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 revie Read More

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 studi Read More

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 rev Read More

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 ca Read More

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 an Read More

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 Read More

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 kosmotropi Read More

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 regar Read More

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 ar Read More

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 Read More

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 bo Read More

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 Read More

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 Read More

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 re Read More

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 use Read More