Subvisible aggregates of proteins are suspected to cause adverse immune response, and a recent FDA guideline has recommended the monitoring of micrometer-sized aggregates (2-10 μm) though recognizing that the underlying mechanism behind aggregation and immunogenicity remains unclear. Here, we report a correlation between the immunogenicity and the size of nanometer-scaled aggregates of a small 6.5 kDa model protein, bovine pancreatic trypsin inhibitor (BPTI) variant. BPTI-19A, a monomeric and nonimmunogenic protein, was oligomerized into subvisible aggregates with hydrodynamic radii (Rh) of 3-4 nm by attaching hydrophobic solubility controlling peptide (SCP) tags to its C-terminus. The results showed that the association of nonimmunogenic BPTI into nanometer-sized subvisible aggregates made it highly immunogenic, as assessed by the IgG antibody titers of the mice's sera. Overall, the study emphasizes that subvisible aggregates, as small as a few nanometers, which are presently ignored, are worth monitoring for deciphering the origin of undesired immunogenicity of therapeutic proteins.

中文翻译：

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使用短的溶解度控制肽标签生成的纳米大小的集合体确实增加了非免疫原性蛋白质的体内免疫原性。

怀疑蛋白质的亚可见聚集体会引起不良的免疫反应，尽管认识到聚集和免疫原性的潜在机制尚不清楚，但最近的FDA指南建议监测微米级的聚集体（2-10μm）。在这里，我们报告免疫原性和小的6.5 kDa模型蛋白，牛胰胰蛋白酶抑制剂（BPTI）变体的纳米级聚集体大小之间的相关性。BPTI-19A是一种单体且非免疫原性蛋白，通过将疏水性溶解度控制肽（SCP）标签连接到其C端，使其低聚为具有3-4 nm流体力学半径（Rh）的亚可见聚集体。结果表明，非免疫原性BPTI与纳米级亚可见聚集体的结合使其具有高度免疫原性，如通过小鼠血清的IgG抗体滴度评估的。总的来说，该研究强调，目前被忽略的小至几纳米的亚可见聚集体，在破译治疗性蛋白质不希望的免疫原性的起源方面值得监测。