重组蛋白中G418残留量超高效液相色谱-蒸发光散射检测方法的建立

目的建立定量检测重组蛋白中G418残留量的超高效液相色谱(ultra performance liquid chromatography,UPLC)-蒸发光散射检测(evaporative light scattering detection,ELSD)法,并进行验证。方法 G418经UPLC洗脱后,通过蒸发光散射检测器检测,首先将柱洗脱液雾化形成气溶胶,然后在加热的漂移管中将溶剂蒸发,余下不挥发性溶质颗粒在光散射检测池中进行检测,通过标准曲线计算质控品中G418浓度。对建立的方法进行专属性、重复性、中间精密度、定量限、耐用性、线性验证。结果 G418浓度在0. 01～0. 20 mg/m L范围内,标准曲线的线性较好,R²> 0. 99,定量限为0. 01 mg/mL;质控品的加样回收率在80%～120%之间;中间精密度试验回收率的RSD为3. 44%;定量限试验回收率的RSD为5. 38%;耐用性试验回收率的RSD为4. 56%。结论 UPLC-ELSD法精密度良好,可用于重组蛋白研发及生产过程中G418残留的监测。

Development of a method for determination of residual G418 content in recombinant protein drugs by ultra performance liquid chromatography-evaporative light scattering detection method

Objective To develop and verify a ultra performance liquid chromatography-evaporative light scattering detection(UPLC-ELSD)method for quantitative determination of residual G418 content in recombinant protein drugs.Methods G418 was eluted by UPLC and detected by ELSD.The eluant was firstly atomized to form an aerosol,then added into the heated drift tube to vaporize the solvent,and the remained non-volatile solute particles were detected in the light scattering detection pool.The G418 content was calculated according to the standard curve.The developed method was verified for specificity,reproducibility,intermediate precision,quantitative limit,durability and linearity.Results The linear range of standard curve was 0.01~0.20 mg/mL,with a R2 value of more than 0.99 and a quantitative limit of 0.01 mg/mL.The recovery rates of quality control were between 80%and 120%.The RSD of recovery rate in intermediate precision test was 3.44%,while that in quantitative limit test was 5.38%,and that in durability test was 4.56%.Conclusion The developed UPLC-ELSD method showed high precision,which might be used for the monitoring of residual G418 content during development and production of recombinant protein drugs.