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Alternating tangential flow filtration (ATF) has become one of the primary methods for cell retention and clarification in perfusion bioreactors. However, product sieving losses due to membrane fouling still limits the performance of these systems. This study used scanning electron microscopy (SEM) and energy dispersive X?ray spectroscopy (EDS) to identify the nature and location of foulants on 0.2 ?m polyethersulfone hollow fiber membranes after use in industrial Chinese Hamster Ovary (CHO) cell perfusion bioreactors for monoclonal antibody production. Membrane fouling was dominated by proteinaceous material, primarily host cell proteins along with some monoclonal antibody. Fouling occurred primarily on the lumen surface with much less protein trapped within the depth of the fiber. Protein deposition was also most pronounced near the inlet / exit of the hollow fibers, which are the regions with the greatest flux (and transmembrane pressure) during the cyclical operation of the ATF. These results provide important insights into the underlying phenomena governing the fouling behavior of ATF systems for continuous bioprocessing.This article is protected by copyright. All rights reserved.