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The novel heavy-chain antibody known as immunoglobulin new antigen receptor (IgNAR) is derived from cartilaginous fishes such as sharks. IgNAR, which binds to antigens with the high specificity and affinity of a conventional IgG antibody and exhibits high resistance to denaturation, has potential as a next-generation antibody in biopharmaceutical and biotechnological applications. High-level expression of recombinant IgNAR in animal cells has been challenging. In our previous study, IgNAR was expressed as a fusion protein with a human IgG Fc region (IgNAR-Fc) in Chinese hamster ovary (CHO) cells, but did not meet the production level required for further research and application. In this study, we sought to identify the production bottleneck in CHO cells as a first step toward achieving abundant production of IgNAR. Using an established IgG high-production CHO cell line as a comparator, we found that the amounts of intracellular dimeric IgNAR-Fc produced in CHO cells were similar to those of intracellular dimeric IgG. Furthermore, the majority of intracellular IgNAR-Fc was retained in the endoplasmic reticulum (ER) and strongly colocalized to ERGIC-53, the cargo receptor for coat protein complex II (COP II)-coated vesicles. These findings suggest that COP II-mediated ER-to-Golgi transport may represent a bottleneck for IgNAR-Fc production in the CHO cell expression system., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)