A.P.L.-authored articles in June 2009
special issue of Current Pharmaceutical Biotechnology on protein aggregation,
removal of aggregates, and prevention of aggregates (volume 10, issue 4):
[PDF files are available for these,
but via e-mail request only.]
Gagnon, P. and Arakawa, T. (2009).
Hot topic: Aggregation detection and removal in biopharmaceutical proteins
(editorial). Curr. Pharm. Biotechnol. 10, 347.
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. 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
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.
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. 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.
(note: the above is the full text of this article)
Philo, J. S. and Arakawa, T. (2009).
Mechanisms of protein aggregation. Curr. Pharm. Biotechnol. 10, 348-351.
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.
Philo, J. S. (2009). A critical
review of methods for size characterization of non-particulate protein
aggregates. Curr. Pharm. Biotechnol. 10, 359-372.
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.
Hamada, H., Arakawa, T., and Shiraki,
K. (2009). Effect of additives on protein aggregation. Curr. Pharm.
Biotechnol. 10, 400-407.
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., MgCl(2)) 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.
Nakakido, M., Kudou, M., Arakawa, T.,
and Tsumoto, K. (2009). To be excluded or to bind, that is the question:
Arginine effects on proteins. Curr. Pharm. Biotechnol. 10, 415-420.
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.
Arakawa, T., Kita, Y., Sato, H., and
Ejima, D. (2009). Stress-free chromatography: affinity chromatography. Curr.
Pharm. Biotechnol. 10, 456-460.
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.
Arakawa, T., Kita, Y., and Ejima, D.
(2009). Stress-free chromatography: IEC and HIC. Curr. Pharm. Biotechnol.
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
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copyright 2009 Bentham Science Publishers)