Expression of Chimeric Antibody in Mammalian Cells using Dicistronic Expression Vector

Xiong, Kun; Liang, Qiwei; Xiong, Hua; Zou, Changxu; Gao, Guodong; Zhao, Zhenwei; Zhang, Hua

doi:10.1007/s10529-005-2736-3

Cited by 8 publications

(4 citation statements)

References 10 publications

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“…In IR/MAR HAC system, it reproducibly took 3–4 weeks to isolate a gene amplificated and recombinant protein produced CHO-DG44 cell line, and 3–4 weeks took to obtain MAR-HAC transfer to CHO-K1 cell line by MMCT (2 months in total). Additionally, those previous studies 30,31 were not been shown to assess stability over long-term protein expression. We showed the protein production rate was stable from the MAR-HAC vector in the long-term culture (Fig.…”

Section: Discussionmentioning

confidence: 92%

“…Recently, the great demand for therapeutic proteins has significantly advanced the manufacturing platform for recombinant protein production using the CHO cell culture system 6 . Several efforts, including cell engineering, optimization of cell line development, and cell culture medium optimization, have resulted in significant improvements in recombinant protein production 30–32 . However, recombinant cell lines developed for therapeutic antibody production often suffer from instability or lose protein expression during long-term culture 4 .…”

Section: Discussionmentioning

confidence: 99%

“…It has been reported that recombinant protein production facilitated by improved the MTX method using CHO cell line 30,31 . A plasmid engineered vector was developed to the increased production of anti-VEGF antibody form CHO dhfr defect cell lines 30 . The engineered vector transfected CHO cells showed more highly sensitive to MTX and increased gene amplification.…”

Section: Discussionmentioning

confidence: 99%

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An efficient protein production system via gene amplification on a human artificial chromosome and the chromosome transfer to CHO cells

Ohira

Miyauchi

Uno

et al. 2019

Sci Rep

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Gene amplification methods play a crucial role in establishment of cells that produce high levels of recombinant protein. However, the stability of such cell lines and the level of recombinant protein produced continue to be suboptimal. Here, we used a combination of a human artificial chromosome (HAC) vector and initiation region (IR)/matrix attachment region (MAR) gene amplification method to establish stable cells that produce high levels of recombinant protein. Amplification of Enhanced green fluorescent protein (EGFP) was induced on a HAC carrying EGFP gene and IR/MAR sequences (EGFP MAR-HAC) in CHO DG44 cells. The expression level of EGFP increased approximately 6-fold compared to the original HAC without IR/MAR sequences. Additionally, anti-vascular endothelial growth factor (VEGF) antibody on a HAC (VEGF MAR-HAC) was also amplified by utilization of this IR/MAR-HAC system, and anti-VEGF antibody levels were approximately 2-fold higher compared with levels in control cells without IR/MAR. Furthermore, the expression of anti-VEGF antibody with VEGF MAR-HAC in CHO-K1 cells increased 2.3-fold compared with that of CHO DG44 cells. Taken together, the IR/MAR-HAC system facilitated amplification of a gene of interest on the HAC vector, and could be used to establish a novel cell line that stably produced protein from mammalian cells.

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Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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An efficient protein production system via gene amplification on a human artificial chromosome and the chromosome transfer to CHO cells

Ohira

Miyauchi

Uno

et al. 2019

Sci Rep

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“…In most humanized antibody cases, chimeric antibodies are generated in the mammalian expression system (Xiong et al, 2005;Kim et al, 2011). However, the yield of transient expression in this expression system is usually low.…”

Section: Introductionmentioning

confidence: 99%

Development of a neutralizing mouse-pig chimeric antibody with therapeutic potential againstHaemophilus parasuisinPichia pastoris

Chai

Jiang

et al. 2014

FEMS Microbiol Lett

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Haemophilus parasuis is one of the most important bacterial diseases of pigs worldwide. The lack of a vaccine against a broad spectrum of strains and the limitation of antimicrobial susceptibility hamper the control of disease. In this study, we cloned the constant regions of gamma heavy chains and kappa light chain of pig lymphocytes in frame with the variable regions of heavy and light chains of mouse monoclonal antibody 1D8, which reacts with all 15 serotypes of H. parasuis and has neutralizing activity. The constructed mouse-pig chimeric antibody was expressed in Pichia pastoris. Results demonstrated that the expressed chimeric antibody inhibited the growth of H. parasuis in vitro. Furthermore, the experiments in mice showed that chimeric antibody increased survival rate of the mice compared with that of the control group (P < 0.05). Importantly, the chimeric antibody partially protected piglets against H. parasuis infection according to the clinical lesion scores and PCR results of H. parasuis in the tissues from piglets of the chimeric antibody-inoculated group and the PBS group. In summary, our results demonstrated that the mouse-pig chimeric antibody could be a therapeutic candidate to prevent the H. parasuis infection and control the prevalence of disease.

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Effects of high passage cultivation on CHO cells: a global analysis

Beckmann

Krämer

Klausing

et al. 2012

Appl Microbiol Biotechnol

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Cell lines for industrial pharmaceutical protein production processes need to be robust, fast-growing, and high-producing. In order to find such cells, we performed a high passage cultivation of monoclonal antibody producing Chinese hamster ovary (CHO) cells in shaking flasks for more than 420 days. Examinations of cell growth, productivity, intracellular protein, and metabolite characteristics as well as product transcript and genomic integrate levels revealed substantial differences between subpopulations that were cryopreserved from long-term cultivation at different time points. Detected growth performance as well as intracellular adenylate energy charge increased during high passage cultivation. In addition, proteome analysis indicated an augmented utilization of glycolysis with higher passage number and an enhanced robustness based on anti-stress proteins. Interestingly, the product formation increased at first but decreased dramatically during the later subcultivations, although selection pressure was applied. Utilizing flow cytometry and quantitative real-time polymerase chain reaction, we further examined the translational, transcriptional, and genomic basis for the observed phenotypes. The detected reduction of antibody expression, in particular of the heavy chain, was ascribed to a decrease of antibody transcript, caused by loss of gene copy number and assumably a malfunctioning splicing mechanism of the dicistronic mRNA. To our knowledge, this is the first systematic approach using process analytics and targeted omic techniques to elucidate the effects of long-term cultivation of CHO cells expressing a therapeutic protein.

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Expression of Chimeric Antibody in Mammalian Cells using Dicistronic Expression Vector

Cited by 8 publications

References 10 publications

An efficient protein production system via gene amplification on a human artificial chromosome and the chromosome transfer to CHO cells

An efficient protein production system via gene amplification on a human artificial chromosome and the chromosome transfer to CHO cells

Development of a neutralizing mouse-pig chimeric antibody with therapeutic potential againstHaemophilus parasuisinPichia pastoris

Effects of high passage cultivation on CHO cells: a global analysis

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