ABSTRACT

The heterogeneity of glycosylation on therapeutic monoclonal antibodies (mAbs) may affect the safety and efficacy of these agents. In particular, glycans of nonhuman origin, such as galactose-alpha-1,3-galactose (gal-α-gal) and N-glycolylneuraminic acid (NGNA), in the Fc region of therapeutic mAbs produced from murine cell lines carry a risk of immunogenicity. Immunogenic glycan structures can have immune-mediated clearance, resulting in faster clearance from in vivo circulation than non-immunogenic structures. To demonstrate the impact of these Fc nonhuman glycans on in vivo clearance, we purified and analyzed the glycan profile of a monoclonal antibody (mAb1) from human serum samples collected from clinical study participants. We purified mAb1 in a three-step chromatographic separation process (protein A, immobilized anti-mAb1 antibody affinity, and weak cation exchange chromatography) and extracted and labeled its N-linked oligosaccharide structures with 2-aminobenzamide acid for analysis on ultrahigh-performance hydrophilic interaction liquid chromatography. A comparison of the glycan profile of mAb1 recovered from human serum on the same day and 4 weeks after dosing revealed no significant differences, indicating similar clearance of mAb1 with nonhuman gal-α-gal or NGNA glycan in the Fc region compared with the human glycans. The relative proportions of the glycans remained similar, and all patients who had already received multiple doses of mAb1 over the course of a year were negative for antidrug antibodies, suggesting that none of the glycans induced an immune response. Therefore, we concluded that mAb1 gal-α-gal and NGNA glycoforms represent a low risk of conferring immunogenicity.

摘要

治疗性单克隆抗体 (mAb) 糖基化的异质性可能会影响这些药物的安全性和有效性。特别是非人类来源的聚糖，例如半乳糖-α-1,3-半乳糖 (gal-α-gal) 和N-羟乙酰神经氨酸 (NGNA)，在由鼠细胞系产生的治疗性 mAb 的 Fc 区具有免疫原性风险。免疫原性聚糖结构可以具有免疫介导的清除，导致比非免疫原性结构更快地从体内循环中清除。为了证明这些 Fc 非人聚糖对体内清除率的影响，我们从临床研究参与者收集的人血清样本中纯化并分析了单克隆抗体 (mAb1) 的聚糖谱。我们通过三步色谱分离过程（蛋白 A、固定化抗 mAb1 抗体亲和力和弱阳离子交换色谱）纯化 mAb1，并用 2-氨基苯甲酰胺酸提取和标记其 N-连接寡糖结构，用于分析超高性能亲水相互作用液相色谱。对同一天和给药后 4 周从人血清中回收的 mAb1 聚糖谱的比较显示没有显着差异，表明与人聚糖相比，mAb1 与非人 gal-α-gal 或 NGNA 聚糖在 Fc 区的清除率相似. 聚糖的相对比例保持相似，并且所有在一年内已​​经接受多剂量 mAb1 的患者的抗药抗体均为阴性，这表明没有一种聚糖诱导免疫反应。因此，我们得出结论，mAb1 gal-α-gal 和 NGNA 糖型代表赋予免疫原性的低风险。表明与人聚糖相比，Fc 区中非人 gal-α-gal 或 NGNA 聚糖对 mAb1 的清除率相似。聚糖的相对比例保持相似，并且所有在一年内已​​经接受多剂量 mAb1 的患者的抗药抗体均为阴性，这表明没有一种聚糖诱导免疫反应。因此，我们得出结论，mAb1 gal-α-gal 和 NGNA 糖型代表赋予免疫原性的低风险。表明与人聚糖相比，Fc 区中非人 gal-α-gal 或 NGNA 聚糖对 mAb1 的清除率相似。聚糖的相对比例保持相似，并且所有在一年内已​​经接受多剂量 mAb1 的患者的抗药抗体均为阴性，这表明没有一种聚糖诱导免疫反应。因此，我们得出结论，mAb1 gal-α-gal 和 NGNA 糖型代表赋予免疫原性的低风险。