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

BackgroundThe 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.MethodsAn 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 natural insulin removal from the products by immunopurification.FindingsAll insulin glargine products elicited at least a minor innate immune response comparable to natural 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 native insulin receptor through the MAPK pathway, and (3) the removal of insulin glargine 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 glargine dimers or others contaminants in these products.ConclusionThe 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 the biochemical composition of non-originator insulin glargine products (dimers, impurities) might be responsible for their greater immunostimulatory potential.

**研究背景**：胰岛素类似物的生产需采用复杂精密的制备工艺，该过程可导致终产物在结构、纯度及其他理化性质上产生差异，进而影响其生物学活性。本研究旨在借助体外人类免疫模型，对比原研（originator）与非原研（non-originator）甘精胰岛素（insulin glargine）的先天免疫活性，以及引发两类产品免疫应答谱差异的潜在机制。

**研究方法**：本研究采用基于内皮细胞/树突状细胞的先天免疫模型，探究原研与非原研甘精胰岛素产品所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变化。机制研究包含以下内容：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受试产品，以及采用免疫纯化法去除产品中的天然胰岛素。

**研究结果**：所有甘精胰岛素产品均可触发至少轻度的先天免疫应答，其水平与天然人胰岛素相当，但部分批次的非原研甘精胰岛素产品可诱导白细胞介素-8（IL-8）与白细胞介素-6（IL-6）的分泌水平升高。在阐明上述产品导致细胞因子生成差异的机制研究中，我们发现：① 炎症应答并非由细菌污染物介导；② 先天免疫应答通过丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）通路由天然胰岛素受体驱动；③ 去除甘精胰岛素可显著降低受试产品诱导先天免疫活性的能力。本研究未检测到产品存在聚集现象，但部分高分子量蛋白的存在提示，此类产品中可能存在甘精胰岛素二聚体或其他污染物。

**研究结论**：本研究数据显示，部分批次的非原研甘精胰岛素产品可在体外引发更强的人类先天免疫应答；同时提供了初步证据，表明非原研甘精胰岛素产品的生化组成变化（如二聚体、杂质含量改变）可能是其免疫刺激潜能更强的原因。