Application Note: Monitoring Protein Aggregation Using Dynamic Light Scattering

The study of protein aggregation encompasses a broad range of interactions and mechanisms. Many studies into this subject area investigate the aggregation of misfolded proteins, which is thought to be responsible for many degenerative diseases. Since aggregation typically leads to a physical change in protein size, particle size analysis has proven to be a useful experimental technique in this field.

Aggregation is a general term that encompasses several types of interactions or characteristics. Aggregates of proteins may arise from several mechanisms and may be classified in numerous ways, including soluble/insoluble, covalent/noncovalent, reversible/irreversible, and native/denatured. For protein therapeutics, the presence of aggregates of any type is typically considered to be undesirable because of the concern that the aggregates may lead to an immunogenic reaction (small aggregates) or may cause adverse events on administration (particulates).

Soluble aggregates refer to those that are not visible as discrete particles and that may not be removed by a filter with a pore size of 0.22 µm. Conversely, insoluble aggregates may be removed by filtration and are often visible to the unaided eye. USP <788> provides clear guidelines and limitations on the number of particles =10 µm and =25 µm in size that may be present in pharmaceutical preparations. Covalent aggregates arise from the formation of a chemical bond between 2 or more monomers. Reversible protein aggregation typically results from relatively weak noncovalent protein interactions. The reversibility is sometimes indicative of the presence of equilibrium between the monomer and higher order forms.

There are also many environmental factors that can lead to protein aggregation. Conditions such as temperature, protein concentration, pH, and the ionic strength may affect the amount of aggregate observed. Stresses to the protein such as freezing, exposure to air, or interactions with metal surfaces may result in surface denaturation, which then leads to the formation of aggregates.