| The aggregation of protein molecules into non-native assemblies in vivo can have profound pathological implications, as in the aggregation of amyloid-beta in Alzheimer's disease and the aggregation of alpha-synclein in Parkinson's disease. In the pharmaceutical industry, protein aggregation is routinely encountered during refolding, purification, sterilization, shipping, storage, and delivery. In addition to reducing efficacy, aggregates in parenterally administered proteins can cause adverse reactions in patients, such as immune response, anaphylactic shock, or even death. Stabilization of protein is particularly challenging because protein molecules are only marginally stable in aqueous solution.; We used recombinant human therapeutic proteins (granulocyte colony stimulating factor, platelet-activating factor acetylhydrolase, and interleukin-1 receptor antagonist) and amyloid disease-associated proteins (alpha-synuclein and immunoglobulin light chains) to probe the fundamental mechanism, thermodynamic driving forces, and kinetics by which proteins aggregate and by which varying solution conditions (e.g., temperature, pH, salts, cosolutes, preservatives, and surfactants) affect this process.; We found that protein aggregation involves at least two critical steps. The first requires perturbation to the protein's native structure to form an aggregation-competent intermediate, and proceeds through a structurally-expanded transition state. The second step involves the assembly of aggregation-competent intermediates to form aggregates. The energy barriers of the two steps are reflected in the values of free energy of unfolding (DeltaG unf) and osmotic second virial coefficient (B 22), respectively. Under conditions where conformational stability dominates, the first step is rate-limiting, and increasing Delta Gunf (e.g., by adding sucrose) decreases aggregation. In solution where colloidal stability is high, the second step is rate-limiting, and increasing repulsive interactions between proteins (e.g., by changing pH and ionic strength) is effective at reducing aggregation.; In addition to conformational and colloidal stability, protein surface activity can emerge to be an important factor controlling aggregation. Protein aggregation is inherently a nucleation and growth process during which soluble proteins assemble and precipitate into insoluble aggregates. Conditions affecting either nucleation or growth can alter protein aggregation behavior. High surface activity coupled with favorable protein-surface contacts can drive aggregation by inducing heterogeneous nucleation. (Abstract shortened by UMI.)... |
