Development and application of an immunogenicity assay against thrombopoiesis stimulating fusion protein (TMP-Fc) based on SPR system

XIE Xin-yao; XU Jing-zhi; LUO Yu; OU Lun; GAO Liu-cun; GAO Chen-yan; WANG Hai-xue; FU Jie; DONG Li-hou; SONG Hai-feng

Abstract：Objective:To develop a validated Biacore 3000 GxP surface plasmon resonance (SPR) immunoassay to study the comparative immunogenicity (IM) of two thrombopoiesis stimulating fusion proteins (TMP-Fc) in cynomolgus monkeys.Methods:Cynomolgus monkeys were administered three successive doses of 100,300,and 1 000 μg·kg-1 LMF (test articles,L) and another successive dose of 300 μg·kg-1 of romiplostim (R) as the control.The measured screening cut point (CP) and specificity cut point (SCP) of these antibodies were converted into signal-to-noise ratio (S/N),and the SCP was validated by further immunoinhibition assays.Validation of the immunoassay included determining the assay sensitivity,quantitative range,Hook effect,drug tolerance,and precision.Results:The CPL and CPR were 1.54 and 1.41,respectively.The threshold of the confirmatory assays for SCPL and SCPR were 37.6％ and 40.9％,respectively.The positive control (PC) for anti-LMF samples was prepared using pooled cynomolgus monkey serum (PCMS) covering the range of 1 ～64 μg·mL-1.The Hook effect was not observed.The sensitivities for anti-LMF and anti-romiplostim in PCMS were 556 and 463 ng·mL-1,respectively.In the drug tolerance experiment,the S/N values of the samples decreased with increasing drug concentration,with the signal inhibition ratio (SIR) or ％ inhibition increasing by contrast.The ％CVs of the negative control (NC),low positive control (LPC),and antibody-positive serum control (rsPC) were lower than 25 ％.A positive rate of anti-drug antibody (ADA) detection was found in all groups.The 300 μg· kg-1 administration of L and R concluded at the same rate (50％).The IM assay results for anti-LMF and anti-romiplostim on R ADA samples were consistent in positive individuals and across time points.Conclusion:The SPR platformbased IM assay developed and validated in this study met the requirements for biological products IM research,and was successfully applied in an immunogenicity evaluation of two TMP-Fc products.