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Abstract

The design of an aqueous formulation for acidic fibroblast growth factor (aFGF) requires an understanding of the type of compounds that can either directly or indirectly stabilize the protein. To this end, spectrophotometric turbidity measurements were initially employed to screen the ability of polyanionic ligands, less specific compounds, and variations in solution conditions (temperature and pH) to stabilize aFGF against heat-induced aggregation. It was found that in addition to the well-known protection of aFGF by heparin, a surprisingly wide variety of polyanions (including small sulfated and phosphorylated compounds) also stabilizes aFGF. These polyanionic ligands are capable of raising the temperature at which the protein unfolds by 15-30 degrees C. Many commonly used excipients were also observed to stabilize aFGF in both the presence and the absence of heparin. High concentrations of some of these less specific agents are also able to increase the temperature of aFGF thermal unfolding by as much as 6-12 degrees C as shown by circular dichroism and differential scanning calorimetry. Other compounds were found which protect the chemically labile cysteine residues of aFGF from oxidation. Aqueous formulations of aFGF were thus designed to contain both a polyanionic ligand that enhances structural integrity by binding to the protein and chelating agents (e.g., EDTA) to prevent metal ion-catalyzed oxidation of cysteine residues. While room-temperature storage (30 degrees C) leads to rapid inactivation of aFGF in physiological buffer alone, several of these aFGF formulations are stable in vitro for at least 3 months at 30 degrees C. Three aFGF topical formulations were examined in an impaired diabetic mouse model and were found to be equally capable of accelerating wound healing.

Biophysical and formulation studies of theSchistosoma mansoniTSP-2 extracellular domain recombinant protein, a lead vaccine candidate antigen for intestinal schistosomiasis

“…These results are consistent with the results of many similar studies with other protein molecules. 25,32,33 Glycerol appeared to be a potential stabilizer; however, due to its much higher osmolarity at a comparable weight amount of sugar molecule, it was not further pursued as a stabilizer. Less effective stabilizing effects were observed for aspartic acid, possibly due to chargecharge interactions between the positive charged side chains of the antigen and the negatively charged carboxylic groups.…”

Lower Inhibitor Development in Hemophilia A Mice following Administration of Recombinant Factor VIII-O-Phospho-L-serine Complex

“…It has also been used to mimic pharmaceutical development conditions (36) and to evaluate excipients for protein formulations (37). The propensity of the protein to aggregate is an indicator of the expected stability, efficacy and immunogenicity of a protein formulation.…”

A Network-based Analysis of Polyanion-binding Proteins Utilizing Human Protein Arrays

“…In some cases it is well established that the interactions of proteins with polyanions involve regulatory functions (e.g. binding of growth factors to HS 2 proteoglycans) (11, 12), genetic information transfer (DNA and RNA), cytoskeleton organization (actin and tubulin microfilaments), chaperone function (9,13,14), protein stabilization (10,15,16) …”