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Endotoxins are a highly pyrogenic and immunogenic contaminant of bacterial origin that must be avoided during the manufacturing of biopharmaceutical products to ensure safety and efficacy. Low endotoxin recovery, also known as a masking effect, is defined as the ability to detect <50% [21] of the expected endotoxin in an endotoxin assay. Masking can be caused by the ability of endotoxins to build aggregates, bind to the protein or organise in micelles or vesicles that in turn inhibit detection of the endotoxin in the solution being tested. Therefore, a masking effect can result from physical parameters of the molecule being tested or from the buffer/environmental conditions of the solution the molecule is in. This can subsequently lead to the underestimation of endotoxin contaminations and lead to a potential false negative test. Tight control over the effectiveness of the downstream process and the use of well-characterised endotoxin testing assays are needed to ensure optimal endotoxin removal. This manuscript demonstrates the capacity to remove the endotoxins within a proven acceptable range by also controlling and evaluating the potential masking effects during downstream process at ambient temperature and also during sample storage condition until the analyse was performed. The endotoxin removal study (ERS) is divided in the initial part to evaluate the process buffers and the conditions of the molecule to avoid the underestimation of endotoxins in process samples in advance. This pre-study is a necessary prerequisite to evaluate the results after the endotoxin spiked downstream unit operations. With those aspects, the removal capacity can be demonstrated. A study was carried out to characterise the endotoxin removal capability of the purification process including controlling of masking effects. The endotoxin removal capacity on ion exchange chromatography and during ultrafiltration/diafiltration unit operations of the downstream processing of an immunoglobulin G1 antibody was conducted using various process parameters to understand their impact on endotoxin removal. In the small-scale study, the processing steps from each tested unit operation were spiked with Escherichia coli endotoxins. The potential masking effect during purification was addressed by controlling the hold time by spiking studies of the different generated pools at ambient temperature. By conducting a masking study, all generated protein pools (flow-through/wash, eluate and regeneration pools) had no masking effect caused through sample handling prior to analysis. Overall, this study showed that endotoxins could be successfully removed by anion exchange chromatography. A partial removal could be achieved by cation exchange chromatography and endotoxins could not be removed with ultrafiltration/diafiltration., Competing Interests: Declaration of Competing Interest None, (Copyright © 2022. Published by Elsevier B.V.)