Generation of monoclonal antibody-producing mammalian cell lines

Ho, Soo Yei; Yang, Yen Wah Tong Yuansheng

doi:10.4155/pbp.13.8

Cited by 35 publications

(36 citation statements)

References 217 publications

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“…1 Chinese hamster ovary (CHO) cells are the dominant host for producing mAb because of their capacity to perform proper folding, assembly and human-like glycosylation. 2 Each IgG molecule consists of 2 identical heavy chain (HC) and 2 identical light chain (LC) polypeptides. Generating a mAb-producing cell line starts with transfecting CHO cells with plasmid vectors carrying the LC, HC and selection marker genes.…”

Section: Introductionmentioning

confidence: 99%

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

Chng

Wang

Nian

et al. 2015

mAbs

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(2015) Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells, mAbs, 7:2, 403-412, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Abbreviations: CHO, Chinese hamster ovary; HC, heavy chain; LC, light chain; mAb, monoclonal antibody; F2A, 2A peptide derived from the foot-and-mouth disease virus; E2A, 2A peptide derived from the equine rhinitis virus; P2A, 2A peptide derived from the porcine teschovirus-1; T2A, 2A peptide derived from the Thosea asigna virus; GF2A, F2A with the GSG linker; GE2A, E2A with the GSG linker; GP2A, P2A with the GSG linker; GT2A, T2A with the GSG linker; IgG, immunoglobulin G; IRES, internal ribosome entry site; G, glycine; P, proline; K, lysine; MTX, methotrexate; SEC, size exclusion chromatography; MS, mass spectrometry; PFM, protein-free medium; HT, hypoxanthine and thymine; GFP, green fluorescence protein; PVDF, polyvinylidene difluorideLinking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.

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Section: Introductionmentioning

confidence: 99%

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

Chng

Wang

Nian

et al. 2015

mAbs

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149

140

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“…Several studies have shown that employing a single vector provides a tighter control over the expression ratio of LC/HC [6, 11, 12]. Co-expression of heavy and light chains under the control of a single promoter can be achieved using either viral internal ribosome entry site (IRES) or viral 2A-self cleavage sequences [13]. …”

Section: Introductionmentioning

confidence: 99%

Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells

Ebadat

Ahmadi

et al. 2017

PLoS ONE

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In recent years, monoclonal antibodies (mAbs) have been developed as powerful therapeutic and diagnostic agents and Chinese hamster ovary (CHO) cells have emerged as the dominant host for the recombinant expression of these proteins. A critical step in recombinant expression is the utilization of strong promoters, such as the Chinese Hamster Elongation Factor-1α (CHEF-1) promoter. To compare the strengths of CHEF with cytomegalovirus (CMV) promoter for mAb expression in CHO cells, four bicistronic vectors bearing either internal ribosome entry site (IRES) or Furin/2A (F2A) sequences were designed. The efficiency of these promoters was evaluated by measuring level of expressed antibody in stable cell pools. Our results indicated that CHEF promoter-based expression of mAbs was 2.5 fold higher than CMV-based expression in F2A-mediated vectors. However, this difference was less significant in IRES-mediated mAb expressing cells. Studying the stability of the F2A expression system in the course of 18 weeks, we observed that the cells having CHEF promoter maintained their antibody expression at higher level than those transfected with CMV promoter. Further analyses showed that both IRES-mediated vectors, expressed mAbs with correct size, whereas in antibodies expressed via F2A system heterogeneity of light chains were detected due to incomplete furin cleavage. Our findings indicated that the CHEF promoter is a viable alternative to CMV promoter-based expression in F2A-mediated vectors by providing both higher expression and level of stability.

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“…This cell line offers several advantages, such as a proven safety profile of products from these cells for humans, less permissive to infection by viruses, easy to obtain high productivity and having capacity to perform human compatible glycosylation . A typical process of cell line development for producing mAbs starts with transfecting the CHO cells with plasmid vectors carrying the recombinant antibody and selection marker genes . Drug selection is next performed to isolate a stably transfected pool of cells having plasmid vectors integrated into the chromosome.…”

Section: Introductionmentioning

confidence: 99%

“…A large number of clones need to be screened to obtain cell lines with high productivity, long term stable production, and good product quality which are required to satisfy the production and regulatory requirements. Much effort has been devoted to improving the process of cell line development, such as optimization of plasmid vectors using strong promoters/enhancers and chromatin regulatory elements, developing high‐throughput clone selection methods using fluorescence‐activated cell sorting (FACS) and automated clone picker ClonePix, and overcoming position effect on gene expression by directing the integration of plasmid vectors into the active sites in genome . As an essential component of the plasmid vectors, engineering the selection markers to enhance the stringency of selection for high producing cells in a stably transfected pool is one of the most effective approaches to improve the efficiency of cell line development …”

Section: Introductionmentioning

confidence: 99%

Optimized Selection Marker and CHO Host Cell Combinations for Generating High Monoclonal Antibody Producing Cell Lines

Yeo

Yang

Mariati

et al. 2017

Biotechnology Journal

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There are several selection markers which are suitable for generating stably transfected Chinese hamster ovary (CHO) cell lines. Due to their different modes of action, each selection marker has its own optimal selection stringency in different host cells for obtaining high productivity. Using an internal ribosome entry site (IRES)-mediated tricistronic vector and a set of IRES variants with different strengths, the expression of five antibiotics resistance genes (ARGs) in CHO-K1 cells and dihydrofolate reductase (DHFR) in CHO DG44 cells is optimized to enhance the stringency of selection for high producing cells. There is an obvious optimal expression level for every selection marker, below or above which, the productivity is significantly lower. The enhanced productivity in ARG generated CHO K1 cells is due to selective integration of active site while the enhanced productivity in the amplified CHO DG44 cells results from increased gene copies. The high producing CHO K1 pools and clones generated using ARG exhibit better production stability than the amplified high producing CHO DG44 pools and clones. Loss of expression for the CHO K1 cell lines is due to loss of gene copies while for CHO DG44 is due to transcriptional silencing. mAb glycan profile also differed significantly between CHO K1 and CHO DG44 cell lines. These results would be helpful when developing optimized vectors for generating high mAb producing CHO cell lines.

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Generation of monoclonal antibody-producing mammalian cell lines

Cited by 35 publications

References 217 publications

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells

Optimized Selection Marker and CHO Host Cell Combinations for Generating High Monoclonal Antibody Producing Cell Lines

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