分光光度法检测重组蛋白药物中PF68残留量

Determination of residual pluronic F-68 content in recombinant protein drugs by colorimetric method

目的建立能够定量检测重组蛋白药物中PF68残留量的分光光度法,并进行验证。方法采用TCA沉淀蛋白方法,上清中PF68与硫氰酸钴铵盐反应形成蓝色复合物,于酶标仪波长624 nm处测定吸光度值,通过标准曲线计算样品中PF68的含量。对方法进行线性、准确度、精密度验证,并确定定量限。结果分光光度法检测重组蛋白中PF68含量,PF68浓度在0.1~1.6 mg/mL范围内,标准曲线的线性较好,相关系数R^2>0.99,定量限低至0.1 mg/mL。各浓度水平验证样品的加样回收率在88%~100%之间;浓度为1.2、0.6、0.3 mg/mL样品重复性试验的RSD分别为4.0%、5.2%、3.9%,中间精密度试验的RSD分别为7.0%、5.7%、4.8%。结论分光光度法准确度和精密度良好,可用于重组蛋白药物研发及生产过程中残留PF68的监测。

Objective To develop and verify a colorimetric method for quantitative determination of residual pluronic F-68（PF68）content in recombinant protein drugs. Methods The colorimetric method is based on the formation of a dark blue complex of PF-68 and cobalt thiocyanate. The absorbance was measured at 624 nm,and the PF68 content was calculated according to the standard curve. The method was verified for linearity,accuracy and precision,of which the limit of quantitation was determined. Results The linear range of standard curve was 0. 1 - 1. 6 mg/mL,with a R2 value of more than 0. 99,while limit of quantitation was 0. 1 mg/mL. The recovery rates of samples at various concentrations were 88% - 100%. The RSDs of reproducibility test results of samples at concentrations of 1. 2,0. 6 and 0. 3 mg/mL were 4. 0%,5. 2% and 3. 9% respectively. Conclusion The developed colorimetric method showed high accuracy and precision,which might be used for the monitoring of residual PF68 content during development and production of recombinant protein drugs.