Abstract
Biopharmaceuticals represented by immunoglobulin G (IgG) are produced by the cultivation of recombinant animal cells, especially Chinese hamster ovary (CHO) cells. It is thought that the intracellular secretion process of IgG is a bottleneck in the production of biopharmaceuticals. Many studies on the regulation of endogenous secretory protein expression levels have shown improved productivity. However, these strategies have not universally improved the productivity of various proteins. A more rational and efficient establishment of high producer cells is required based on an understanding of the secretory processes in IgG producing CHO cells. In this study, a CHO cell line producing humanized IgG1, which was genetically fused with fluorescent proteins, was established to directly analyze intracellular secretion. The relationship between the amount of intracellular and secreted IgG was analyzed at the single cell level by an automated single-cell analysis and isolation system equipped with dual color fluorescent filters. The amounts of intracellular and secreted IgG showed a weak positive correlation. The amount of secreted IgG analyzed by the system showed a weak negative linear correlation with the specific growth of isolated clones. An immunofluorescent microscopy study showed that the established clones could be used to analyze the intracellular secretion bottleneck. This is the first study to report the use of fluorescent protein fusion IgG as a tool to analyze the secretion of recombinant CHO cells.
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Acknowledgements
The authors would like to thank Dr. Onitsuka in Tokushima University for providing plasmids. We also thank Dr. Yu and Mr. Sakata from AS ONE Corporation for technical assistance with experiments using the automated single-cell analysis and isolation system. We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
Funding
This work was financially supported by the Project Focused on Developing Key Technology of Discovering and Manufacturing Drugs for Next-Generation Treatment and Diagnosis from the Ministry of Economy, Trade and Industry of Japan and from the Japan Agency for Medical Research and Development (AMED) under Grant Number 17ae0101003 and by Grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS; JP26630433, JP26249125, JP17H06157, and JP17J00927).
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Kaneyoshi, K., Yamano-Adachi, N., Koga, Y. et al. Analysis of the immunoglobulin G (IgG) secretion efficiency in recombinant Chinese hamster ovary (CHO) cells by using Citrine-fusion IgG. Cytotechnology 71, 193–207 (2019). https://doi.org/10.1007/s10616-018-0276-7
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DOI: https://doi.org/10.1007/s10616-018-0276-7