Current Pharmaceutical Biotechnology - Volume 10, Issue 4, 2009

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.