两种基于RAJI细胞的抗CD20单抗新型定量方法的比较及在药物代谢动力学中的应用

Comparative Analysis of Two Novel Quantitative Methodologies for Measuring Recombiant-anti-CD20zumab Based on RAJI Cells and Their Application in Pharmacokinetic Study

建立和比较基于RAJI细胞的流式细胞法(Flow cytometry assay,FCA)和细胞放射免疫法(Immuno-radiametric assay,IRA)测定重组抗CD20人源化单克隆抗体(r-anti-CD20zumAb)的浓度。两种方法均利用标记后抗体和被检测抗体竞争性结合RAJI细胞表面的CD20抗原,由标记后抗体的荧光或放射性变化而间接反应检测抗体的浓度。FCA和IRA定量范围分别为0.1～100mg/L和0.04～20mg/L,FCA的日内及日间精密度分别小于4.0%和3.0%,准确度为-1.7%～1.1%,IRA的日内及日间精密度值均小于7.0%,准确度为-8.9%～13.2%。方法学确证研究表明,两种方法均具有良好的特异性、灵敏度、精密度和准确度。血浆样品分析结果显示,两种方法具有良好的一致性,是检测猕猴血浆中人源化单克隆抗体浓度的理想方法。

To establish sensitive methods for measuring recombinant anti-CD20 humanized monoclonal antibody（rh-anti-CD20zumab）,a flow cytometry assay（FCA） and a cell immunoradiometric assay（IRA） were developed and compared.The concentrations of rh-anti-CD20 zumab were calculated by the FITC or radio labeled antibody that was specific binding to CD20 antigen on RAJI cells.The quantitative ranges of FCA and IRA were 0.1-100 mg/L and 0.04-20 mg/L,respectively.For FCA,the precisions of intra-day and inter-day were 〈4.0% and 〈3.0%,the accuracy was-1.7%-1.1%.For IRA,the precisions of intra-day and inter-day were both 〈7.0%,the accuracy was-8.9%-13.2%.The method validation results showed that these two methods have sufficient sensitivity,precision,accuracy and specificity,which could satisfy with the requirement of pharmacokinetic study.The quantification results of plasma samples show that these two methods have excellent consistency.Therefore,the new methods basing on RAJI cells were ideal methods to determine concentrations of rh-anti-CD20zumAb in rhesus monkeys plasma samples.