Mechanistic Complexity of Subvisible Particle Formation: Links to Protein Aggregation are Highly Specific

There is little knowledge available on the mechanistic features of the protein aggregation pathway, which lead to subvisible particles (SVPs) (0.1-100 µm in size). Additionally, the relationship between soluble aggregates (SAs) (those that are less than 0.1 µm in size) and SVP formation is largely unknown. To better understand these relationships and the mechanism of SVP formation, we conducted agitation experiments on three different classes of proteins; two antibodies [an immunoglobulin G (IgG) 1 and an IgG4] and a glycoprotein. A quantification of SVPs, using the Brightwell Microfluidics Instrument, and levels of SAs by size-exclusion chromatography were determined as a function of agitation time. Not surprisingly, the propensity to aggregate and particulate was different for each protein. However, integrated mass analysis in these studies showed that the relationship between SA and SVP formation is also protein and formulation dependent, and can vary greatly between molecules. Morphological and statistical analysis of SVPs in agitated and nonagitated samples revealed that changes in both the shape and the size distribution of the SVPs population are also protein dependent and highly defined. Collectively, these results suggest/illustrate the complexity of elucidating an aggregation mechanism that encompasses both SAs and SVPs. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:4140-4154, 2012