Santana Héctor, García Gerardo, Vega Maribel, Beldarraín Alejandro, Páez Rolando
Pharmaceutical Development Department, Center for Genetic Engineering and Biotechnology, Havana, Cuba;
Quality Control Department, Center for Genetic Engineering and Biotechnology, Havana, Cuba;
PDA J Pharm Sci Technol. 2015 May-Jun;69(3):399-416. doi: 10.5731/pdajpst.2015.01052.
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.
我们报告了一种用于伤口愈合的重组人表皮生长因子（rhEGF）冻干制剂通过病灶内注射的稳定性评估。在经过强化暴露条件后，评估了包装材料对成品干粉的光保护适用性。研究了注射用粉末在浓度为25 - 250μg/瓶以及温度为45、60和70°C时的降解动力学。在25±2°C/相对湿度60±5%（6个月）和2 - 8°C（24个月）黑暗条件下储存后，在多个时间点进行分析，评估了长期稳定性。在2 - 8°C储存24小时后，还评估了用各种稀释剂复溶后的稳定性。通过反相高效液相色谱（RP - HPLC）、尺寸排阻HPLC和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS - PAGE）分析rhEGF样品的结构完整性。通过测量小鼠成纤维细胞系中的细胞增殖来研究生物活性。结果表明，仅初级包装中的冻干rhEGF对光敏感，因为反相高效液相色谱显示的降解被二级纸箱包装完全抑制。随着蛋白浓度从25μg/瓶增加到250μg/瓶，冻干rhEGF的稳定性增加。长期稳定性研究表明，在含有25或250μg/瓶rhEGF的冻干制剂中，rhEGF没有明显降解或物理变化。在所研究的储存条件下，rhEGF在注射用水或0.9%氯化钠中复溶后未观察到物理、化学或生物学变化。
评估了一种用于伤口愈合的重组人表皮生长因子（rhEGF）冻干制剂通过病灶内注射的稳定性。在经过强化暴露条件后，评估了包装材料对成品干粉的光保护适用性。研究了注射用粉末在浓度为25 - 250μg/瓶以及温度为45、60和70°C时的降解动力学。根据国际协调会议（ICH）指南检查加速、长期和复溶稳定性及其使用时间。通过不同的化学、物理和生物活性测定分析rhEGF样品的稳定性。结果表明，仅初级包装中的冻干rhEGF对光敏感，因为反相高效液相色谱显示的降解被二级纸箱包装完全抑制。随着蛋白浓度增加，冻干rhEGF的稳定性增加。在25±2°C/相对湿度60±5%储存6个月或2 - 8°C储存24个月后，冻干制剂中未观察到rhEGF明显降解或物理变化。在2 - 8°C储存24小时后，rhEGF在注射用水或0.9%氯化钠中复溶后未观察到物理、化学或生物学变化。
