Stabilization of recombinant human basic fibroblast growth factor by chemical modifications of cysteine residues

Abstract

The production of recombinant human basic fibroblast growth factor (rhbFGF) in Escherichia coli cells yielded active forms of this polypeptide which, however, displayed a high degree of instability towards oxidative processes. Biochemical studies in our laboratory and those of others indicated that the reactivity of the four cysteine residues was the main cause of the observed instability. Several attempts to obtain more stable derivatives of rhbFGF were carried out by modification of the sulfhydryl groups. Among these, treatment of rhbFGF with iodoacetic acid led to the isolation of a partially carboxymethylated form (Cm-FGF). Peptide mapping analysis of the modified protein showed that two cysteines (78 and 96) were blocked by a carboxymethyl group. The remaining cysteines (34 and 101) were not modified under the conditions used and were found to be in the reduced form. Cm-FGF and unmodified rhbFGF showed similar affinity both for heparin and for high-affinity receptors. Cm-FGF was more stable than the unmodified molecule as measured by HPLC and SDSjPAGE analysis. Interestingly, Cm-FGF was more active than unmodified rhbFGF in stimulating proliferation of endothelial cells and DNA synthesis in 3T3 fibroblasts. This new derivative could represent a desirable complementation to rhbFGF for the development of more stable pharmaceutical formulations in wound healing applications.

Refinement of the structure of human basic fibroblast growth factor at 1.6 Å resolution and analysis of presumed heparin binding sites by selenate substitution

“…In addition these two nonconserved cysteines are also blocked by carboxymethyl groups if the protein is treated with iodoacetic acid (Caccia et al, 1992). The formation of an adduct between BME and Cys 69, indicated by the electron density maps, is consistent with the accessibility of this residue to other ligands.…”

Development of inhalable dry powder formulation of basic fibroblast growth factor

“…However, the development of a dry powder formulation of bFGF is constrained by the relatively poor stability of bFGF and the uncertainty in its compatibility with carrier excipients. First, bFGF has four cysteine residues, which are highly reactive under oxidative conditions (Caccia et al, 1992). Its stability is also negatively affected by heat, organic solvent, and pH (Yu et al, 2007).…”

“…Here, we used the spray-drying method to prepare inhalable dry powders containing bFGF and studied the effects of carrier excipients on the aerodynamic properties of the dry powder and the integrity of bFGF. The results of our study show that, albeit the poor stability profile (Caccia et al, 1992;Yu et al, 2007), bFGF can be successfully formulated as an inhalable powder when the carrier excipients are judiciously selected.…”

Coordinated fibroblast growth factor and heparan sulfate regulation of osteogenesis

“…Growth factors are rapidly cleared in vivo as well as being highly susceptible to proteolytic degradation (Babensee et al, 2000). In addition, large quantities of growth factors are usually required in order to achieve a successful outcome, as their exposure to sterilisation procedures in the unbound state, as well as exposure to organic solvents, alkaline pH or storage at room temperature all appear to reduce their bioactivity (Westall et al, 1983;Caccia et al, 1992;Berscht et al, 1994). Furthermore, all of the aforementioned growth factors are proto-oncogenic, which is increasingly becoming an important translational problem (Hunter and Avalos, 2000).…”