目的 验证检测注射用重组人凝血因子Ⅶa（recombinant human coagulation factor Ⅶa, rFⅦa）纯度的分子排阻高效液相色谱法，并应用于rFⅦa成品纯度的稳定性分析。方法 利用rFⅦa标准品、原液、成品，验证采用分子排阻高效液相色谱法检测rFⅦa的系统适用性与专属性、上样量耐用性、准确度、重复性、中间精密度和色谱柱耐用性，观察rFⅦa分别置于2～8 ℃和40 ℃储存6个月的稳定性。结果 rFⅦa国际标准品中二聚体的峰谷比为1.43，单体的拖尾因子为1.08。制剂缓冲液在单体峰位置出现的辅料峰最大占比0.4%。在10~50 μg蛋白上样量范围内，rFⅦa原液和成品单体峰面积百分比含量的相对标准偏差（relative standard deviation, RSD）分别为0.03%和0.07%。采用强制热降解实验验证准确度，强制热降解处理前后多聚体、二聚体/寡聚体和单体的峰面积百分比变化的回收率为97.5%～100.0%，样品总峰面积的回收率为98.5%～100.6%。重复性验证rFⅦa原液和成品的单体峰面积百分比含量的RSD分别为0.03%和0.02%。中间精密度验证不同操作人员、分析设备和检测日期的差异无统计学意义（F=0.13~2.09， P值均>0.05），原液及成品单体峰面积百分比的RSD分别为0.2%和0.9%。不同批次色谱柱的耐用性结果差异无统计学意义（F=0.01， P>0.05）。在2～8 ℃储存6个月后，注射用rFⅦa成品的分子大小分布无明显变化，纯度保持稳定；在40 ℃储存6个月后，单体减少3.1%～4.0%，二聚/寡聚体增加1.9%～2.3%，多聚体增加1.2%～1.7%。结论 分子排阻高效液相色谱法高效、准确、重现性好，适用于注射用rFⅦa的纯度检测。注射用rFⅦa成品在2～8 ℃或40 ℃条件下储存6个月稳定性良好。

Objective To validate a size-exclusion high-performance liquid chromatography (SEC-HPLC) method for determining the purity of recombinant human coagulation factor Ⅶa (rFⅦa) for injection, and apply it to stability analysis of the purity of rFⅦa finished products.Methods The SEC-HPLC method was validated for the system suitability and specificity, sample loading robustness, accuracy, repeatability, intermediate precision, and column durability using rFⅦa standard， bulk and finished product. The stability of rFⅦa stored at 2-8 °C and 40 °C for 6 months was analyzed using this method.Results The peak-to-valley ratio of dimer in the rFⅦa international standard was 1.43, and the tailing factor of monomer was 1.08. An excipient peak accounting for up to 0.4% at the monomer position in the formulation buffer. Within the protein loading range of 10-50 μg, the relative standard deviations (RSDs) of the monomer peak area percentage for rFⅦa bulk and finished product were 0.03% and 0.07%, respectively. Accuracy was evaluated through forced thermal degradation testing, which demonstrated recovery rates of 97.5%- 100.0% for percentage changes in the peak areas of polymers, dimers/oligomers, and monomers, and 98.5%-100.6% for the total peak area. The RSDs for the monomer peak area percentages in the repeatability verification of rFⅦa bulk and finished product were 0.03% and 0.02%, respectively. Intermediate precision verification showed no statistically significant differences across operators, analytical equipments, or testing dates (F=0.13-2.09,P all＞0.05), with RSDs of 0.2% for bulk and 0.9% for finished products. Column durability results showed no statistically significant differences among column batches (F=0.01, P＞0.05). After 6 months of storage at 2-8 °C, the molecular size distribution of rFⅦa finished product remained stable. Following 6 months of accelerated stability conditions at 40 °C, the monomer decreased by 3.1%-4.0%, dimer/oligomer increased by 1.9%-2.3%, and polymer increased by 1.2%-1.7%.Conclusions The validated SEC-HPLC method is efficient, accurate, and reproducible for purity determination of rFⅦa for injection. The finished product of rFⅦa for injection exhibits good stability when stored at 2–8 °C or 40 °C for 6 months.