Analysis of the immunoglobulin G (IgG) secretion efficiency in recombinant Chinese hamster ovary (CHO) cells by using Citrine-fusion IgG

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. Keywords Chinese hamster ovary cell Á Therapeutic antibody production Á Single clone analysis and isolation Á Fluorescent protein fusion antibody Á Yellow fluorescent protein (YFP) Á Intracellular IgG secretion

Secretion analysis of intracellular “difficult-to-express” immunoglobulin G (IgG) in Chinese hamster ovary (CHO) cells

“…These antibody components remained mainly within the ER for several hours, indicating that the post-translational modification of proteins in the ER and/or transportation of IgG between the ER and Golgi apparatus may be bottlenecks in IgG secretion. We also established cell lines producing IgG1 in which the LC is fused with fluorescent proteins to directly investigate the secretion process; results from these cells revealed that the IgG remained in the ER and cis-Golgi (Kaneyoshi et al 2018b). However, we did not analyze the secretion of so-called ''difficult-to-express'' (DTE) IgG in those works.…”

Artificial induction of chromosome aneuploidy in CHO cells alters their function as host cells

“…Human Reference Serum (Bethyl Laboratories, Inc.) was used as the standard. In other experiments, recombinant protein concentrations in the culture supernatants were determined by a sandwich enzyme‐linked immunosorbent assay as described previously (Kaneyoshi et al, 2019). Briefly, goat anti‐human IgG‐Fc (Bethyl Laboratories, Inc.) was used as the capture antibody and HRP‐conjugated goat anti‐human IgG (Bethyl Laboratories, Inc.) was used as the detection antibody.…”

Establishment of fast-growing serum-free immortalised cells from Chinese hamster lung tissues for biopharmaceutical production

“…Glucose, lactate, glutamine, glutamic acid, and ammonium ion concentrations in the cell supernatants were determined by BioProfile 400 (Nova Biomedical Corp., Waltham, MA, USA). Recombinant protein concentrations in the cell supernatants were determined by a sandwich enzyme-linked immunosorbent assay (ELISA), as described in a previous report 30 . Briefly, a goat anti-human IgG-Fc fragment (Bethyl Laboratories, Montgomery, TX, USA) was used as the capture antibody, and a horseradish peroxidase (HRP)-conjugated goat anti-human IgG-Fc fragment (Bethyl Laboratories) was used as the detection antibody.…”