Jurnal Imunologi
Jurnal Imunologi
Jurnal Imunologi
Website: www.ijetae.com (ISSN 2250-2459, ISO 9001:2008 Certified Journal, Volume 3, Issue 10, October 2013)
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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Fig. 1: Commonly used chromatographic methods for monoclonal antibody purification and some novel alternatives
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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Resin
Type
Vendor
Functional Group
Backbone
Particle Size
(in micron)
Strong
Merck
Sulfoisobutyl
Methacrylate
40-90
Strong
Merck
Sulfoisobutyl
Methacrylate
20-40
Weak
Merck
Carboxyethyl
Methacrylate
40-90
ESHMUNO S
Strong
Merck
Sulfoisobutyl
50-120
CM Sepharose
Weak
GE Healthcare
Carboxymethyl
45-165
Capto S
Strong
GE Healthcare
Sulfoethyl
90 (average)
Capto SP ImpRes
Strong
GE Healthcare
Sulfopropyl
36-44
S Ceramic HyperD
Strong
Pall
Sulfopropyl
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.
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Resin
Type
Vendor
Functional Group
Backbone
Particle Size
(in micron)
Capto Q ImpRes
Strong
GE Healthcare
Quaternary amine
High-flow agarose
36-44
Weak
GE Healthcare
Diethylaminoethyl
6% cross-linked agarose
45-165
Strong
GE Healthcare
Quaternary amine
6% cross-linked agarose
45-165
Weak
Merck
Diethylaminoethyl
Methacrylate
40-90
Strong
Merck
Trimethylammoniumethyl
Methacrylate
40-90
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.
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Technique
Importance
Distinct Advantages
Available Resins
Limitations
Protein A
chromatography
MabSelect SuRe,
ProSep A High
Capacity, POROS
Mabcapture A, nProtein
A Sepharose 4 Fast
Flow
Cation
Exchange
Chromatography
S Ceramic HyperD, CM
Ceramic HyperD, SP
Sepharose Fast Flow,
POROS XS, Capto SP
ImpRes
Anion Exchange
Chromatography
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
Hydrophobic
Interaction
Chromatography
Multimodal
Chromatography
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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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