title: Development of gel-forming lyophilized formulation with recombinant human thrombin.
abstract: The objective of this work was development and evaluation of gel-forming lyophilized formulation with recombinant human thrombin for topical administration. The influence of pH, ionic strength and buffer type on protein stability was evaluated as part of the pre-formulation screening studies. Results indicated an optimal pH from 6.0 to 7.0 and increased stability with increasing content of sodium chloride. The tested buffer types had no significant effect on thrombin stability. For further development, thermosensitive Pluronic® F-127 was employed as a bulking and gelling agent. Physical and mechanical characterization and viscosity measurement confirmed the gel-forming properties of the formulation at the application temperature of 32 °C. Several techniques (addition of well-soluble polyols, different freezing protocols and reconstitution under vacuum) were tested to decrease the reconstitution time. The obtained results revealed that a vacuum in the vial headspace is crucial for acceptable reconstitution. The freeze drying process has no negative impact on recombinant thrombin stability, and this was confirmed by reverse-phase-HPLC, activity assay and optical density measurements.
authors.affiliation: a hameln rds a.s. , Horná , Modra , Slovak Republic and.
authors.forename: Andrej
authors.initials: A
authors.lastname: Murányi
authors.forename: Peter
authors.initials: P
authors.lastname: Bartoš
authors.forename: Eduard
authors.initials: E
authors.lastname: Tichý
authors.forename: Jana
authors.initials: J
authors.lastname: Lazová
authors.forename: Jana
authors.initials: J
authors.lastname: Pšenková
authors.forename: Marián
authors.initials: M
authors.lastname: Žabka
mesh_terms: D002626:Chemistry, Pharmaceutical / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D004339:Drug Compounding / Q000379:methods*; D005612:Freeze Drying; D005782:Gels; D006490:Hemostatics / Q000008:administration & dosage* / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D020442:Poloxamer / Q000737:chemistry*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000008:administration & dosage* / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D013696:Temperature; D013917:Thrombin / Q000008:administration & dosage* / Q000737:chemistry; D013997:Time Factors; D014783:Viscosity
chemical_list: D005782:Gels; D006490:Hemostatics; D011994:Recombinant Proteins; D020442:Poloxamer; D012965:Sodium Chloride; D013917:Thrombin; C513462:recombinant human thrombin
keywords: Gel forming; Pluronic F-127; recombinant; reconstitution time; thrombin