摘要
目的:探讨重组抗人表皮生长因子受体2(human epidermal growth factor receptor 2,Her2)单克隆抗体(单抗)的酸碱电荷异质体对其生物活性和亲和力的影响。方法:分别用过氧化氢、肽-N-糖苷酶F、羧肽酶B对抗Her2单抗进行氧化、脱糖、酶切处理。用阳离子交换高效液相色谱法和毛细管等电聚焦电泳分析抗Her2单抗的电荷异质体,使用微量差示扫描荧光法分析抗Her2单抗的热稳定性,用细胞增殖抑制法和表面等离激元共振技术分析抗Her2单抗的亲和力。结果:脱糖处理抗Her2单抗的酸性电荷异质体增加了5.58%,与免疫球蛋白G Fc受体1(high affinity immunoglobulin gamma Fc receptorⅠ,FcGR1A)的亲和力明显减弱,亲和力常数为9.032×10^-8 mol/L。氧化和酶切处理抗Her2单抗的热稳定性降低,去折叠温度分别为63.6和59.5℃,均低于未处理抗Her2单抗(66.7℃)。结论:3种处理均会使抗Her2单抗产生电荷异质性,脱糖处理抗Her2单抗与FcGR1A的亲和力减弱,氧化和酶切处理抗Her2单抗的热稳定性降低。
Objective:To investigate the influence of the acidic/basic charge variants of recombinant anti-human epidermal growth factor receptor 2(Her2)monoclonal antibody(mAb)on its biological activity and affinity.Methods:The anti-Her2 mAb was oxidized with hydrogen peroxide,deglycosylated with peptide-N-glycosidase F(PNGase F),and removed of the lysine in heavy chain C-terminus by carboxypeptidase B.Cation exchange-high performance liquid chromatography and capillary isoelectric focusing electrophoresis were used to analyze the charge variants of anti-Her2 mAb.The thermal stability was analyzed by nano differential scanning fluorimetry.Cell proliferation inhibition test and surface plasmon resonance were used to analyze the biological activity and affinity of anti-Her2 mAb.Results:The acidic charge variants of deglycosylated anti-Her2 mAb increased by 5.58%.Its affinity to high affinity immunoglobulin gamma Fc receptorⅠ(FcGR1A)was significantly reduced,with affinity constant of 9.032×10^-8 mol/L.The thermal stabilities of oxidized and digested anti-Her2 mAbs both decreased,with unfolding temperatures of 63.6℃and 59.5℃,respectively,lower than that of untreated anti-Her2 mAb(66.7℃).Conclusions:All 3 treatments can cause charge heterogeneity in anti-Her2 mAb.The affinity of deglycosylated anti-Her2 mAb to FcGR1A,and the thermal stabilities of both oxidized and digested anti-Her2 mAbs decrease.
作者
王海旗
王斌
李永东
孙洪秀
王朋辉
毕利利
Wang Haiqi;Wang Bin;Li Yongdong;Sun Hongxiu;Wang Penghui;Bi Lili(Department of Research and Development,Hualan Genetic Engineering Co.,Ltd.,Xinxiang 453000,China)
出处
《国际生物制品学杂志》
CAS
2020年第5期214-218,共5页
International Journal of Biologicals
关键词
抗体
单克隆
抗体亲和力
动力学
电荷异质体
Antibodies,monoclonal
Antibody affinity
Kinetics
Charge variants