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.

Stability of rhbFGF as determined by UV spectroscopic measurements of turbidity.

Loss of potency of a protein formulation due to precipitation of the protein is a major concern to the pharmaceutical scientist. A simple screening method was developed to study the effect of excipients on protein precipitation. It will not provide accurate stability data but it allows the rejection of excipients that may interfere with the stability of a protein formulation. The method is based on measuring the increase in turbidity at 277 nm by UV-spectroscopy and was sensitive and reproducible enough to obtain data within 15 hr at 30 degrees C or 40 degrees C, which will allow prediction of precipitation behavior that would need with conventional methods 2-3 years. Human recombinant basic fibroblast growth factor (rhbFGF or bFGF) was formulated at various pH-values as well as in the presence of various concentrations of preservatives, surfactants, gelling agent, EDTA, NaCl, sodium sulfate, sucrose, and glycosaminoglycans (GAG). Most excipients increased bFGF aggregation rate when their concentrations were increased. Exceptions were heparin and some of its derivatives, and sodium sulfate; high concentrations of sucrose and sodium chloride suppressed aggregation.