贝伐珠单克隆抗体的HPLC定量检测方法的建立

发布时间：2019-03-11 19:06

【摘要】：目的:建立HPLC-Protein A定量检测贝伐珠单抗的方法,用于该单抗生产过程的含量测定和质量控制。方法:采用Agilent Bio-Monolith Protein A Column(5.2 mm×4.95 mm,0.10 m L)色谱柱,以PBS(含0.12 mol·L-1氯化钠,p H 7.4)为流动相A,0.5 mol·L-1醋酸(p H 2.5)为流动相B。上样后用流动相A冲平,然后用流动相B进行洗脱,流速为1.0 m L·min-1,柱温为25℃,检测波长为280 nm。结果:以质量浓度为横坐标,峰面积为纵坐标绘制标准曲线,在0.078~10.0 mg·m L-1浓度范围内,R20.999;回收率在98.32%~101.7%之间;批内精密度RSD≤1.5%;批间精密度RSD≤1.6%;检测限0.30μg,定量限0.90μg;在p H变化±0.2、流动相A中氯化钠浓度变化±10.0%、柱温变化±5.0℃、流速变化±20.0%条件下,峰面积相对标准差RSD为0.39%~1.0%;发酵液样品的保留时间为1.350 min,与对照品的保留时间一致;在18 h内,同一批发酵液共检测27次,峰面积的RSD为0.44%。结论:方法学验证结果显示HPLC-Protein A法可以用于贝伐珠单抗生产过程的含量测定和质量控制。
[Abstract]:Aim: to establish a method for the quantitative determination of bevacizumab by HPLC-Protein A, and to apply it to the determination and quality control of the mAb in the production process. Methods: a Agilent Bio-Monolith Protein A Column (5.2 mm 脳 4.95 mm,0.10 m L) column was used with PBS (containing 0.12 mol 路L-1 sodium chloride, p H 7.4) as the mobile phase A. 0.5 mol 路L-1 (p H 2.5) was used as mobile phase B. The sample was flushed with mobile phase A and eluted with mobile phase B, the flow rate was 1.0ml 路min-1, the column temperature was 25 鈩，

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