Potential inaccurate quantitation and sizing of protein aggregates by size exclusion chromatography: essential need to use orthogonal methods to assure the quality of therapeutic protein products.

摘要

The levels, types, and sizes of aggregates in therapeutic protein products are important quality attributes that have received considerable attention from the biotechnology industry and regulatory agencies. In the context of therapeutic proteins, the term "aggregates" typically refers to assemblies of protein molecules that are considered degradation products and can include a range of sizes (from dimers to visible particles) and types (e.g., covalently cross-linked or noncovalent). Aggregation can occur at all steps in the manufacturing process, during product shipping and storage and even during administration to the patient (e.g., due to interactions with intrave- nous tubing).3 Moreover, aggregates of therapeutic proteins may cause adverse effects in patients such as injection site reactions, anaphylaxis, and immuno-genicity, with consequences ranging from patient discomfort to permanent harm and potentially death. Importantly, the amount, type, size and other characteristics (e.g., the conformation of protein molecules within the aggregate) of aggregates needed to induce adverse effects cannot be predicted for a given product. It is thought, however, that even trace amounts (e.g., <1% by mass) of certain aggregates might be responsible for adverse effects in patients, and that larger-sized aggregates are more prone to induce immunogenicity than smaller oligomers. Therefore, tremendous effort is put into minimizing protein aggregation during manufacturing steps and in the final formulation/dosage form.