The production of biopharmaceutical immunoglobulin G (IgG) using cultured mammalian cells, especially Chinese hamster ovary (CHO) cells is well established and has been markedly improved through the modification of cells and cell culture engineering technologies. The establishment of high-production cell lines remains a challenge. The intracellular secretion of IgG has been investigated to identify and solve the rate-limiting steps in antibody production. However, strategies that regulate the expression of proteins that are related to antibody secretory pathway have not consistently improved their production. In this study, key features and limitations of the antibody secretion process in recombinant CHO cells were analyzed to develop more efficient approaches for establishing high-production cells. By chase assay with protein translation inhibitors, IgG secretion reached a plateau when at least 20% of IgG remained in the cells. The secretion kinetics and retention ratio of IgG varied between IgG subclasses (two types of IgG1 and an IgG3 subclass). Immunofluorescent microscopy and size exclusion chromatography showed that the remaining intracellular IgG localized mainly within the endoplasmic reticulum (ER) and less with the cis-Golgi network, despite the formation of fully assembled IgG. These results show that remaining intracellular IgG is a target for enhancing antibody secretion, even in high-production CHO cells. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.