重组抗人表皮生长因子受体2单克隆抗体的电荷异质体分析

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 receptor Ⅰ，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。

Analysis of charge variants for recombinant anti-human epidermal growth factor receptor 2 monoclonal antibody#br#

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 receptor 1 of immunoglobulin G (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.