Aggregation Analysis of Therapeutic Proteins, Part 1: General Aspects and Techniques for Assessment

stable in solution, undergoing conformational changes due to various stresses during purification, processing and storage. Such stresses can include elevated temperature, shear strain, surface adsorption, and high protein concentration (1). Structurally altered proteins have a strong tendency to aggregate, often leading to eventual precipitation (2). Irreversible aggregation is a major problem for long-term storage stability of therapeutic proteins and for their shipping and handling. Aggregation problems have been implicated in adverse reactions and other safety issues since the beginning of clinical applications of protein pharmaceuticals. Immunoglobulin aggregates have long been known to cause anaphylactoid reactions (3, 4). In recent years, a serious concern about aggregation was raised by an upsurge in incidents of pure red-cell aplasia (PRCA) in patients receiving recombinant erythropoietin, although it appears that aggregation is not involved in PRCA (5, 6). To minimize such risks from therapeutic proteins in clinical applications, formulations must be optimized to reduce aggregation during storage, handling, and shipping (7). Size-exclusion chromatography (SEC) has been a workhorse for detecting and quantifying protein aggregation (8, 9). But SEC is often questioned because of possible loss of proteins (soluble aggregates, in particular) by their nonspecific binding to the columns (10). Native gels have also been used to observe protein aggregation, but only qualitatively. Column-free techniques such as analytical ultracentrifugation (AUC), field-flow fractionation (FFF), and dynamic light scattering (DLS) now find increasing application in aggregation analysis. Here, we review advantages and disadvantages of various techniques for assessing protein aggregation. In part 1, we discuss general aspects of protein aggregation and present two techniques mentioned above: native gel electrophoresis and SEC.