Aggregation Of Therapeutic Proteins: Causes, Control, Consequences And Challenges

By Dr. John Carpenter, University Of Colorado, School Of Pharmacy

Aggregation is one of the most problematic and challenging issues for development of safe and effective therapeutic protein products. Aggregate amounts and types are critical quality issues. Moreover, even relatively low levels of aggregates potentially can cause immunogenicity in patients. And aggregate formation can occur at any step in protein production and use, from fermentation to delivery to the patient. Furthermore, there can be major gaps in the analytical approaches used for aggregates.

Proteins usually aggregate from partially unfolded molecules, which can be part of the native state ensemble of substates. Even when a formulation is developed that greatly stabilizes the native state, a significant amount of aggregates can form; especially over pharmaceutically-relevant time scales. Furthermore, exposure to interfaces (e.g., air-liquid and solid-liquid) can induce aggregation. Such exposure can occur during processing steps, as well as in the final product container.

Protein aggregation in aqueous solution can be slowed by maximizing the stability of the native conformation (“conformational stability) and the energetics of intermolecular repulsion (“colloidal stability”). Because of the importance of charge-charge interactions in protein aggregation, determining the optimum pH is a critical early step in formulation development. Optimized conformational and colloidal stability may help to reduce interfaceinduced aggregation, but often a non-ionic surfactant is also required to minimize this type of damage. Particle formation caused by foreign materials shed from filling pumps and containers/closures is one of the most difficult problems for formulation development. Specific studies are needed to assess and optimize formulation compatibility with pumps and container/closure, and additional formulation studies are needed if these systems are changed for a given product.