CPB Protein Aggregation Special Issue
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 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. 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. 10, 461-463.

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.