Impact of gene vector design on the control of recombinant monoclonal antibody production by chinese hamster ovary cells

Davies, Sarah L.; O'Callaghan, Peter M.; McLeod, Jane; Pybus, Leon P.; Sung, Yun Hee; Rance, James; Wilkinson, Stephen J.; Racher, Andrew J.; Young, Robert J.; James, David C.

doi:10.1002/btpr.692

Cited by 33 publications

(24 citation statements)

References 48 publications

(71 reference statements)

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“…Productivity of clones from a F2A-based vector was comparable with clones generated using a reference vector using separate expression unit design [104]. Interestingly, Davies et al recently reported that LC was still detected in excess in higher producing clones generated using the F2A system due to increased HC degradation [110]. One drawback of the F2A system is the possible formation of unwanted residues or fusion proteins [111,112].…”

Section: » Co-expression Of Lc and Hc Genesmentioning

confidence: 95%

Generation of monoclonal antibody-producing mammalian cell lines

Ho¹,

Yang²

2013

Pharmaceutical Bioprocessing

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Section: » Co-expression Of Lc and Hc Genesmentioning

confidence: 95%

Generation of monoclonal antibody-producing mammalian cell lines

Ho¹,

Yang²

2013

Pharmaceutical Bioprocessing

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“…26 F2A from the foot-andmouth disease virus, which is the most studied 2A, has been used for mAb expression in mammalian cells and for in vivo gene therapy. 18,21,[27][28][29][30][31] 2A peptides have approximately 20 amino acids and "self-cleavage" occurs between the last 2 amino acids, glycine (G) and proline (P). Adding a furin recognition sequence between the first gene and 2A aids in removing 2A residues from the upstream gene.…”

Section: Introductionmentioning

confidence: 99%

“…18,21 However, incomplete cleavage of F2A was observed in some studies, resulting in HC-F2A-LC or LC-F2A-HC fusion proteins and LC and HC attached with F2A remnants. 18,27,30 The incorrectly processed peptides formed aggregates that could not be removed by protein A purification. 18 Besides F2A, E2A from equine rhinitis A virus, P2A from porcine teschovirus-1 and T2A from Thosea asigna virus have also been used widely in biomedical research.…”

Section: Introductionmentioning

confidence: 99%

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

Chng

Wang

Nian

et al. 2015

mAbs

132

140

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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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“…More important we showed that expression of H chain in the pOptiVEC vector, which contains the MTX regulatory element, severely suppressed gene multiplication. The importance of disproportional H and L chain production for optimal Ab assembly was studied by Davies et al (2011). The authors used dual or single open reading frame expression vectors to drive H and L chains expression independently or jointly.…”

Section: Discussionmentioning

confidence: 99%

Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains

et al. 2014

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Tumor necrosis factor-a (TNF-a) plays a key role in rheumatoid arthritis and some other autoimmune diseases. Therapy with anti-TNF-a recombinant antibodies (Ab) appears to be highly effective. Production of new hyper-producing eukaryotic cell lines can decrease the treatment cost, which currently is very high. However, due to the complexity of protein transcription, translation, processing, and secretion in mammalian cells, the stages at which antibody expression is affected are still poorly determined. The aim of this work was to compare the productivity of two cell lines developed in CHO DG44 cells, deficient in dihydrofolate reductase, transfected with vectors carrying either heavy (H) or light (L) chains of chimeric antibody under different combinations of selective elements. Both H and L chains were cloned either in pOptiVEC or pcDNA3.3 vectors and different combinations were used to produce HL and LH cell lines. We have shown that Ab production has been low and comparable between HL and LH cells until selection on methotrexate (MTX) when LH but not HL cells have responded with 3.5 times increased productivity. Flow cytometry analysis has demonstrated that intracellular concentration of full size Abs in LH cells was 5.6 times higher than in HL ones due to higher amount of H chain synthesis. No differences in viability between HL and LH cells have been found. We have concluded that the expression of H chain in the pOptiVEC vector, which is responsible for MTX resistance, has led to the suppression of H chain synthesis and limitation in full Ab assembly.

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Impact of gene vector design on the control of recombinant monoclonal antibody production by chinese hamster ovary cells

Cited by 33 publications

References 48 publications

Generation of monoclonal antibody-producing mammalian cell lines

Generation of monoclonal antibody-producing mammalian cell lines

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

Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains

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