title: Stability Studies of a Freeze-Dried Recombinant Human Epidermal Growth Factor Formulation for Wound Healing.
abstract: We report on the stability assessment of a recombinant human epidermal growth factor (rhEGF) freeze-dried formulation for wound healing by intra-lesional injections. The suitability of packaging material for the light protection of finished dried powder was evaluated after stressed exposure conditions. Degradation kinetics of powder for injection was investigated at concentrations of 25-250 μg/vial and temperatures of 45, 60, and 70 °C. The long-term stability was evaluated after storage at 25 ± 2 °C/60 ± 5% relative humidity (6 months) and 2-8 °C (24 months) in the dark and analyzed at several time points. The stability after reconstitution with various diluents was also assessed after 24 h storage at 2-8 °C. The rhEGF samples were analyzed for structural integrity by reversed-phase high-performance liquid chromatography (RP-HPLC), size-exclusion HPLC, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Biological activity was investigated by measuring the cell proliferation in a murine fibroblast cell line. Results show that freeze-dried rhEGF in primary packaging only was photosensitive, as degradation by RP-HPLC that was completely suppressed by the secondary carton package was revealed. An increase in freeze-dried rhEGF stability was observed with the increase in protein concentration from 25 to 250 μg/vial. The long-term stability study showed no significant rhEGF degradation or physical change within the freeze-dried formulations containing 25 or 250 μg/vial of rhEGF. No physical, chemical or biological changes were observed for rhEGF after reconstitution in water for injection or 0.9% sodium chloride during the storage conditions studied.
  
  
  
  The stability of a recombinant human epidermal growth factor (rhEGF) freeze-dried formulation for wound healing by intra-lesional injections was assessed. The suitability of packaging material for the light protection of finished dried powder was evaluated after stressed exposure conditions. Degradation kinetics of powder for injection was investigated at concentrations of 25-250 μg/vial and temperatures of 45, 60, and 70 °C. The accelerated, long-term, and reconstitution stabilities were examined according to ICH guidelines for their utility time. The stability of rhEGF samples was analyzed by different chemical, physical, and biological activity assays. Results show that freeze-dried rhEGF in primary packaging only was photosensitive, as degradation by reversed-phase high performance liquid chromatography that was completely suppressed by the secondary carton package was revealed. An increase in freeze-dried rhEGF stability was observed with the increase in protein concentration. No significant rhEGF degradation or physical changes were observed within the freeze-dried formulations after 6 months storage at 25 ± 2 °C/60 ± 5% relative humidity or 24 months storage at 2-8 °C. No physical, chemical, or biological changes were observed for rhEGF after reconstitution in water for injection or 0.9% sodium chloride after 24 h storage at 2-8 °C.
authors.affiliation: Pharmaceutical Development Department, Center for Genetic Engineering and Biotechnology, Havana, Cuba; hector.santana@cigb.edu.cu santanahector14@gmail.com.
authors.forename: Héctor
authors.initials: H
authors.lastname: Santana
authors.affiliation: Quality Control Department, Center for Genetic Engineering and Biotechnology, Havana, Cuba;
authors.forename: Gerardo
authors.initials: G
authors.lastname: García
authors.affiliation: Quality Control Department, Center for Genetic Engineering and Biotechnology, Havana, Cuba;
authors.forename: Maribel
authors.initials: M
authors.lastname: Vega
authors.affiliation: Central Office for Scientific Pole, Havana, Cuba.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Beldarraín
authors.affiliation: Pharmaceutical Development Department, Center for Genetic Engineering and Biotechnology, Havana, Cuba;
authors.forename: Rolando
authors.initials: R
authors.lastname: Páez
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004349:Drug Packaging; D004355:Drug Stability; D004356:Drug Storage; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000737:chemistry*; D005079:Excipients / Q000737:chemistry; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005612:Freeze Drying; D006168:Guinea Pigs; D006801:Humans; D051379:Mice; D011994:Recombinant Proteins; D012965:Sodium Chloride / Q000737:chemistry; D013696:Temperature; D014867:Water / Q000737:chemistry; D014945:Wound Healing / Q000187:drug effects*
chemical_list: D005079:Excipients; D011994:Recombinant Proteins; D014867:Water; D012965:Sodium Chloride; D004815:Epidermal Growth Factor
keywords: Epidermal growth factor; ICH; Lyophilized formulation; Reconstitution; Stability