FMU100抗体的超高效液相色谱法定量检测方法的建立

发布时间：2018-01-06 09:35

  本文关键词：FMU100抗体的超高效液相色谱法定量检测方法的建立　出处：《福建医科大学学报》2017年05期 　论文类型：期刊论文

  更多相关文章： 色谱法 高压液相 抗体

【摘要】：目的建立UPLC-protein A定量检测FMU100单克隆抗体的方法。方法使用Waters UPLC超高效液相色谱仪,Applied Biosystem的PA ImmunoDetection Sensor Cartridge(2.1×30mm,20μm)色谱柱,柱温30℃,检测波长280nm,进样体积10μL,以流动相A(10mmol/L PBS含0.15mmol/L NaCl,pH 7.2)-流动相B(0.15mmol/L NaCl,pH 2.6)进行梯度洗脱,流速0.5mL/min。结果在0.034~2.200mg/mL浓度,线性相关系数r~2为0.999 9,浓度与峰面积间呈良好的线性关系;检测限为0.01mg/mL,定量限为0.07mg/mL;回收率为98.18%~99.16%;重复性RSD为0.1%~1.9%;在8h内,每个2h测定发酵液中抗体的含量,FMU100抗体峰面积RSD为1.8%;在流动相A pH变化±0.2、流动相B NaCl比例变化±5%、柱温变化±5℃、检测波长变化±5nm、流速相对值变化±20%时,FMU100抗体峰峰面积RSD为1.7%(n=12);检测方法符合系统适应性要求。结论建立的UPLC-protein A定量检测FMU100抗体的方法简单快速、方法可靠,符合方法学验证要求。
[Abstract]:Objective to establish a UPLC-protein method for quantitative detection of A FMU100 monoclonal antibody. Methods using Waters UPLC ultra high performance liquid chromatography, Applied Biosystem PA ImmunoDetection Sensor Cartridge (2.1 x 30mm, 20 m) column, the column temperature was 30 degrees, the detection wavelength of 280nm, sample volume 10 L, mobile phase in A (10mmol/L PBS with 0.15mmol/L NaCl, pH 7.2 B (0.15mmol/L) - mobile phase NaCl, pH 2.6) by gradient elution, flow rate of 0.5mL/min. results in the concentration of 0.034~2.200mg/mL, the linear correlation coefficient r~2 is 0.9999, the concentration and peak area showed a good linear relationship; the detection limit is 0.01mg/mL, the limit of quantitation was 0.07mg/mL; the recovery rate is 98.18%~99.16%; repeat RSD 0.1%~1.9%; in 8h, determination of antibody in fermentation broth of each 2h, FMU100 antibody peak area RSD was 1.8%; in the mobile phase A pH + 0.2, NaCl + 5% B phase change in the proportion of flow, column temperature of - 5 DEG C, detection wavelength When the relative value of change is + 20%, the peak area of FMU100 antibody is 1.7% (n=12), and the detection method accords with the requirement of system adaptability. Conclusion the method of UPLC-protein A for the quantitative detection of FMU100 antibody is simple, fast, reliable, and accords with the requirement of 5nm validation.

【作者单位】： 福建医科大学药学院免疫治疗研究所;
【基金】：福建省自然科学基金(2016J01154)
【分类号】：R392-33
【正文快照】： 活化的T细胞上表达有许多的抑制受体[1],在肿瘤组织中可能被肿瘤利用形成免疫逃逸[2],利用封闭性抗体能调节T细胞活性,对肿瘤的治疗有非常好的效果[3]。FMU100为本单位基于此原理研制的新型人源化单克隆抗体。高效液相色谱法(high performance liquidchromatography,HPLC)因具，

本文编号：1387317