Data from: Evaluation of the innate immunostimulatory potential of originator and non-originator copies of insulin glargine in an in vitro human immune model

Background: The manufacture of insulin analogs requires sophisticated production procedures which can lead to differences in the structure, purity, and/or other physiochemical properties of resultant products that can affect their biologic activity. Here, we sought to compare originator and non-originator copies of insulin glargine for innate immune activity and mechanisms leading to differences in these response profiles in an in vitro model of human immunity. Methods: An endothelial/dendritic cell-based innate immune model was used to study antigen-presenting cell activation, cytokine secretion, and insulin receptor signalling pathways induced by originator and non-originator insulin glargine products. Mechanistic studies included signalling pathway blockade with specific inhibitors, analysis of the products in a Toll-like receptor reporter cell line assay, and insulin removal from the products by immunopurification. Findings: All insulin glargine products elicited at least a minor innate immune response comparable to human insulin, but some lots of a non-originator copy product induced the elevated secretion of the cytokines, IL-8 and IL-6. In studies aimed at addressing the mechanisms leading to differential cytokine production by these products, we found (1) the inflammatory response was not mediated by bacterial contaminants, (2) the innate response was driven by the insulin receptor through the MAPK pathway, and (3) the removal of insulin significantly reduced their capacity to induce innate activity. No evidence of product aggregates was detected, though the presence of some high molecular weight proteins argues for the presence of insulin dimers or others contaminants in these products. Conclusion: The data presented here suggests some non-originator insulin glargine product lots drive heightened in vitro human innate activity and provides preliminary evidence that changes in their biochemical composition (dimers, impurities) might be responsible for their greater immunostimulatory potential.

背景：胰岛素类似物（insulin analogs）的生产需借助复杂的制备工艺，该过程可能导致终产品在结构、纯度及其他理化性质上出现差异，进而影响其生物学活性。本研究旨在对比甘精胰岛素（insulin glargine）的原研产品与仿制产品的先天免疫活性，并在人类体外免疫模型中探究两类产品应答谱产生差异的潜在机制。

方法：本研究采用基于内皮细胞-树突状细胞的先天免疫模型，分析原研与仿制甘精胰岛素诱导的抗原呈递细胞活化、细胞因子分泌及胰岛素受体信号通路。机制研究环节包括：使用特异性抑制剂阻断信号通路、通过Toll样受体（Toll-like receptor）报告细胞系实验分析产品特性，以及通过免疫纯化去除产品中的胰岛素组分。

结果：所有受试甘精胰岛素产品均可引发至少轻度的先天免疫应答，其应答水平与人胰岛素相当；但某款仿制甘精胰岛素的部分批次可诱导IL-8与IL-6的分泌水平升高。针对两类产品诱导细胞因子产生差异的机制研究显示：(1) 炎症应答并非由细菌污染物介导；(2) 先天免疫应答通过胰岛素受体依赖丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）通路激活；(3) 去除产品中的胰岛素可显著降低其诱导先天免疫活性的能力。本研究未检测到明显的产品聚集物，但部分高分子量蛋白的存在提示产品中可能存在胰岛素二聚体或其他污染物。

结论：本研究数据表明，部分甘精胰岛素仿制药批次可引发更强的体外人类先天免疫活性，且初步证据显示其生化组成（如二聚体、杂质）的差异可能是其免疫刺激潜能更高的原因。