Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, Okazaki 444-8787, Japan.
J Pharm Sci. 2019 Jul;108(7):2323-2333. doi: 10.1016/j.xphs.2019.02.023. Epub 2019 Mar 6.
Antibody aggregates are a potential risk for immunogenicity; therefore, rational approaches to improve associated aggregation properties need to be developed. Here, we report the amino acid region responsible for aggregation initiation. Two types of therapeutic IgG1 antibody monomer samples were prepared: IgG1 mAb40-3M stored at 40°C for 3 months, which existed in monodisperse state, and the monomer mAb65-5m, which was dissociated from small soluble aggregates by heating at 65°C for 5 min. Hydrogen deuterium exchange mass spectrometry of mAb40-3M identified 2 sites in the Fc region (site 1, F239-M256; site 2, S428-G450) with increased exchange rates. Site 1 includes a region reported as being susceptible to structural change induced by stress. Exposure of site 1 was undetected after 2 months of storage at 40°C but was subsequently detectable after 3 months. As site 2 is spatially close to site 1, the structural change of site 1 could propagate site 2. Besides these 2 regions, hydrogen deuterium exchange mass spectrometry of mAb65-5m identified an exposure of I257-W281 in Fc (site 3), within which a peptide sequence with high aggregation tendency was discovered. We thus concluded that exposure of site 3 is a trigger for the association of a partially denatured antibody.
抗体聚集是免疫原性的潜在风险；因此，需要开发合理的方法来改善相关的聚集特性。在这里，我们报告了导致聚集起始的氨基酸区域。制备了两种类型的治疗性 IgG1 抗体单体样品：在 40°C 下储存 3 个月的 IgG1 mAb40-3M，其处于单分散状态，以及通过在 65°C 加热 5 分钟从小可溶性聚集体中解离的单体 mAb65-5m。mAb40-3M 的氢氘交换质谱鉴定出 Fc 区域中的 2 个位点（位点 1，F239-M256；位点 2，S428-G450）的交换率增加。位点 1 包括一个据报道易受应激引起的结构变化影响的区域。在 40°C 下储存 2 个月后，未检测到位点 1 的暴露，但在 3 个月后可检测到。由于位点 2 与位点 1 空间上接近，因此位点 1 的结构变化可能会传播到位点 2。除了这两个区域之外，mAb65-5m 的氢氘交换质谱鉴定出 Fc 中 I257-W281 的暴露（位点 3），在其中发现了一个具有高聚集倾向的肽序列。因此，我们得出结论，位点 3 的暴露是部分变性抗体缔合的触发因素。
