International Journal of Emerging Technology and Advanced Engineering

Website: www.ijetae.com (ISSN 2250-2459, ISO 9001:2008 Certified Journal, Volume 3, Issue 10, October 2013)

Chromatographic Methods for the Purification of Monoclonal

Antibodies and their Alternatives:A Review
Ishan Arora1
1

Department of Chemical Engineering, Indian Institute of Technology Delhi

The purification process must be capable of removing
both product related impurities which include high
molecular weight aggregates and several isoforms that may
be formed as a consequence of deamidation, oxidation or
shuffling of disulfide bonds and process related impurities
which include leached protein A, DNA, certain cell culture
media additives and host cell protein [3]-[5]. It is essential
to remove these impurities since they may seriously
hamper the biological activity of the biopharmaceutical.
The cost effectiveness and ease of scale up of the
purification process are also crucial considerations.
Currently, packed bed chromatography is the backbone
of downstream processing. We typically use three
chromatography steps the first being Protein A
chromatography
followed
by
two
polishing
chromatography steps which may be anion exchange
chromatography, cation exchange chromatography,
hydrophobic interaction chromatography, mixed mode
chromatography etc. [1],[6]. The choice of the appropriate
polishing steps is mainly governed by the nature of the
impurities that need to be removed. Generally, one of the
polishing chromatography steps involves the use of ion
exchange chromatography. Protein A chromatography is
extremely effective and selective in removing host cell
proteins, virus particles, DNA and other impurities and at
the same time gives a very good yield [5],[7]. But it does
have a few drawbacks such as the possibility of formation
of aggregates due to the elution at low pH and inability to
remove aggregates formed during earlier processing steps
[6],[7]. The polishing chromatography steps are typically
used to remove the remaining aggregates, viruses, host cell
protein and leached protein A [8]. However, the use of
chromatographic separations has certain demerits such as
the requirement of large liquid volumes, high cost and
comparatively long cycle times. Hence, there is significant
interest in alternatives to chromatography such as aqueous
two phase extraction, precipitation and charged
ultrafiltration membranes [6],[9],[10].

Abstract Monoclonal Antibodies (mAbs) have emerged as

a unique and exciting group of biological products. They
represent a rapidly growing biopharmaceutical market
segment with interesting and extremely useful medical
applications in the treatment of numerous diseases such as
cancer and immunological disorders. The monoclonal
antibody purification process that is adopted should be
reliable, robust and must efficiently remove various
impurities such as aggregates, host cell protein, DNA, some
cell culture media additives, clipped/low molecular weight
species, viruses etc. The currently established purification
platform typically employs three chromatography steps with
Protein A chromatography as the initial capture step followed
by two polishing steps which may include cation exchange
chromatography,
anion
exchange
chromatography,
hydrophobic
interaction
chromatography
etc.
But
conventional packed bed chromatography suffers from
certain serious drawbacks. As a result, alternatives to
chromatography are also becoming important. In this paper,
various techniques used for the purification of monoclonal
antibodies are discussed and the merits and demerits of each
technique are critically analyzed besides discussing about the
typical applications of each of these methods.
KeywordsAqueous two phase systems, Chromatography,
Impurities, Monoclonal antibodies, Resin, Yield

I. INTRODUCTION
Monoclonal antibodies (mAbs) have emerged as an
extremely important and valuable class of therapeutic
products. They have been successfully introduced as
therapies to myriad diseases such as rheumatoid arthritis,
auto-inflammatory disease, colorectal cancer, allergic
asthma and multiple sclerosis [1],[2]. This is primarily due
to their high specificity and amazing versatility [3]. As a
result, monoclonal antibodies represent a rapidly growing
biotechnology field.
A pertinent concern is the development of a reliable
purification process that can efficiently remove the
different types of impurities in order to produce products
suitable for human use. Also, the loss of yield of the
product during purification should be minimum.

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International Journal of Emerging Technology and Advanced Engineering

Website: www.ijetae.com (ISSN 2250-2459, ISO 9001:2008 Certified Journal, Volume 3, Issue 10, October 2013)

Fig. 1: Commonly used chromatographic methods for monoclonal antibody purification and some novel alternatives

In Fig.1, HIC refers to Hydrophobic Interaction

Chromatography, IEC refers to Ion Exchange
Chromatography and ATPS refers to Aqueous Two Phase
Systems

There has been a lot of research to address the important

problem of aggregate formation during Protein A elution
and it has been demonstrated that the addition of stabilizers
such as arginine to the Protein A elution buffer can help
reduce aggregation [3].

II. DISCUSSION OF DIFFERENT CHROMATOGRAPHY

TECHNIQUES

B. Ion Exchange Chromatography (IEC)

It is the most common polishing step used in the
purification of monoclonal antibodies to remove the trace
amounts of impurities remaining after the Protein A
chromatography step. As the name suggests, Ion Exchange
Chromatography implies separation on the basis of charge.
The separation is on account of differential electrostatic
affinities of the biomolecules for a charged stationary phase
material [12]. It is a very common practice to classify ion
exchange stationary phases as strong and weak exchangers.
This is on the basis of the pH range over which these ion
exchangers retain their charge. Those ion exchangers which
have the ability to retain their charge over a wide pH range
are classified as strong exchangers while those which can
do so over a narrow pH range are known as weak
exchangers [11],[12]. Ion Exchange Chromatography is
classified into two categories: Cation Exchange
Chromatography and Anion Exchange Chromatography.

A. Protein A Chromatography
It is a mode of affinity chromatography that makes use
of the specific interactions which take place between the Fc
region of mAbs and immobilized protein A [3]. Protein A
chromatography is an unparalleled and versatile technique
since it allows the removal of more than 95% impurities in
a single step [8]. As a result, it is almost unanimously
selected as the first step (capture step) in the monoclonal
antibody purification process. Another remarkable feature
of this technique is its simplicity and ease of operation.
However, as mentioned earlier, the low pH elution
conditions used in Protein A chromatography are a serious
cause of concern since they can lead to aggregation and
loss of biological activity [11]. The leaching out of Protein
A in small amounts from its support matrix is also a major
problem since the leachate and its fragments are
immunotoxic [7].

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TABLE I
PROPERTIES OF SOME COMMON CATION EXCHANGE CHROMATOGRAPHY RESINS

Resin

Type

Vendor

Functional Group

Backbone

Particle Size
(in micron)

Fractogel EMD SO3-(M)

Strong

Merck

Sulfoisobutyl

Methacrylate

40-90

Fractogel EMD SO3-(S)

Strong

Merck

Sulfoisobutyl

Methacrylate

20-40

Fractogel EMD COO-(M)

Weak

Merck

Carboxyethyl

Methacrylate

40-90

ESHMUNO S

Strong

Merck

Sulfoisobutyl

Surface grafted rigid polyvinyl

ether hydrophilic polymer

50-120

CM Sepharose

Weak

GE Healthcare

Carboxymethyl

Cross linked agarose

45-165

Capto S

Strong

GE Healthcare

Sulfoethyl

Cross linked agarose with

dextran surface extender

90 (average)

Capto SP ImpRes

Strong

GE Healthcare

Sulfopropyl

High flow agarose

36-44

S Ceramic HyperD

Strong

Pall

Sulfopropyl

Polystyrene shell and hydrogel

50 (average)

POROS XS

Strong

Applied
Biosystems

Sulfopropyl

Cross-linked poly(styrenedivinylbenzene)

50 (average)

1)
Cation Exchange Chromatography: In Cation
Exchange Chromatography, negatively charged functional
groups such as carboxymethyl, sulfopropyl and
sulfoisobutyl are immobilized to the resin so that it has
affinity for positively charged ions (cations) [5]. It is an
extremely useful technique for removing certain impurities
such as aggregates, deamidated (acidic material), oxidized
species which would be present even after Protein A
chromatography [4]. Table I highlights the properties of
some commonly used cation exchange chromatography
resins.

2) Anion Exchange Chromatography:

It retains
negatively charged ions (anions) because the stationary
phase contains positively charged functional groups such as
Diethylaminoethyl (DEAE), Trimethylammoniumethyl
(TMAE) etc. It is conventionally operated in flow through
mode or bind and elute mode but recently a new mode of
anion exchange chromatography known as weak
partitioning mode has become popular which can provide
an enhanced clearance of impurities since the pH and
conductivity are selected in such a way that weaker binding
impurities can be removed more efficiently as compared to
the flow through mode [1]. Some commonly used anion
exchangers are Fractogel EMD DEAE(M), Q Hyper D and
Q Sepharose Fast Flow [12],[13]. The properties of some
common anion exchangers are depicted in Table II.

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TABLE II
PROPERTIES OF SOME COMMON ANION EXCHANGE CHROMATOGRAPHY RESINS

Resin

Type

Vendor

Functional Group

Backbone

Particle Size
(in micron)

Capto Q ImpRes

Strong

GE Healthcare

Quaternary amine

High-flow agarose

36-44

DEAE Sepharose Fast Flow

Weak

GE Healthcare

Diethylaminoethyl

6% cross-linked agarose

45-165

Q Sepharose Fast Flow

Strong

GE Healthcare

Quaternary amine

6% cross-linked agarose

45-165

Fractogel EMD DEAE(M)

Weak

Merck

Diethylaminoethyl

Methacrylate

40-90

Fractogel EMD TMAE(M)

Strong

Merck

Trimethylammoniumethyl

Methacrylate

40-90

C. Hydrophobic Interaction Chromatography (HIC)

The distinct advantage offered by hydrophobic
interaction chromatography over other techniques is its
ability to efficiently remove aggregates. It can also remove
other impurities such as host cell protein, DNA, leached
Protein A etc. making it a good candidate for being used
after ion exchange chromatography as a polishing step [5]
The basic principle is that proteins and other molecules
which have hydrophobic surface properties get
preferentially attached (bound) to HIC resins containing
ligands such as phenyl and butyl under aqueous conditions
with a high salt concentration in the buffer but get eluted
when a gradient of decreasing salt concentration is used
[14]. But there are some limitations associated with using
HIC in bind and elute mode which hinder its widespread
application. It has relatively lower yield compared to other
chromatography steps and the elution product pool may
contain fair amount of salt since high concentrations of
binding salts are typically required in order to achieve good
capacities [4], [15]. Recently, there has been a lot of work
on resin pore size optimization which can lead to a
considerable improvement in the binding capacity of HIC
resins [15].

D.

Multimodal
Chromatography
(Mixed
Mode
Chromatography)
As the name suggests, Multimodal Chromatography
possesses the unique ability to combine various types of
interactions such as hydrophobic interaction, hydrogen
bonding and ionic interaction within one single resin
[5],[16]. It can lead to dramatic improvements in the
monoclonal antibody purification process by providing
improved selectivity, new separation mechanisms, salt
tolerant adsorption and a remarkably high loading capacity
[17],[18],[19]. It can prove to be an extremely important
technique for removing aggregates and enhancing the
process efficiency for downstream processing of mAbs
[20]. In certain cases, using multimodal chromatography as
a purification step after Protein A chromatography may
help to reduce the number of chromatography steps from
three to two, thus increasing the productivity of the process
[21]. Examples of multimodal chromatographic resins are
Capto adhere, Capto MMC, HEA HyperCel, MEP
HyperCel and PPA HyperCel [19]. It is interesting that if
we can combine an externally controlled pH gradient with
such resins, we may be successful in carrying out certain
separations that cannot be achieved by conventional
methods [16]. But there are certain issues that need careful
study such as the complexity of interactions involved and
resin lifetime.

478

International Journal of Emerging Technology and Advanced Engineering

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TABLE III
SUMMARY OF THE VARIOUS CHROMATOGRAPHIC METHODS USED FOR MONOCLONAL ANTIBODY PURIFICATION

Technique

Importance

Distinct Advantages

Available Resins

Limitations

Protein A
chromatography

Primarily used as capture step to

remove DNA, host cell protein,
cell culture media components
and various other impurities

Extremely efficient, highly

selective, removal of most
of the impurities achieved
in a single step, very robust

MabSelect SuRe,
ProSep A High
Capacity, POROS
Mabcapture A, nProtein
A Sepharose 4 Fast
Flow

Elution at low pH leads to

aggregate formation, high cost of
resins, leached Protein A ligand
added as impurity

Cation
Exchange
Chromatography

Effective in removing both

process and product related
impurities such as leached Protein
A, host cell protein, aggregates

High step yield, relatively

less expensive resins, very
significant reduction of host
cell protein level, high
capacity

S Ceramic HyperD, CM
Ceramic HyperD, SP
Sepharose Fast Flow,
POROS XS, Capto SP
ImpRes

Sample preparation needs buffer

exchange if conductivity is not
low, partial insolubility of some
mAbs under low pH and low
conductivity conditions used for
achieving high binding capacity

Anion Exchange
Chromatography

Generally employed as the last

chromatography step in order to
remove residual host cell proteins,
DNA, endotoxin, leached Protein
A and viruses

Excellent removal of
endotoxin, high loading
capacity in flow through
mode, inexpensive resins

Capto Q ImpRes,
Fractogel EMD
TMAE(S), DEAE
Ceramic HyperD,
DEAE Sepharose Fast
Flow

Requirement for low loading buffer

conductivity, large column volume
needed for fast flow

Hydrophobic
Interaction
Chromatography

Used as a polishing step for

removing high molecular weight
aggregates, host cell protein

Very effective for aggregate

removal, easily removes
those contaminants which
are most difficult for other
chromatography techniques

Octyl Sepharose 4 Fast

Flow, Fractogel EMD
Phenyl(S), SOURCE
15ETH, Capto Butyl

High salt concentration in elution

product pool, relatively lower
binding capacity in bind and elute
mode, not very effective for
removal of DNA and leached
Protein A in flow through mode

Multimodal
Chromatography

Mostly used after the Protein A

chromatography step to
selectively remove DNA,
aggregates, host cell proteins and
viruses and can convert a
conventional three step
purification process to an
extremely productive two step
process

Resin can bind through

hydrogen bonding,
hydrophobic and
electrostatic interactions,
unique selectivity, stronger
binding, savings in
operational costs by using
only two chromatography
steps

MEP HyperCel, Capto

MMC, ESHMUNO
HCX, Capto adhere

Interactions involved are complex,

lack of understanding about nature
of binding, extensive optimization
required

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III. NOVEL ALTERNATIVES TO COLUMN
CHROMATOGRAPHY

Also, another big advantage is that membrane

chromatography allows us to use much higher flow rates
than column chromatography. Hydrophobic Interaction
Membrane Chromatography has been demonstrated to be
useful in simultaneously removing leached protein A and
aggregates from monoclonal antibodies, a remarkable
breakthrough indeed! [7].
However, this is just one side of the coin. Membrane
chromatography does suffer from certain limitations which
need to be overcome before the potential of this incredible
technique can be fully tapped. These include membrane
fouling, lower throughput on account of lower unit surface
areas, lower binding capacity, uneven flow distribution etc.
[5],[6],[29],[30]. We need to study more about the various
aspects of membrane chromatography and come up with
ingenious solutions to combat these challenging problems.

E. Aqueous Two Phase Systems (ATPS)

Aqueous two phase systems are spontaneously formed
when aqueous solutions of two mutually incompatible
components are mixed such as two polymers which are
structurally different or a polymer (typically PEG) and a
salt (generally citrate, phosphate or sulphate) above a
certain critical concentration [2],[22],[23]. Aqueous two
phase extraction can serve as an important and novel
technique for the purification of monoclonal antibodies
owing to its cost effectiveness, high capacity,
biocompatibility and scale up potential [22], [23]. The
selective partitioning between the two phases in ATPS is
governed by numerous factors which include intrinsic
properties such as surface hydrophobicity, charge,
conformational characteristics and system properties such
as pH, ionic strength, polymer type, salt type, concentration
of salt etc. [2],[24]. An amazing feature of this technique is
that it has the potential to integrate clarification,
concentration and partial purification of mAbs into just one
single step [25]. But certain challenges need to be
overcome before its full potential can be tapped such as the
problems associated with the handling and disposal of large
quantities of raw materials needed for the process and the
limited understanding we have about the complex
interactions between the different components in the
system [2],[10].

IV. CONCLUSION
Although a wide range of techniques are available for
the purification of monoclonal antibodies, most of them
rely on the use of Protein A chromatography as the capture
step followed by one or two polishing steps which are
generally selected based on the particular impurity
clearance challenge [1]. Chromatography has been the
workhorse of downstream processing primarily due to its
simplicity and high resolving power [2]. Protein A
chromatography is almost unanimously selected as the
initial capture step but it has certain serious drawbacks
which have attracted significant interest and lot of research
has been done to overcome these shortcomings as far as
possible. Cation exchange chromatography, anion
exchange chromatography and hydrophobic interaction
chromatography are generally used as polishing steps for
the removal of product and process related impurities and
viruses [5]. Hydrophobic Interaction Chromatography
(HIC) is very useful for the removal of aggregates. Anion
exchange chromatography provides excellent removal of
endotoxin while Cation exchange chromatography is
particularly good for removal of host cell protein besides
some other impurities. It has been demonstrated that
Multimodal chromatography, when used after Protein A
chromatography, can help reduce the number of
chromatographic steps for monoclonal antibody
purification from three to two, thus improving the yield and
shortening the process time [21]. However, extensive
optimization and proper understanding of the nature of
interactions are required before we can make the best use
of the amazing potential of Multimodal chromatography.

F. Membrane Chromatography
This is an innovative and special technique in which we
typically use microporous membranes consisting of a
polymeric substrate to which certain functional ligands are
coupled [5]. Membrane chromatography has a remarkable
potential and is rapidly emerging as an alternative to
conventional column chromatography. This is primarily
because membrane chromatography offers certain distinct
benefits. These include reduction of buffer consumption,
quick operation and low space requirement [26]. Here,
convection is the main mechanism of transport of
molecules to their binding sites with negligible pore
diffusion and hence, the total mass transfer resistance in
such a case is substantially lower than traditional
chromatography columns [27]. It eliminates the need for
column packing which is strenuous and unreliable [28].
This method helps us overcome the problem of high
pressure drop which is prevalent in conventional column
chromatography [27].

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phase
systems
(ATPS),
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