SEC-HPLC法测定rhTNK-tPA单链含量

SEC-HPLC chromatography method for determination of rhTNK-tPA's single-chain content

目的:测定重组人组织型纤溶酶原激活剂改构体(combinant human mutant-threonine mutant-asparagine mutant-lysine tissue type plasminogen activator,rhTNK-tPA)单链含量。方法:将样品用二硫苏糖醇(DTT)打开二硫键,采用分子排阻色谱(size-exclusion high performance liquid chromatograph,SEC-HPLC)法测定。色谱柱:Tsk-Gel2000SWxl;流动相:0.2 mol·L-1PB-0.1%SDS水溶液;流速:0.5 mL·min-1;检测波长:214 nm;柱温:23℃;检测时间:30 min。结果:试验精密度和重复性良好;分离度不低于1.1;3批rhTNK-tPA原液的单链含量分别为74.41%、75.39%、74.43%,3批成品的单链含量分别为73.82%、74.76%、74.91%。结论:本方法经方法学验证可用于rhTNK-tPA产品的常规检定。

Objective： To establish a method for the determination of the single chain content of recombinant human tenecteplase（ TNK- tPA）. Methods： Disulfide bond of the samples were first opened by dithiothreitol（ DTT）,and then the HPLC- SEC method was adopted for the analysis in this study. The column was Tsk- Gel2000 SWxl,and the mobile phase was consisted of 0. 2 mol·L- 1PB and 0. 1% SDS solution; The flow rate was 0. 5 mL·min- 1,the detection wavelength was set at 214 nm,and the column temperature was 23 ℃. The detection time was 30 min. Results： The precision and repeatability of the tests were very good,the resolution between the single chain and two- chain alteplase peaks was not less than 1. 1. The single chain content of rhTNK- tPA was 74. 41%,75. 39%,74. 43%（ three batches of bulk） and 73. 82%,74. 76%,74. 91%（ three batches of final products）,respectively. Conclusion： The established method can be used for the routine quality control of rhTNK- tPA.