Newly established cell lines derived from Chinese hamster for production of biologics

The Chinese hamster (Cricetulus griseus) is a species of hamster that was used as a laboratory animal more than 50 years ago. The Chinese hamster ovary (CHO) cell line was established in 1957 by Puck et al. and maintained in ex vivo conditions (1). CHO cells are now a workhorse for recombinant biopharmaceutical production. Puck et al. continuously cultivated lung, kidney, spleen and ovary-derived cells for more than 10 months using serum medium. In this study, we established a cell line from female Chinese hamster tissues: lung, kidney and ovary. Primary cells were obtained from these tissues and maintained for several months or more (Figure 1). We were ultimately able to construct three immortal cell lines, CHL-YN (fibroblast) from lung, CHK-Q (epitheliocyte) from kidney and CHO-MK (epitheliocyte) from ovary, respectively. Infinite proliferation of these cell lines is obtained by spontaneous transformation. Please click Additional Files below to see the full abstract

Newly-established Chinese hamster-derived cell line for protein production

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Varied productivity according to the differences between targeted locations of antibody expression vectors in Chinese Hamster ovary cells

Chinese hamster ovary (CHO) cell lines are widely used in the pharmaceutical industry to produce therapeutic antibodies. However, trial and error cell selection methods are still used to construct high-producing cell lines. Exogenous genes are predicted to express differently depending on the expression vector integration sites. Chromosomal instability is one of the characteristics in CHO cells. We have previously constructed the CHO genomic bacterial artificial chromosome (BAC) library that is expected to cover entire the CHO-DG44 genome (Omasa et al., Biotechnol. Bioeng., 104, 986-994, 2009). The BAC-based physical map is a powerful tool to identify each chromosome and analyze chromosome rearrangement of CHO cell lines. According to the previous results, stability of each chromosome in a CHO cell differs. In this study, we constructed antibody producing cell lines using gene-targeting methods, and investigated the effect of targeting sites differences on the protein production. IgG1 expression vectors and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated proteins) vectors, which cut target sites, were co-transfected into CHO-DG44 cell line. The stably conserved chromosome and the other chromosome were selected as targeting sites. The targeting sequences were obtained from the CHO genomic BAC library: Cg0160L03 (Cao et al., Biotechnol. Bioeng., 109, 1357-1367, 2012) for the conserved chromosome; Cg0031N14, identified to contain exogenous gene amplified region with a large palindrome structure (Park et al., J. Biosci. Bioeng., 109, 504-511, 2010), for the other chromosome. The result showed that the specific antibody production rates were about 15 times higher in cell lines where the expression vectors were targeted into the conserved chromosomes than in the cell lines where expression vectors were targeted into the other chromosome. Our results indicated that the productivity varied according to the differences between targeted chromosomes

Metabolomic characterization of monoclonal antibody-producing Chinese hamster lung (CHL)-YN cells in glucose-controlled serum-free fed-batch operation

Sukwattananipaat P., Kuroda H., Yamano-Adachi N., et al. Metabolomic characterization of monoclonal antibody-producing Chinese hamster lung (CHL)-YN cells in glucose-controlled serum-free fed-batch operation. Biotechnology and Bioengineering, (2024); https://doi.org/10.1002/bit.28777.The fast-growing Chinese hamster lung (CHL)-YN cell line was recently developed for monoclonal antibody production. In this study, we applied a serum-free fed-batch cultivation process to immunoglobulin (Ig)G1-producing CHL-YN cells, which were then used to design a dynamic glucose supply system to stabilize the extracellular glucose concentration based on glucose consumption. Glucose consumption of the cultures rapidly oscillated following three phases of glutamine metabolism: consumption, production, and re-consumption. Use of the dynamic glucose supply prolonged the viability of the CHL-YN-IgG1 cell cultures and increased IgG1 production. Liquid chromatography with tandem mass spectrometry-based target metabolomics analysis of the extracellular metabolites during the first glutamine shift was conducted to search for depleted compounds. The results suggest that the levels of four amino acids, namely arginine, aspartate, methionine, and serine, were sharply decreased in CHL-YN cells during glutamine production. Supporting evidence from metabolic and gene expression analyses also suggest that CHL-YN cells acquired ornithine- and cystathionine-production abilities that differed from those in Chinese hamster ovary-K1 cells, potentially leading to proline and cysteine biosynthesis

Intracellular secretion analysis of therapeutic antibodies in engineered high- producible CHO cells

The Chinese Hamster Ovary (CHO) cell is the most commonly used cell line for the production of therapeutic recombinant proteins. The improvements in target gene amplification and culture method have contributed in achieving a very high productivity. Some studies have focused on post-translational secretion processes, and overexpression of proteins which work in the secretion pathway successfully increased the productivity [1]. However, those studies were performed based on the knowledge obtained from the normal, adherent cultured cells, and the detailed secretion processes of recombinant proteins in engineered, suspension cultured cells is still unclear. To clarify problems and to find new targets for a more efficient establishment of high producers, the basic analyses about the secretion in engineered, high-producible CHO cells were performed. Please click Additional Files below to see the full abstract

Construction of a novel kinetic model for the production process of a CVA6 VLP vaccine in CHO cells

Bioprocess development benefits from kinetic models in many aspects, including scale-up, optimization, and process understanding. However, current models are unable to simulate the production process of a coxsackievirus A6 (CVA6) virus-like particle (VLP) vaccine using Chinese hamster ovary cell culture. In this study, a novel kinetic model was constructed, correlating (1) cell growth, death, and lysis kinetics, (2) metabolism of major metabolites, and (3) CVA6 VLP production. To construct the model, two batches of a laboratory-scale 2 L bioreactor cell culture were prepared and various pH shift strategies were applied to examine the effect of pH shift. The proposed model described the experimental data under various conditions with high accuracy and quantified the effect of pH shift. Next, cell culture performance with various pH shift timings was predicted by the calibrated model. A trade-off relationship was found between product yield and quality. Consequently, multiple objective optimization was performed by integrating desirability methodology with model simulation. Finally, the optimal operating conditions that balanced product yield and quality were predicted. In general, the proposed model improved the process understanding and enabled in silico process development of a CVA6 VLP vaccine.The version of record of this article, first published in Cytotechnology, is available online at Publisher’s website: https://doi.org/10.1007/s10616-023-00598-

Characterization of Mouse Tissue Kallikrein 5

Mouse tissue kallikreins (Klks) are members of a large, multigene family consisting of 37 genes, 26 of which can code for functional proteins. Mouse tissue kallikrein 5 (KIk5) has long been thought to be one of these functional genes, but the gene product, mK5, has not been isolated and characterized. In the present study, we prepared active recombinant mK5 using an Escherichia coli expression system, followed by column chromatography. We then determined the biochemical and enzymatic properties of purified mK5. mK5 had trypsin-like activity for Arg at the P1 position, and its activity was inhibited by typical serine protease inhibitors. mK5 degraded gelatin, fibronectin, collagen type IV, high-molecular-weight kininogen, and insulin-like growth factor binding protein-3. Our data suggest that mK5 may be implicated in the process of extracellular matrix remodeling

Synthesis of Human Antibodies Against HBsAg in Newly Established Chinese Hamster Lung (CHL-YN) Cell Line

Hepatitis B immunoglobulin (HBIG) is an effective treatment for hepatitis B, including postexposure prophylaxis of HBV infection, prevention of HBV reinfection in liver transplant patients, and reducing sexual transmission. This study investigated the synthesis of human IgG antibodies that specifically target HBsAg subtype adr in CHL-YN cells, a newly established cell line that grows faster than CHO-K1. To achieve the synthesis of human IgG antibodies, a plasmid vector encoding DNA sequences for human IgG antibodies against HBsAg was constructed and then transiently transfected into CHL-YN cells. The expression and antigen-binding capacity of the recombinant human IgG antibodies were analyzed using western blot and ELISA. The results showed successful expression and secretion of human IgG antibodies that recognize HBsAg subtype adr in CHL-YN cells. The ELISA test confirmed the specificity of the human IgG antibodies towards HBsAg subtype adr. Thus, this study concluded that human IgG antibodies that target HBsAg subtype adr were transiently expressed in CHL-YN cells