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目的:建立基于生物膜干涉(Biolayer interferometry,BLI)技术检测治疗性单抗与新生儿Fc受体(neonatal Fc receptor,FcRn)亲和力的方法。方法:链霉亲和素(streptavidin,SA)偶联传感器结合生物素标记FcRn后,使用稀释液(0.1%BSA/0.05%Tween20/PBS,p H 6.0)封闭传感器后采用BLI方法检测,以稀释液1∶2系列稀释样品(浓度范围为125~16 000 nmol·L~(-1)),空白对照采用未结合FcRn的传感器检测上述系列稀释样品。结果采用Steady state analysis方式拟合,计算样品平衡常数(KD)值,计算样品各自的95%可信区间(confidence interval,CI),对不同抗体与FcRn的亲和力进行比较。结果:Ig G-FcRn结合曲线呈现明显的快结合-快解离形态。应用建立的方法检测不同单抗与FcRn的亲和力结果:bevacizumab与FcRn的亲和力较trastuzumab高95%,存在显著性差异;bevacizumab生物类似药(similar biotherapeutic products,SBP)-1与FcRn亲和力较原研药(reference biotherapeutic products,RBP)有32%的升高,且有显著性差异。其他Cys-偶联药物前后、Lys-偶联药物前后、表达细胞系由SP2/0更换为CHO均未对Ig G-FcRn亲和力产生影响。结论:建立了优化的基于生物膜干涉技术检测Ig G抗体与FcRn亲和力测定方法,可用于单抗及抗体偶联药物与FcRn亲和力的评价。
OBJECTIVE: To establish a method for detecting the affinity of therapeutic monoclonal antibody (McAb) with neonatal Fc receptor (FcRn) based on biofilm interferometry (BLI). METHODS: After binding the biotin-labeled FcRn to streptavidin (SA) -conjugated sensor, the BLI method was used to block the sensor with a diluent (0.1% BSA / 0.05% Tween20 / PBS, p H 6.0) 1: 2 dilution series (concentration range 125 ~ 16 000 nmol·L -1), and the blank control was used to detect the above series of diluted samples with non-FcRn-binding sensor. Results Steady state analysis was used to calculate the equilibrium constants (KD) of samples. The 95% confidence interval (CI) of each sample was calculated and the affinity of different antibodies to FcRn was compared. RESULTS: The Ig G-FcRn binding curve showed a distinctly fast binding-fast dissociation pattern. The established method was used to test the affinity of different monoclonal antibodies to FcRn. Results: The affinity of bevacizumab with FcRn was 95% higher than that of trastuzumab, with significant difference. The similarities of bevacizumab biosimilar drugs (SBP) There was a 32% increase in reference biotherapeutic products (RBP) with significant differences. Before and after other Cys-coupled drugs, the change of the expression cell line from SP2 / 0 to CHO before and after Lys-coupled drug did not affect Ig G-FcRn affinity. CONCLUSION: An optimized biofilm-based interferometric technique for the determination of the affinity of Ig G antibody to FcRn has been established and can be used to evaluate the affinity of monoclonal antibody and antibody-conjugated FcRn.