贝伐珠单克隆抗体的HPLC定量检测方法的建立

Establishment of an HPLC quantitative method for bevacizumab

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,R2〉0.999;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。

Objective：To establish an HPLC-Protein A quantitative method for the content determination and quality control of bevacizumab in the production process.Methods：An Agilent Bio-Monolith Protein A chromatographic column（5.2mm × 4.95 mm,0.10 m L） was adopted with PBS（containing 0.12 mol·L-1sodium chloride,p H 7.4）as the mobile phase A and 0.5 mol·L-1acetic acid（p H 2.5） as the mobile phase B.After loading the sample,the mobile phase A was first used for washing,followed by elution with the mobile phase B.The flow rate was 1.0 m L·min-1,the column temperature was 25 ℃ and the detection wavelength was 280 nm.Results：The standard curve was created using the concentration of bevacizumab as X axis and the peak area as Y axis.Within the concentration range of 0.078-10.0 mg·m L-1,the curve fitting was good（R2 0.999）.The recovery was 98.32%-101.7%.The RSD of intra batch precision was ≤ 1.5% and RSD of inter batch precision was ≤ 1.6%.The LOQ was 0.3μg and the DOQ was 0.9 μg.In the condition that p H varied ± 0.2,sodium chloride concentration in the mobile phase A varied ± 10%,the column temperature varied ± 5.0 ℃,and the flow rate varied ± 20%,the RSD of peak area was 0.39%-1.01%.The retention time of bevacizumab in the fermentation broth was 1.350 min,which was consistent with the reference standards.Within 18 hours,the same batch of fermentation liquid was detected 27 times,and RSD of the peak area was 0.44%.Conclusion：The result proved that the HPLC-Protein A method is suitable for the quantitative determination and quality control of bevacizumab.