Approaches to analysis of aggregates and demonstrating mass balance in pharmaceutical protein (basic fibroblast growth factor) formulations.

Denaturation, aggregation, and precipitation, which are common events in protein aging, limit the development of pharmaceutical protein formulations. Using the example of basic fibroblast growth factor (bFGF), we describe a systematic approach for quantitative recovery of soluble and insoluble aggregates in aged samples to achieve mass balance in three analytical methods, UV spectroscopy, size exclusion HPLC (HP-SEC), and reverse phase HPLC. Soluble aggregates were evaluated by UV spectroscopy and HP-SEC; the latter method was modified to include 2 M guanidine hydrochloride (GnHCl) in the mobile phase in order to differentiate and simultaneously analyze native and denatured protein. Insoluble aggregates were first solubilized with GnHCl and then recovered quantitatively with the modified HP-SEC method. Chaotrope treatment did not affect the UV peak absorbance but altered the HPLC profiles. The changes were consistent with dissociation of disulfide-linked multimers to monomers with an intramolecular disulfide linkage. This phenomenon was used for estimation of the monomer-multimer distribution in the untreated aggregated sample. These methods established approaches for quantitative recovery and characterization of bFGF aggregates.