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Current
Pharmaceutical Biotechnology
ISSN: 1389-2010
Current Pharmaceutical Biotechnology
Volume 10, Number 4, June 2009
Contents
Aggregation Detection and Removal Biopharmaceutical
Proteins
Guest Editors: Pete Gagnon &
Tsutomu Arakawa
Editorial Pp. 347
[PMID:
19519408 PubMed - indexed for MEDLINE]
Chapter 1: Mechanisms of Protein Aggregation Pp.
348-351
John S. Philo and Tsutomu Arakawa
[Abstract] [Purchase
Article] [PMID:
19519409 PubMed - indexed for MEDLINE]
Chapter 2: Immunogencity Assessment of
Therapeutic Proteins and Peptides Pp. 352-358
Arunan Kaliyaperumal and Shuqian Jing
[Abstract] [Purchase
Article] [PMID:
19519410 PubMed - indexed for MEDLINE]
Chapter 3.1: A Critical Review of Methods
for Size Characterization of Non-Particulate Protein Aggregates
Pp. 359-372
John S. Philo
[Abstract] [Purchase
Article] [PMID:
19519411 PubMed - indexed for MEDLINE]
Chapter 3.2: A Critical Review of Analytical
Methods for Subvisible and Visible Particles Pp.
373-381
Linda Narhi, Yijia Jiang, Shawn Cao, Kalman Benedek
and Deborah Shnek
[Abstract] [Purchase
Article] [PMID:
19519412 PubMed - indexed for MEDLINE]
Chapter 3.3: Separation and Characterization
of Protein Aggregates and Particles by Field Flow Fractionation
Pp. 382-390
Shaw Cao, Joey Pollastrini and Yijia
Jiang
[Abstract] [Purchase
Article] [PMID:
19519413 PubMed - indexed for MEDLINE]
Chapter 3.4: Application of Vibrational
Spectroscopy to the Structural Characterization of Human Monoclonal
Antibody and Its Aggregate Pp. 391-399
Cynthia H. Li and Tiansheng Li
[Abstract] [Purchase
Article] [PMID:
19519414 PubMed - indexed for MEDLINE]
Chapter 4.1: Effect of Additives on Protein
Aggregation Pp. 400-407
Hiroyuki Hamada, Tsutomu Arakawa and
Kentaro Shiraki
[Abstract] [Purchase
Article] [PMID:
19519415 PubMed - indexed for MEDLINE]
Chapter 4.2: Suppression of Protein Aggregation
by L-Arginine Pp. 408-414
Christian Lange and Rainer Rudolph
[Abstract] [Purchase
Article] [PMID:
19519416 PubMed - indexed for MEDLINE]
Chapter 4.3: To be Excluded or to Bind, that is the Question:
Arginine Effects on Proteins Pp. 415-420
Makoto Nakakido, Motonori Kudou, Tsutomu Arakawa and
Kouhei Tsumoto
[Abstract] [Purchase
Article] [PMID:
19519417 PubMed - indexed for MEDLINE]
Chapter 5.1: Ion Exchange Chromatography of Proteins
and Clearance of Aggregates Pp. 421-426
Y. Yigzaw, P. Hinckley, A. Hewig and
G. Vedantham
[Abstract] [Purchase
Article] [PMID:
19519418 PubMed - indexed for MEDLINE]
Chapter 5.2: Recent Advancement in Application of
Hydrophobic Interaction Chromatography for Antibody Aggregate
Removal in Industrial Purification Process Pp. 427-433
Yuefeng Lu, Brian Williamson and Ronald
Gillespie
[Abstract] [Purchase
Article] [PMID:
19519419 PubMed - indexed for MEDLINE]
Chapter 5.3: IgG Aggregate Removal by Charged-Hydrophobic
Mixed Mode Chromatography Pp. 434-439
Pete Gagnon
[Abstract] [Purchase
Article] [PMID:
19519420 PubMed - indexed for MEDLINE]
Chapter 5.4: Antibody Aggregate Removal by Hydroxyapatite
Chromatography Pp. 440-446
Pete Gagnon and Kevin Beam
[Abstract] [Purchase
Article] [PMID:
19519421 PubMed - indexed for MEDLINE]
Chapter 5.5: Application of High Hydrostatic Pressure
to Dissociate Aggregates and Refolding Proteins Pp.
447-455
Matthew B. Seefeldt, Mary S. Rosendahl, Jeffrey
L. Cleland and Lyndal K. Hesterberg
[Abstract] [Purchase
Article] [PMID:
19519422 PubMed - indexed for MEDLINE]
Chapter 6.1: Stress-Free Chromatography: Affinity
Chromatography Pp. 456-460
Tsutomu Arakawa, Yoshiko Kita, Haruna Sato and
Daisuke Ejima
[Abstract] [Purchase
Article] [PMID:
19519423 PubMed - indexed for MEDLINE]
Chapter 6.2: Stress-Free Chromatography: IEC and HIC
Pp. 461-463
Tsutomu Arakawa, Yoshiko Kita and Daisuke
Ejima
[Abstract] [Purchase
Article] [PMID:
19519424 PubMed - indexed for MEDLINE]
Abstracts
[Back to top]
[PMID:
19519408 PubMed - indexed for MEDLINE]
Editorial:
Protein aggregation is a major concern in the field of
recombinant and plasma-derived pharmaceutical products. Aggregation
of proteins occurs via a number of different mechanisms, as
briefly summarized in Chapter 1. Aggregation has long been
recognized as a contributor to the problem of product immunogenicity
[1, 2]. The immunogenicity problem of protein biopharmaceuticals
and its relation to aggregates is briefly reviewed in Chapter
2. The ability to detect whether a particular protein is immunogenic
or not depends greatly on the sensitivity and specificity
of the assays used to measure the antibodies generated against
the protein in patient serum. It is also important to determine
whether the generated antibodies neutralize the activity of
both exogenous and endogenous protein. Thus, assays to distinguish
non-neutralizing from neutralizing antibodies are also needed.
The development of assays for antibody detection and characterization
is also described in Chapter 2.
Aggregates comprise a wide range of different sizes and structures.
Detailed characterization may shed light on the cause of their
formation, provide correlations between the type of aggregates
and their respective immunogenicity, and help in identifying
purification methods suitable for aggregate removal. A number
of techniques are available for the analysis of aggregate
size. Aggregates may be formally grouped into 2 classes, i.e.,
particulates and “soluble” aggregates. Entirely
different techniques are used to detect these different classes
of aggregates. For particulates particle counting and microscopic
techniques are typically used. For soluble aggregates SEC
is the most critical technique for quantitation and size characterization,
but matrix-free techniques such as dynamic light scattering,
analytical ultracentrifugation, and field flow fractionation
offer valuable complements [3-5]. Three chapters, Chapter
3.1, 3.2 and 3.3, are dedicated to characterization of aggregate
size. FT-IR has been the main technique for the analysis of
secondary structure of protein aggregates. Raman spectroscopy
can be also used to measure the secondary structure as well
as tertiary structure of both particulates and soluble aggregates,
as described in Chapter 3.4.
Low molecular weight agents are sometimes added to protein
solutions to suppress aggregation during production, purification,
storage and freezing, or lyophilization. A comprehensive review
of these additives is given in Chapter 4.1. Arginine appears
to be the most effective and versatile in suppression of protein
aggregation. The discovery that arginine is an aggregation
suppressor and its mechanism are described in Chapters 4.2
and 4.3.
It may not be possible in all cases to completely prevent
or suppress aggregation. This makes effective removal methods
essential for overall aggregate management. Size exclusion
chromatography seems a natural choice since it is so widely
used for aggregate measurement. No chapter about this approach
is included here but it has been discussed occasionally in
the literature [6, 7]. Besides its effectiveness for aggregate
removal, it offers the benefit of buffer exchanging the product
into final formulation, and the chief development tasks are
largely limited to determination of loading capacity and flow
rate. Its low capacity and flow rate however impose an economic
burden on industrial applications, and the resultant dilution
of product is usually highly undesirable. Consequently the
trend has been toward the use of adsorptive chromatography
methods. Chapters 5.1-5.4 address removal of soluble aggregates
by ion exchange, hydrophobic interaction, mixed ion exchange/hydrophobic
ligands, and hydroxyapatite chromatography. The focus of these
chapters is primarily on monoclonal antibodies because they
have been more thoroughly studied and represent such a large
fraction of biotechnology products currently under development,
but similar lessons should also apply to process chromatography
of other protein products.
Aggregate removal naturally entails a loss of product, not
only in the form of the aggregates themselves, but also in
the form of neighboring native product fractions that must
be sacrificed to ensure adequately low aggregate content in
the final product. Application of high pressure has been revealed
as an effective means to reverse aggregation and restore proteins
to their native conformation. Examples describing the technology
and its prospects for industrial application are discussed
in Chapter 5.5.
Ironically, aggregation may be caused by elements of the protein
purification process itself. Most fractionation methods involve
exposure to ranges of pH, conductivity, or shear that can
impose stress on a protein. In some cases, chromatography
ligands themselves can compound that stress. It would be preferable
if these stresses could be eliminated, or at least ameliorated.
The last two chapters, Chapter 6.1 and 6.2, explore options
for minimizing purification-associated aggregation through
“stress-free” chromatography.
REFERENCES
[1] Rosenberg, A.S. (2006) AAPS J., 8,
E501-E507.
[2] Schellekens, H. (2008) Biotechnol. Annu. Rev.,
14, 191-202.
[3] Giddings, J.C. (1993) Science, 260,
1456-1465.
[4] Arakawa, T.; Philo, J.S.; Ejima, D.; Sato, H. and Tsumoto,
K. (2007) BioProcess Int., 5, 52-70.
[5] Williams, S.K. and Lee, D. (2006) J. Sep. Sci.,
29, 1720-1732.
[6] Vandeveyer, C. and Freitag, R. (2004) in Antibodies:
production and purification. (Subramanian, G., Ed.),
Kluwer Academic/Plenum Publishers, New York, Vol. 9.
pg. 133.
[7] Aldington, S. and Bonnerjea, J. (2006) J. Chromatogr.
B, 848, 64-78
Pete Gagnon
Guest Editor
Current Pharmaceutical Biotechnology
Chief Scientific Officer, Validated Biosystems
240 Avenida Vista Montana, Ste. 7F, San Clemente, CA 92672
USA
Tel: 949-276-7477; Fax: 949-606-1904
E-mail: pete@validated.com
Tsutomu Arakawa
Guest Editor
Current Pharmaceutical Biotechnology
President, Alliance Protein Laboratories
3957 Corte Cancion, Thousand Oaks, CA 91360
USA
Tel: 805-388-1074; Fax: 805-388-8929
E-mail: tarakawa2@aol.com
[Back to top] [Purchase
Article] [PMID:
19519409 PubMed - indexed for MEDLINE]
Mechanisms of Protein Aggregation
John S. Philo and Tsutomu
Arakawa
Aggregation or reversible self-association of protein
therapeutics can arise through a number of different mechanisms.
Five common aggregation mechanisms are described and their
relations to manufacturing processes to suppress and remove
aggregates are discussed.
[Back to top]
[Purchase Article] [PMID:
19519410 PubMed - indexed for MEDLINE]
Immunogencity Assessment of Therapeutic Proteins
and Peptides
Arunan Kaliyaperumal and Shuqian
Jing
Assessment of immunogenicity is a major aspect in evaluating
the safety of biological therapeutic proteins. It is important
to evaluate the immunogenic potential of the biologics in
an appropriate fashion using clearly defined strategy and
clinical trials. The studies must include the appropriate
risk assessment procedures using validated methods. The immune
responses against the therapeutic biologics can be studied
using various methodologies. These include enzyme linked immunoassays
(ELISA), surface plasmon resonance (SPR), chemiluminescence,
and flowcytometry assays for binding antibodies and cell based
assays for neutralizing antibodies. The immune responses to
the biologics can widely vary in various cross section of
the population, thus a combination of techniques are necessary
to fully evaluate the immunogenic potential of the biologics.
This review outlines various commonly used technology platforms,
its merits and shortcomings for the evaluation of the immune
responses.
[Back to top]
[Purchase Article] [PMID:
19519411 PubMed - indexed for MEDLINE]
A Critical Review of Methods for Size Characterization of
Non Particulate Protein Aggregates
John S. Philo
Although size exclusion chromatography (SEC) has been,
and will continue to be, the primary analytical tool for characterization
of the content and size distribution of non-particulate aggregates
in protein pharmaceuticals, regulatory concerns are driving
increased use of alternative and complementary methods such
as analytical ultracentrifugation and light scattering techniques.
This review will highlight and critically review the capabilities,
advantages, and drawbacks of SEC, analytical ultracentrifugation,
and light scattering methods for characterizing aggregates
with sizes below about 0.3 microns. The physical principles
of the biophysical methods are briefly described and examples
of data for real samples and how that data is interpreted
are given to help clarify capabilities and weaknesses.
[Back to top]
[Purchase Article] [PMID:
19519412 PubMed - indexed for MEDLINE]
A Critical Review of Analytical Methods for Subvisible and
Visible Particles
Linda Narhi, Yijia Jiang, Shawn Cao, Kalman
Benedek and Deborah Shnek
The subvisible and visible particles present in a solution
are often classified based on size, and are quantified by
the actual number of particles present rather than by weight
or molar amounts. The analysis of these particles in protein
therapeutics are governed by compendial methods and the regulatory
agencies, and the methods available to measure them originally
evolved focusing on potential safety issues, including capillary
occlusion and immunogenicity, that might arise from their
presence. Ultracentrifugation, size exclusion chromatography,
etc., discussed in previous articles, can be used to analyze
aggregates of less than 0.10 microns. This article will focus
on methods for analyzing and quantitating sub visible particles
(SbVP) of 2 microns or larger. At the present time there is
no routine method for quantitating sub visible particles (SbVP)
between 0.1 microns and 2 microns. The most common technique
for quantitating the amount of subvisible particles between
2 and 100 microns is the light obscuration method. This technique
can determine size and amount of particles, but cannot differentiate
between the types of particles, such as protein particles,
foreign material, micro bubbles or silicone oil droplets,
that can be present in protein solutions. The difficulties
in adapting this method, originally developed for small molecule
drugs for IV administration, to protein therapeutics delivered
subcutaneously is discussed. The flow imaging techniques can
determine morphology and optical characteristics of the particles,
but still not identify the chemical composition. Other methods
that can also be used, but are applicable for characterization
purposes only, are discussed. The primary method for quantitating
visible particles is visual inspection, a method that can
be subjective and relies on adequate training of the human
inspectors. Automated methods for visible particle determination
are being developed. Identification of the chemical composition
of isolated particles greater than about 50 microns is possible
using several micro-spectroscopic methods, and these will
also be discussed.
[Back to top]
[Purchase Article] [PMID:
19519413 PubMed - indexed for MEDLINE]
Separation and Characterization of Protein Aggregates and
Particles by Field Flow Fractionation
Shaw Cao, Joey Pollastrini and
Yijia Jiang
Field flow fractionation (FFF) is a technique that holds
great promise for the analysis and characterization of protein
aggregates and particles, due to its wide dynamic range and
matrix-free separation mechanism. FFF can be routinely used
to achieve good monomer-oligomer separation and quantification
for a variety of protein types, and is a reasonable choice
for an orthogonal method for size exclusion chromatography
and analytical ultracentrifugation. Quantifying sub-micrometer
particles in protein therapeutics is a potential of the FFF
technique that is yet to be realized, due to the lack of detection
with sufficient sensitivity. In this article the effect of
several important parameters on the optimization of FFF analyses
are explored, and the strengths, weaknesses, and potential
new applications of the technique are discussed.
[Back to top]
[Purchase Article] [PMID:
19519414 PubMed - indexed for MEDLINE]
Application of Vibrational Spectroscopy to the Structural
Characterization of Human Monoclonal Antibody and Its Aggregate
Cynthia H. Li and Tiansheng
Li
Aggregation is often the major issue during formulation
and manufacturing development of therapeutic proteins, in
particular human monoclonal antibody. Currently, there is
a lack of structural information of aggregates of such large
protein as human antibodies, due to the large molecular sizes
of the aggregates. In this article, we shall discuss the application
of vibrational spectroscopies including FT-IR, Raman and Raman
Optical Activity (ROA), to characterize the structures of
various types of monoclonal antibody aggregates formed under
different stresses. Two different classes of human monoclonal
antibodies, namely IgG1 and IgG2, have been subjected to this
structural investigation. The common stresses leading to antibody
aggregation, mis-folding or unfolding during manufacturing
and formulation include exposure to acidic pHs, heat and shear
stress. The effect of different types of stresses on the structure
and aggregate formation of human monoclonal antibodies has
been investigated by employing vibrational spectroscopy. While
data present only monoclonal antibody, the same technology
can be used for any protein aggregates.
[Back to top]
[Purchase Article] [PMID:
19519415 PubMed - indexed for MEDLINE]
Effect of Additives on Protein Aggregation
Hiroyuki Hamada, Tsutomu Arakawa and
Kentaro Shiraki
This paper overviews solution additives that affect protein
stability and aggregation during refolding, heating, and freezing
processes. Solution additives are mainly grouped into two
classes, i.e., protein denaturants and stabilizers. The former
includes guanidine, urea, strong ionic detergents, and certain
chaotropic salts; the latter includes certain amino acids,
sugars, polyhydric alcohols, osmolytes, and kosmotropic salts.
However, there are solution additives that are not unambiguously
placed into these two classes, including arginine, certain
divalent cation salts (e.g., MgCl2)
and certain polyhydric alcohols (e.g., ethylene glycol). Certain
non-ionic or non-detergent surfactants, ionic liquids, amino
acid derivatives, polyamines, and certain amphiphilic polymers
may belong to this class. They have marginal effects on protein
structure and stability, but are able to disrupt protein interactions.
Information on additives that do not catalyze chemical reactions
nor affect protein functions helps us to design protein solutions
for increased stability or reduced aggregation.
[Back to top]
[Purchase Article] [PMID:
19519416 PubMed - indexed for MEDLINE]
Suppression of Protein Aggregation by L-Arginine
Christian Lange and Rainer
Rudolph
L-Arginine is one of the most commonly used and most
generally applicable suppressors of protein aggregation. Its
effect as enhancer of in vitro protein refolding
was serendipitously discovered two decades ago. This article
aims at giving a brief overview about the discovery of the
arginine effect, the range of its applications that have been
explored over the past two decades, and of the current state
of the discussion regarding the mechanisms responsible for
the action of L-arginine as suppressor of aggregation.
[Back to top]
[Purchase Article] [PMID:
19519417 PubMed - indexed for MEDLINE]
To be Excluded or to Bind, that is the Question: Arginine
Effects on Proteins
Makoto Nakakido, Motonori Kudou, Tsutomu Arakawa and
Kouhei Tsumoto
In spite of its wide application to protein refolding,
purification, and storage, we have not yet addressed a general
solution to the mechanism of the effects of arginine hydrochloride
on proteins. To elucidate the mechanism of the effects on
proteins, several attempts have been reported. In this review,
we would review the attempts from thermodynamic and kinetic
viewpoints.
[Back to top]
[Purchase
Article] [PMID:
19519418 PubMed - indexed for MEDLINE]
Ion Exchange Chromatography of Proteins and Clearance
of Aggregates
Y. Yigzaw, P. Hinckley, A. Hewig and
G. Vedantham
Clearance of product related aggregates in therapeutic
proteins is a major focus of purification process development.
A typical purification process will have one or two chromatographic
steps that remove these product related aggregates to an acceptable
level. Both cation exchange and anion exchange chromatography
can provide robust clearance of aggregates. The primary factors
that are critical for aggregate clearance are: resin chemistry,
binding and elution condition, peak collection and column
load factor. This review covers how these factors can be optimized
to increase the effectiveness of ion exchange chromatography
in removing aggregates.
[Back to top]
[Purchase
Article] [PMID:
19519419 PubMed - indexed for MEDLINE]
Recent Advancement in Application of Hydrophobic
Interaction Chromatography for Antibody Aggregate Removal
in Industrial Purification Process
Yuefeng Lu, Brian Williamson and Ronald
Gillespie
Hydrophobic interaction chromatography (HIC) is a classic
purification tool applied in protein and antibody, laboratory
and industrial production process. It has been mainly used
for the removal of both product-related impurities such as
aggregates, as well as process contaminants such as host cell
proteins. This review will focus on the recent development
of HIC in its applications in the industrial purification
processes. The process economy and requirements of high product
purity and quality have driven much of the recent advancement
in HIC chromatography in terms of increased throughput and
enhanced selectivity or resolution. Meanwhile, high throughput
screening (HTS), design of experiments (DoE) and platform
approach for process development have been applied to shorten
the development time. The throughput improvement has been
achieved through new resins with increased binding capacity,
using dual salts for load conditioning, and operating in the
flow-through mode. In addition, hydrophobic interaction membrane
filter chromatography technology reduces bed volumes and buffer
usage and potentially improves process throughput by reducing
cycle time. Selectivity and/or resolution enhancements have
been achieved through optimization of operation parameters
such as temperature and efforts such as application of solvent
additives.
[Back to top]
[Purchase
Article] [PMID:
19519420 PubMed - indexed for MEDLINE]
IgG Aggregate Removal by Charged-Hydrophobic Mixed
Mode Chromatography
Pete Gagnon
Charged-hydrophobic mixed mode chromatography methods
have been applied to antibody purification for decades and
have focused more recently on the specific task of aggregate
removal. They exploit various combinations of alkyl and aromatic
hydrophobic groups with positively and/or negatively charged
residues. Charge and hydrophobicity remain relatively constant
as function of pH for some ligands; one or both vary for others.
All of these compound selectivities and their associated elution
strategies are intended to achieve purification of native
IgG through preferential retention of aggregates. This review
focuses on the two members of this family that have shown
the most promise for aggregate removal: MEP HyperCel™
and Capto™
adhere. It defines how they work, how they interact with various
classes of biomolecules, how those interactions are controlled
by different elution strategies, and how to determine which
may be most effective for a particular antibody. Consideration
is also given to their specific strengths and limitations
from an industrial perspective.
[Back to top]
[Purchase Article] [PMID:
19519421 PubMed - indexed for MEDLINE]
Antibody Aggregate Removal by Hydroxyapatite Chromatography
Pete Gagnon and Kevin Beam
Hydroxyapatite (HA) has proven in recent years to be
one of the most versatile and powerful methods for removing
aggregates from antibody preparations. It is effective with
IgA, IgG and IgM, and it reduces aggregate levels from above
60% to less than 0.1%. Three basic elution strategies have
evolved, one that removes aggregates from a modest proportion
of clones, another from the majority, and one that appears
to be universally effective. Each has distinct development
and process ramifications. This review defines what HA is,
how it interacts with various classes of biomolecules, how
those interactions are controlled by different elution strategies,
and how to determine which approach may be most effective
for a particular antibody. Consideration is also given to
HA’s specific strengths and limitations from an industrial
perspective.
[Back to top]
[Purchase Article] [PMID:
19519422 PubMed - indexed for MEDLINE]
Application of High Hydrostatic Pressure to Dissociate Aggregates
and Refolding Proteins
Matthew B. Seefeldt, Mary S. Rosendahl, Jeffrey
L. Cleland and Lyndal K. Hesterberg
Non-denaturing pressures of around 2000 bar are effective
for eliminating and refolding protein aggregates and may be
applicable in various phases of protein manufacturing to decrease
aggregate levels in products and improve process yields. Lower
aggregate levels can result in reduced immunogenicity of proteins
and enable the correct refolding of proteins that might not
be recovered with traditional techniques. High pressure treatment
can also be used to conduct selective PEGylation and protease
cleavage reactions while minimizing protein aggregation. High
pressure processes have been used in the food industry for
over 50 years and large scale (300 L) systems are commercially
available, enabling production of proteins on the kilogram
scale. This review summarizes the utility of high pressure
refolding to remove and refold protein aggregates, enhance
therapeutic proteins, and facilitate manufacturing improvements
at industrial scales.
[Back to top]
[Purchase
Article] [PMID:
19519423 PubMed - indexed for MEDLINE]
Stress-Free Chromatography: Affinity Chromatography
Tsutomu Arakawa, Yoshiko Kita, Haruna Sato and
Daisuke Ejima
A number of approaches are available in minimizing aggregation
of the final protein products. This chapter describes one
such approach, i.e., an attempt to avoid stressful conditions
that may eventually lead to protein aggregation. Affinity
chromatography uses specific interaction between protein to
be purified and ligand attached to the column. Due to high
affinity, dissociation of such interaction and hence elution
often require harsh solvent conditions. Ion exchange and hydrophobic
interaction chromatography also pose certain stressful conditions
on proteins. Here we describe development of mild elution
buffer using arginine. This chapter covers Protein-A, dye,
Protein-A mimetic and antigen affinity chromatography.
[Back to top]
[Purchase
Article] [PMID:
19519424 PubMed - indexed for MEDLINE]
Stress-Free Chromatography: IEC and HIC
Tsutomu Arakawa, Yoshiko Kita and Daisuke
Ejima
Ion exchange chromatography (IEC) poses stresses on proteins
in both binding and elution steps. Proteins often bind to
the column with high affinity, resulting in concentration
of the protein upon binding. Elution often requires high salt
concentration, leading to high protein concentration with
high salt concentration. Although hydrophobic interaction
chromatography (HIC) involves weak interaction, salting-out
salts are used for binding. These conditions may cause protein
aggregation. This short article describes an approach to reduce
such aggregation in IEC and HIC. This was achieved by adding
small amount of salt or arginine in the loading sample or
elution solvent, resulting in elution of proteins with less
aggregation or higher recovery.
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