Purpose

To evaluate the physical and chemical degradation of monoclonal antibodies in the presence of Zn²⁺.

Methods

A full length IgG1 monoclonal antibody (mAb1) was formulated with various amounts of Zn²⁺. The resulting mixture was incubated for several weeks at room temperature and analyzed using a variety of biochemical techniques to look for various physical (e.g. aggregation) and chemical (e.g. fragmentation) degradation pathways.

Results

mAb1 of the IgG1 subclass undergoes aggregation in the presence of Zn²⁺ in a concentration dependent manner. Up to hexamers were characterized using SEC-MALS. No fragmentation was noticed in the presence of Zn²⁺ as opposed to that found in our previous report when IgG1 mAbs were incubated in the presence of Cu²⁺ ions. Site directed mutagenesis indicated the involvement of Fc histidine (His 310) in Zn²⁺ mediated aggregation.

Conclusions

A novel metal ion mediated isodesmic aggregation mechanism was found in IgG1 class of monoclonal antibodies. Histidine residues in the Fc region were determined to be the binding site and implicated in Zn²⁺ mediated aggregation.

中文翻译：

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金属离子与 mAb 的相互作用：第 2 部分。锌介导的 IgG1 单克隆抗体聚集

目的

评估在 Zn ²⁺存在下单克隆抗体的物理和化学降解。

方法

^{用不同量的 Zn 2+}配制全长 IgG1 单克隆抗体 (mAb1) 。将所得混合物在室温下孵育数周，并使用多种生化技术进行分析，以寻找各种物理（例如聚集）和化学（例如碎裂）降解途径。

结果

IgG1 亚类的 mAb1 在 Zn ²⁺存在下以浓度依赖性方式发生聚集。使用 SEC-MALS 表征多达六聚体。^{在存在 Zn 2+}的情况下没有发现碎片，这与我们之前的报告中在存在 Cu ²⁺离子的情况下孵育 IgG1 mAb 时发现的情况相反。定点诱变表明 Fc 组氨酸 (His 310) 参与 Zn ²⁺介导的聚集。

结论

在 IgG1 类单克隆抗体中发现了一种新的金属离子介导的等桥聚集机制。Fc 区中的组氨酸残基被确定为结合位点并涉及Zn ²⁺介导的聚集。