单抗制剂中聚山梨酯80含量气相色谱检测方法的建立

Development of a gas chromatography method for determination of polysorbate 80 content in monoclonal antibody preparations

目的建立测定单抗制剂中聚山梨酯80含量的气相色谱(gas chromatography,GC)方法,并进行方法的优化及验证。方法利用优化后的聚山梨酯80水解条件,用1 mol/L的KOH溶液水解样品中聚山梨酯80,脂肪酸酯键碱水解后定量释放出单油酸,油酸经甲酯化后,采用GC法检测油酸甲酯,根据内标法对样品中聚山梨酯80进行定量分析。对建立的方法进行专属性、系统适应性、线性、精密度、准确度及稳定性验证,并检测3批抗TNFα单抗注射剂中聚山梨酯80的含量。结果当聚山梨酯80和KOH的比例为1∶1时,在40℃水解4 h,水解效果最佳。该方法系统适应性良好,检测聚山梨酯80含量具有专属性;当聚山梨酯80的进样浓度在0.1~4.0 mg/ml范围内,呈良好的线性关系,R^2=0.999 9;两名实验人员制备的共12份供试品的聚山梨酯80浓度的RSD为3.72%;聚山梨酯80低、中、高3个加标浓度的平均回收率分别为105.52%、104.67%和102.51%,RSD为2.37%;处理后样品在27 h内稳定性良好。3批样品中聚山梨酯80的含量分别为1.032、1.016和1.022 mg/ml,分别为标示量的103.2%、101.6%和102.2%。结论该方法简便、准确、可靠,可用于单抗制剂中聚山梨酯80含量的测定。

Objective To develop, optimize and validate a gas chromatography（GC ） method for determination of polysorbate 80 content in monoclonal antibody （MeAb） preparations. Methods Under the optimized hydrolysis condition, the polysorbate 80 in test samples was hydrolyzed with 1 tool/L potassium hydroxide solution to obtain oleic acid which was subsequently methyl esterified and determined by GC, based on which the polysorbated 80 content was analyzed using internal standard. The developed method was validated for specificity, systemic suitability, linearity, precision, accuracy and stability, and used for determination of polysorbate 80 contents in three batches of McAb against TNFa. Results When polysorbate 80 and alkali was mixed at a volume ratio of 1 ： 1 at 40 ~C for 4 h, the hydrolysis reaction was complete. The method showed good system suitability and specificity for determination of polysorbate 80 content, and good linearity at a polysorbated 80 concentration of 0. 1 - 4. 0 nag / ml, with a R2 value of 0. 999 9. The RSD of polysorbated 80 contents in twelve samples prepared by two staff was 3. 72%. The mean spike recoveries of polysorbate at low, medium and high concentrations were 105.52%, 104. 67% and 102. 51% respectively, with a RSD of 2. 37%. The treated samples showed high stability within 27 h. The polysorbate 80 contents in three batches of samples were 1. 032, 1. 016 and 1. 022 mg/ml, which were 103. 2%, 101. 6% and 102. 2% of the labeled contents, respectively. Conclusion The developed GC method was simple, accurate, reliable and might be used for the determination of polysorbate 80 content in McAb preparations.