Abstract
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.
Highlights
Insulin glargine is an analog of the native insulin peptide hormone that has a well-established role in regulating carbohydrate metabolism via its ability to increase glucose transport across cell membranes, enhance cellular glycolysis, and trigger glycogen synthesis in various cell types [1]
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
It is perhaps less well appreciated that insulin glargine, like any peptide, can affect the immune system: Given the ubiquitous expression of insulin receptors (IRs) on immunocytes that will interact with insulin glargine, insulin glargine can modulate a variety of immune processes, such as chemotaxis, phagocytosis, and chemokine/cytokine production [2,3]
Summary
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. 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
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