturer data

Table II: Study outline; column height = 100 mm, column inner diameter = 10 mm, run time = 5 h, CV is column volume.
Step
Equilibrium

Solution
20 mM citrate-phosphate buffer pH 7.5, 1 mol/L sodium
chloride (NaCl)

5 CV

300 cm/h

Load

Harvested cell-culture fluid

50 g/L resin (residence time 6 min)

100 cm/h

20 mM citrate-phosphate buffer pH 7.5, 1 mol/L NaCl

5 CV

300 cm/h

Wash 2

10 mM citrate-phosphate buffer pH 7.7

5 CV

300 cm/h

Elution

50 mM acetic acid

6 CV

300 cm/h

Regeneration

0.1 mol sodium hydroxide

3 CV

120 cm/h

Storage (per 4 cycles)

2% benzyl alcohol, 50 mM sodium acetate, pH 5.0

5 CV

120 cm/h

COMMERCIAL-SCALE
PRODUCTION CHALLENGES

ALL FIGURES ARE COURTESY OF THE AUTHORS

Flow rate

Wash 1

cessful as targeted therapeutics

for a variety of diseases, includi n g s e ve r a l for m s of c a nc e r,
multiple sclerosis, and immunological disorders such as rheumatoid arthritis and psoriasis. In
2007, mAbs accounted for almost
half of the top -20 best-selling
biotechnolog y drugs in the US
alone, establishing them as an
important group of molecules (3).
Today, mAbs constitute the single
largest class of biological drugs
and accounts for about 36% of
the total biologics market with
an annual sales growth rate of
approximately 10% (4).

The rapid growth in mAb demand

has triggered industry efforts to
increase manufacturing capacity,
with the consequence that the
antibody titers in mammalian
cell culture have increased dramatically. Today, a typical process
accumulates titers of 15 g/L, but
expression levels as high as 1013
g/L have been reported (5). The
increase in upstream productivity

Volume

creates a subsequent demand on

downstream processing to address
high-titer HCCF.
Commercial-scale purification
of m Abs usua lly conta ins t wo
or three chromatographic steps.
Protein A is the affinit y chro matography ligand of choice for
the first antibody capture step,
because its high selectivity gives
excellent purity (typically > 99%)
and high y ields. Fur ther more,
Protein A-based resins form the
basis of almost all mAb-purification platforms as they are easy
to use at both small and large
scale with generic experimental
protocols.
Increased antibody titers create a potential purification challe nge b e c au s e of t he l i m ite d
capacity of current Protein A resins. To handle the high titers,
new resi ns w it h sig n i f ic a nt ly
greater capacity are needed. In
addition, Protein A resins with
the ability to withstand repeated
cleaning-in-place (CIP) with lowcost sodium hydroxide (NaOH)
considerably improves process
economics.

PROTEIN A RESINS

Protein A is a bacterial protein

f r o m S t aphy lo c o c c u s au r e u s ,
with the capacity to bind mammalian antibodies of class immunoglobulin G (IgG) w it h high
affinity. The gene for Protein A
has been cloned and expressed
in Escherichia coli (6, 7) allowing for the production of large
qua nt it ies of recombi na nt
Protein A.
Although recombinant Protein
A is widely used as an affinity
ligand for the capture and purif ication of antibodies, its sensit iv it y to a lka line cond it ions
prevents the use of rigorous and
cost-effective CIP and sanitization protocols based on NaOH.
C omp a r e d to c onve nt ion a l
Protein A resins, one of the affinity chromatography resins investigated, Resin 2 (MabSelect SuRe,
GE Hea lt hca re) is based on a
modified alkali-tolerant Protein
A ligand. Through protein engineering, the amino acids in one
of the IgG-binding domains particularly sensitive to alkali were
identified and substituted with

December 2013 www.biopharminternational.com BioPharm International

mAb Purification

Figure 1: Resin lifetime study using harvested cell-culture fluid (HCCF). Step yield
is >95 %, elution volume is 2.2 +/- 0.7 column volumes (CV), and carry-over is <1/1000;
all show no trend. Impurities are as follows: DNA, 3.2 log; HCP, 3.1 log; Protein A,
approximately 10 ppm (using data from GE Healthcare obtained from commercially
available resin); all show no trend.

Figure 2: Cost-performance of Resin 3 prototype compared to conventional resins

(Resin 1 and Resin 2). Product amount is 500 kg, fermenter size is 10,000 L (for 1 g/L)
or 5000 L (for 3.5 g/L), column size is 20 cm bed height, column lifetime is 120 cycles
for Resin 1 and 200 cycles for Resin 2 and 3, process time is 10-15 h.

more stable ones.

A novel protot y pe Resin
3 offers an increased dy namic
b i nd i n g c ap ac it y ( DB C) at a
slightly longer residence time.

EVALUATING
CAPACITY AND REUSABILITY

Table I shows the Protein A resins that were compared for perfor ma nce a nd cost- ef f ic ienc y.
According to the vendor, Resin 3
exhibits higher DBC than Resin
2 at longer residence times (8).
A residence t ime of 6 min is

expected to give a DBC (at 10%

breakthrough) of approximately
60 g antibody per liter resin.
In this study, the authors were
a ble to c on f i r m t h i s b e h avior, a nd for f u r t her st ud ies a
residence t i me of 6 m i n a nd
a loading of 50 g antibody per liter
resin, corresponding to approximately 80% of the DBC (at 10%
breakthrough), were selected.
Table II presents the outline of
the lifetime st udy. A n amount
of cell-culture supernatant corresponding to 50-g antibody per

BioPharm International www.biopharminternational.com December 2013

liter resin was applied to the column at 6 min. residence time.

This was followed by a two-step
wash i ng procedu re to remove
unbound particles. Bound antibody was eluted w it h 50 m M
acetic acid and the column resin

The engineered
Protein A ligand allows
clean-in-place and
sanitization protocols
based on sodium
hydroxide.
was cleaned in place with five
column volumes (CV) of 0.1 M
NaOH.
The results show that the step
yield was consistently over 95%,
and high log-reduction factors
of host-cell proteins (HCP) and
DNA were achieved (see Figure 1).
In this study, carryover was evaluated each 28th cycle and was
found to be less than 0.1% (i.e.,
after cycle 28, 56, 84, and 112).
The lifetime study with mAb-containing feedstock demonstrates
that the product qualit y, DBC,
and yield with Resin 3 were stable
for more than 100 purification
cycles. No increase in pressure
was observed during the study.

COST PERFORMANCE

Cost performance is dependent

on product amount produced per
year, batch size, column size, and
acceptable process time. In this
study, the cost of Protein A-based
production was calculated titer
by titer by the use of conventional resins (Resin 1, which is GE
Healthcares rProtein A Sepharose
4 Fast Flow, and Resin 2) and

mAb Purification

Resin 3 prototype resin. The calculations are based on an annual

mAb production amount of 500
kg and bioreactor size of 10,000 L
for 1 g/L titer or 5000 L for 3 g/L
and 5 g/L titers. Column diameter was estimated by considering
a process time within 10 to 15 h.
T he tot a l p u r i f ic at ion co st
per kilogram of produced antibody from 1 g/L and 3 g/L titers,
including chemicals and water,
u s i n g R e s i n 1 ($13 , 0 0 0 a n d
$14,0 0 0 respec t ively) ca n be
reduced by 29% and 46% respectively by using Resin 2 instead, as
shown in Figure 2 .
With Resin 3, in the case of a
titer level of 3 g/L, the authors
found that the overall purificat ion cost can be even f ur t her
reduced by 26%. With a lower
titer level of 1 g/L, however, the
decrease was only 4%.
Under the selected conditions,
Resin 1 was not suitable for purification of antibody from a 5 g/L
titer. The purification cost per
kilogram of produced antibody
from a 5 g/L titer using Resin
2 (70 0 0 USD) ca n be reduced
by 32% by using Resin 3 (see
Figure 2).
T hese resu lts show t hat t he
use of Resin 3 in purification of

Rapid growth in mAb demand has triggered

industry efforts to increase manufacturing
capacity, with the consequence that the
antibody titers in mammalian cell culture
have increased dramatically.
antibodies from high-titer feeds
sig nif icantly improves process
economy (see Figure 2).

SUMMARY

These data demonstrate that the

Resin 3 prototype has high capacit y and reusabilit y with stable
step y ield and impurit y clearance (e.g., DNA, HCP) for more
than 100 cycles. The engineered
Protein A ligand allows for the
use of rigorous and cost-effective
CIP and sanitization protocols
based on NaOH. Furthermore,
t he l iga nd is protease stable,
which leads to lower ligand leakage, and the highly cross-linked
agarose matrix allows for high flow
velocities at production scale.
In conclusion, process economy
can be significantly improved by
the use of Resin 3 in purification of

monoclonal antibodies from hightiter cell culture supernatants.

ACKNOWLEDGEMENTS

T he aut hors w ish to t ha n k

G E H e a lt h c a r e L i f e S c i e n c e s
(Uppsala, Sweden) for providing
Resin 3 prototype resin.

REFERENCES
1. G. Khler and C. Milstein, Nature 256
(5517) 495-497 (1975).
2. S. Kozlowski and P. Swann, Adv. Drug
Deliv. Rev. 58 (5-6) 707 722 (2006).
3. P.A. Scolnik, mAbs 1 (2), 179-184 (2009).
4. S. Aggarwal, Nat. Biotechnol. 29 (12)
1083-1089 (2011).
5. B. Kelley, mAbs 1 (5), 443-452 (2009).
6. S. Lofdahl, et al., Proc. Natl. Acad. Sci.
USA 80 (3) 697-701 (1983).
7. D. Colbert, et al., J. Biol. Response Mod. 3
(3) 255-259 (1984).
8. GE Healthcare, Dynamic binding
capacity study on MabSelect SuRe
LX for capturing high-titer monoclonal
antibodies, Application Note, 28-987525, Edition AA.

Posted with permission from the December 2013 issue of BioPharm International www.biopharminternational.com. Copyright 2014, Advanstar Communications, Inc. All rights reserved.
For more information on the use of this content, contact Wrights Media at 877-652-5295.

December 2013 www.biopharminternational.com BioPharm International

107491