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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 establish a gas chromatography (GC) method for the determination of polysorbate 80 in the preparation of monoclonal antibody and to optimize and validate the method. Methods Polysorbate 80 was hydrolyzed by 1 mol / L KOH solution using optimized conditions of polysorbate 80. The mono-oleic acid was released quantitatively after the hydrolysis of fatty acid ester bond, and the oleic acid was methylated. Methyl oleate was detected by GC method and polysorbate 80 was quantitatively analyzed by internal standard method. The established methods were validated for specificity, system suitability, linearity, precision, accuracy and stability, and the polysorbate 80 content of three batches of anti-TNFα mAb injections was tested. Results When the ratio of polysorbate 80 to KOH was 1: 1, hydrolysis was best at 40 ℃ for 4 h. The method has a good adaptability to the system, and the detection of the content of polysorbate 80 is specific. When the concentration of polysorbate 80 is in the range of 0.1-4.0 mg / ml, a good linear relationship is obtained, R 2 = 0.999 9; The RSDs of 12 polysorbate 80 samples prepared by the experimentalists were 3.72%. The average recoveries of polysorbate 80 at the three spiked concentrations were 105.52%, 104.67% and 102.51 %, RSD was 2.37%. The treated samples showed good stability within 27 h. The polysorbate 80 content in three batches of samples was 1.032, 1.016 and 1.022 mg / ml, respectively, 103.2%, 101.6% and 102.2% of the indicated amounts. Conclusion The method is simple, accurate and reliable and can be used for the determination of polysorbate 80 in monoclonal antibody preparations.