Abstract
Basic fibroblast growth factor (FGF-2) is a highly labile protein with strong potential for tissue engineering. The aim of this study was to develop FGF-2 formulations that are stable against physical stressors encountered in pharmaceutical processing and evaluation. Pharmaceutical excipients, alone or in combination, were added to aqueous FGF-2 (770 ng/mL) solution and the stability of the resulting solutions on storage at 4–37 °C was evaluated. Stability of the solutions to repeated freeze-thaw cycles and lyophilisation was also evaluated, as well as the stability of the lyophilised stabilised protein to storage at −4, 4 and 18 °C for up to 12 months. In all of these experiments FGF-2 was quantified by ELISA assay. The as-received FGF-2, when dissolved in water, was highly unstable, retaining only 50% of baseline protein content after 30 min at 37 °C or 1 h at 25 °C. By contrast, FGF-2 solutions prepared with 0.5% w/v methylcellulose (MC) and 20 mM alanine (formulation F5) or with 0.5% w/v MC and 1 mg/mL human serum albumin (HSA) (formulation F6) were highly stable, having residual FGF-2 content comparable to baseline levels even after 2 h at 37 °C and 5 h at 25 °C. F5 and F6 were also highly stable to repeated freeze-thaw cycles, with >99% of FGF-2 load remaining after the third cycle. In addition, F5 and F6 were stable to lyophilisation, and the lyophilised products could be stored at −4, 4 or 18 °C for at least 12 months, with less than 1% loss in mean FGF-2 content. Thus, FGF-2 solution is effectively stabilised against both thermal and processing stressors in the presence of MC and alanine (F5), or MC and HSA (F6). The resultant FGF-2 solutions may be applied as medicinal products or further processed into more advanced medicinal products, e.g., scaffolds, for wound healing and tissue regeneration.
Highlights
Basic fibroblast growth factor (FGF-2) is an endogenous, 18-kDa heparin-binding protein that promotes cellular proliferation, migration and differentiation of a variety of tissues [1,2]
Samples exposed to 4 ◦ C were stable for only 2 h while those exposed to 25 ◦ C were stable for 30 min
To render the FGF-2 solution sufficiently stable for processing into acceptable medicinal products that could be transported and stored without requiring −20 ◦ C storage, this study explored a range of excipients which were expected to act via different mechanisms
Summary
Basic fibroblast growth factor (FGF-2) is an endogenous, 18-kDa heparin-binding protein that promotes cellular proliferation, migration and differentiation of a variety of tissues [1,2]. Several therapeutic applications for FGF-2 have been identified in the literature [3], with the efficacy of FGF-2 for the treatment of a wide variety of conditions, such as burns [4], mouth ulcers [5], fractures [6], pressure and diabetic ulcers [7,8] and critical limb ischemia [9] evaluated in various clinical trials. One of the largest areas of research relates to the use of FGF-2 for wound healing applications. Chronic wounds often have reduced FGF-2 concentrations, which may account for their poor rates of healing and revascularisation [10,11]. FGF-2 has been extensively evaluated for chronic wound management, and some success has been demonstrated in vitro [12,13,14]. The translation of FGF-2 into clinical use has been severely limited by its inherent instability, in particular in aqueous solutions
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