Editorial [Hot Topic: Aggregation Detection and Removal Biopharmaceutical Proteins (Guest Editors: Pete Gagnon & Tsutomu Arakawa)]

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.