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.

• Gel forming
• Pluronic F-127
• recombinant
• reconstitution time
• thrombin

Acknowledgements

The authors are grateful to Anastázia Poláková and Helena Katonová for technical assistance with viscosity and structure measurements.

Declaration of interest

This work was supported by project “Industrial research for new drugs on the basis of recombinant proteins”, code ITMS: 26240220034. We support research activities in Slovakia. This project is being co-financed by the European Union.