Production of recombinant protein therapeutics in cultivated mammalian cells

doi:10.1038/NBT1026

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Production of recombinant protein therapeutics in cultivated mammalian cells

Journal Article•DOI•

Production of recombinant protein therapeutics in cultivated mammalian cells

Florian M. Wurm¹•Institutions (1)

École Polytechnique Fédérale de Lausanne¹

01 Nov 2004-Nature Biotechnology (Nature Publishing Group)-Vol. 22, Iss: 11, pp 1393-1398

TL;DR: Recently, the productivity of mammalian cells cultivated in bioreactors has reached the gram per liter range in a number of cases, a more than 100-fold yield improvement over titers seen for similar processes in the mid-1980s.

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Abstract: Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications because of their capacity for proper protein folding, assembly and post-translational modification. Thus, the quality and efficacy of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria, plants and yeast. Recently, the productivity of mammalian cells cultivated in bioreactors has reached the gram per liter range in a number of cases, a more than 100-fold yield improvement over titers seen for similar processes in the mid-1980s. This increase in volumetric productivity has resulted mainly from improvements in media composition and process control. Opportunities still exist for improving mammalian cell systems through further advancements in production systems as well as through vector and host cell engineering.

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Journal Article•DOI•

The therapeutic monoclonal antibody market

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Dawn M Ecker, Susan Dana Jones, Howard L. Levine

14 Jan 2015-mAbs

TL;DR: Since the commercialization of the first therapeutic monoclonal antibody product in 1986, this class of biopharmaceutical products has grown significantly so that, as of November 10, 2014, forty-seven monoclotal antibody products have been approved in the US or Europe for the treatment of a variety of diseases.

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Abstract: Since the commercialization of the first therapeutic monoclonal antibody product in 1986, this class of biopharmaceutical products has grown significantly so that, as of November 10, 2014, forty-seven monoclonal antibody products have been approved in the US or Europe for the treatment of a variety of diseases, and many of these products have also been approved for other global markets. At the current approval rate of ∼ four new products per year, ∼ 70 monoclonal antibody products will be on the market by 2020, and combined world-wide sales will be nearly $125 billion.

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1,099 citations

Patent•

Dual variable domain immunoglobulins and uses thereof

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Chengbin Wu¹, Tariq Ghayur¹, Richard W. Dixon, Jochen G. Salfeld•Institutions (1)

AbbVie¹

18 Aug 2006

TL;DR: The present invention relates to engineered multivalent and multispecific binding proteins, methods of making, and specifically to their uses in the prevention, diagnosis, and/or treatment of disease.

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Abstract: The present invention relates to engineered multivalent and multispecific binding proteins, methods of making, and specifically to their uses in the prevention, diagnosis, and/or treatment of disease.

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900 citations

Journal Article•DOI•

Downstream processing of monoclonal antibodies--application of platform approaches.

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Abhinav A. Shukla¹, Brian Hubbard¹, Tim Tressel¹, Sam Guhan¹, Duncan Low¹ - Show less +1 more•Institutions (1)

Amgen¹

15 Mar 2007-Journal of Chromatography B

TL;DR: This paper describes the key elements of a flexible, generic downstream process platform for mAbs that has been adopted at Amgen and consists of a well-defined sequence of unit operations with most operating parameters being pre-defined and a small subset of parameters requiring development effort.

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825 citations

Journal Article•DOI•

The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

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Xun Xu, Harish Nagarajan, Nathan E. Lewis, Shengkai Pan, Zhiming Cai¹, Xin Liu, Wenbin Chen, Min Xie, Wenliang Wang, Stephanie Hammond², Mikael Rørdam Andersen³, Norma F. Neff⁴, Benedetto Passarelli⁴, Winston Koh⁴, H. Christina Fan⁴, Jianbin Wang⁴, Yaoting Gui¹, Kelvin H. Lee², Michael J. Betenbaugh³, Michael J. Betenbaugh⁵, Stephen R. Quake⁴, Iman Famili, Bernhard O. Palsson³, Jun Wang⁶ - Show less +20 more•Institutions (6)

Peking University¹, Delaware Biotechnology Institute², Technical University of Denmark³, Howard Hughes Medical Institute⁴, Johns Hopkins University⁵, University of Copenhagen⁶

01 Aug 2011-Nature Biotechnology

TL;DR: A draft genomic sequence of the CHO-K1 ancestral cell line is presented and it is discussed how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production.

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Abstract: Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of the assembled scaffolds with 21 chromosomes isolated by microfluidics to identify chromosomal locations of genes. Furthermore, we investigate genes involved in glycosylation, which affect therapeutic protein quality, and viral susceptibility genes, which are relevant to cell engineering and regulatory concerns. Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome, although 141 of these homologs are not expressed under exponential growth conditions. Many important viral entry genes are also present in the genome but not expressed, which may explain the unusual viral resistance property of CHO cell lines. We discuss how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production.

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759 citations

Cites background from "Production of recombinant protein t..."

...This is consistent with the observation that CHO cells do not normally show ST6Gal activity19, whereas terminal α(2,3)-linked sialic acid residues are abundant....
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...Fucosylation Most mammals have five primary types of fucosyltransferases, classified by the linkages between fucose and their substrates: α(1,2), α(1,3), α(1,4), α(1,6) and protein O-fucosyltransferases (Supplementary Table 14 for the glycans fucosylated by each class)....
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...(iii) Sialylation of a terminal galactose can occur through α(2,3) or α(2,6) linkages in human....
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...Hence, the property of resistance to viral infection demonstrated by CHO cells Arylsulfatases *Fucosyltransferases Galactosyltransferase *GalNAc−transferases Galactosidase Glucosyltransferases GlcNAc−transferases UDP-glucuronosyltransferases Hexosaminidases Hyaluronan synthase Iduronidases Sialidases Sulfatases *Sulfotransferases Xylosyltransferases **Lysosomal enzymes **Mannosyltransferases Mannosidases **Nuc. sugars transporters Nucleotide synthesis Sialyltransferases Expressed n = 159 Not expressed n = 141 GalN Ac−t ransf erase s Fucosyltransferases Sul fotr ans fera ses Nucleotide synthesis Lysosomal enzymes Mannosyltransferases All expressed: Fucosidases Gal-T & GalNAc-T bifunctional HSGlcNAc/GlcA transferases Heparanases **Hyaluronoglucosaminidases Miscellaneous N-glycan transferases Sulfohydrolases ER lumen Golgi b2 a2 b2 a3 a6 b2 a3 b4 a3 a6 a3 a6 a3 b4 a2 a2 b4 b4 a2 a2 a2 a3 a2 b4 a3 a3 a3 a3 b4 a6 b4 a6 a6 a2 a2 a3 a6 a3 b4 b2 b6 b2 b4 a3 b4 a2 a6 a3 a3 b4 a6 b4 b4 b4 b4 a6 b4 a3 a3 a3 a3 a2a2 a3 a2 a6 a2 b4 a3 a2 b4 a2 b4 a2 b2 a3 b4 a6 a6 a3 a6 b4 a6 a3 b2 b2 b2 a3 a3 a2 b4 b4 a3 b4 b4 a3 a6 b4 b4 a2a6 b2 a6 b4 b4 a3 a2 a2 a3 b4 b4 a6 a3 b2 b4 a3 b4 b4 a2 a3 b4 b4 b4 b4 a6 b4 b4 a6 b4 b2 a6 a6 b4 b4 a6 a6 b4 a3 a6 a3 b4 a6 b4 b4 b4 b4 b4 b4 a6 a3 a3 a6 a2 b6 a6 b2 a2 a3 b4 b4 a3 a3 b4 a6 a3 a3 a6 a2 b2 b4 a6 b4 a6 a3 a6 b2 b4 b2 b4 b4 a2 a6 b2 a6 b4 a6 b4 a3 a3 a2a2 a6 b4 a3 a3 b2 a2 b4 b4 b4 a3 a6 b4 a3 b4 a3 a2 b4 a3 b4 b6 a3 b2 a2 b4 a2 a2 a3 a6 b4 b2b2 a3 a2 b6 a6 b2 b4 b4 a3 a3 b2 b2 a3 b4 b2 a6 a6 b4 b4 b4 a3 a6 a6 b4 b2 gdp[g] Asn-X-Ser/Thr[r] man[g] cmp[g] man[g] H2O[g] man[g] H+[g] H2O[r] H2O[r] H+[g] H+[g] H+[g] udp[g] man[g] uacgam[g] uacgam[g] uacgam[g] cmp[g] man[r] udp[g] H2O[g] gdpfuc[g] H+[g] H2O[g] man[g] glc-D[r] H2O[g] H2O[r] man[g] H+[r] H+[g] H2O[r] H2O[g] H2O[g] H+[g] doldp[r] man[g] glc-D[r] udp[g] cmp[g] uacgam[g] H2O[g] udp[g] glc-D[r] H2O[g] H+[g] uacgam[g] H+[g] udp[g] udp[g] man[g] udpgal[g] udpgal[g] H+[g] udp[g] H+[g] cmp[g] S23Tg MM7B1g MM5cg MM6B1ag DOLASNT MM6B2g MM8Ber G14Tg S23Tg M13N2Tg M16NTg MM6B1bg G14Tg F6Tg M1316Mg MM7B2g MG1er MG2er MG3er M16N6Tg MM5bg S23Tg S23Tg M7MASNBterg M13N4Tg cmp[g] H+[g] S26Tg cmpacna focytb5 H2O H+ O2 cmpgcna CMAH ctp ppi acnam CMPSAS ficytb5 CMP- CMP- CMP- CMP-CMP- CMP- CMP- iii. iv. i. ii....
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...The gene responsible for producing this epitope, glycoprotein α(1,3) galactosyltransferase (Ggta1), is not expressed in human, but is active in mouse....
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Journal Article•DOI•

Optimal and consistent protein glycosylation in mammalian cell culture.

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Patrick Hossler¹, Sarwat F. Khattak¹, Zheng Jian Li¹•Institutions (1)

Bristol-Myers Squibb¹

01 Sep 2009-Glycobiology

TL;DR: This review will focus on the advancements made in glycosylation control in a manufacturing process, as well as the next steps in understanding and controlling protein glycosYLation.

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Abstract: In the biopharmaceutical industry, mammalian cell culture systems, especially Chinese hamster ovary (CHO) cells, are predominantly used for the production of therapeutic glycoproteins. Glycosylation is a critical protein quality attribute that can modulate the efficacy of a commercial therapeutic glycoprotein. Obtaining a consistent glycoform profile in production is desired due to regulatory concerns because a molecule can be defined by its carbohydrate structures. An optimal profile may involve a spectrum of product glycans that confers a desired therapeutic efficacy, or a homogeneous glycoform profile that can be systemically screened for. Studies have shown some degree of protein glycosylation control in mammalian cell culture, through cellular, media, and process effects. Studies upon our own bioprocesses to produce fusion proteins and monoclonal antibodies have shown an intricate relationship between these variables and the resulting protein quality. Glycosylation optimization will improve therapeutic efficacy and is an ongoing goal for researchers in academia and industry alike. This review will focus on the advancements made in glycosylation control in a manufacturing process, as well as the next steps in understanding and controlling protein glycosylation.

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691 citations

Cites background from "Production of recombinant protein t..."

...…therapeutics, other cell lines such as human embryonic kidney cells (HEK) (Durocher et al. 2007), baby hamster kidney (BHK), mouse myeloma (NS0), and human retinal cells (PERC.6) (Jones et al. 2003; Wurm 2004; Petricciani and Sheets 2008) are being developed as high producing cell lines....
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References

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Journal Article•DOI•

A new technique for the assay of infectivity of human adenovirus 5 DNA.

[...]

Frank L. Graham¹, A. J. Van Der Eb¹•Institutions (1)

Leiden University¹

01 Apr 1973-Virology

TL;DR: A new technique for assaying infectivity of adenovirus 5 DNA has been developed and a reproducible relationship between amounts of DNA inoculated per culture and numbers of plaques produced was demonstrated.

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9,230 citations

"Production of recombinant protein t..." refers background in this paper

...In 1973, Graham and van der Eb showed that exposing cells to nanoparticles of DNA and calcium phosphate makes possible DNA transfer into cultivated mammalian cell...
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Journal Article•DOI•

Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity

[...]

Pablo Umaña¹, Joel Jean-Mairet¹, R Moudry², H Amstutz², James E. Bailey¹ - Show less +1 more•Institutions (2)

ETH Zurich¹, Swiss Red Cross²

01 Feb 1999-Nature Biotechnology

TL;DR: The glycosylation pattern of chCE7 was engineered in Chinese hamster ovary cells with tetracycline–regulated expression of GnTIII to optimize the ADCC activity, and this activity correlated with the level of constant region–associated, bisected complex oligosaccharides determined by matrix–assisted laser desorption/ionization time–of–flight mass spectrometry.

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Abstract: The glycosylation pattern of chCE7, an antineuroblastoma chimeric IgG1, was engineered in Chinese hamster ovary cells with tetracycline-regulated expression of beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisected oligosaccharides that have been implicated in antibody-dependent cellular cytotoxicity (ADCC). Measurement of the ADCC activity of chCE7 produced at different tetracycline levels showed an optimal range of GnTIII expression for maximal chCE7 in vitro ADCC activity, and this activity correlated with the level of constant region-associated, bisected complex oligosaccharides determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The new optimized variants of chCE7 exhibit substantial ADCC activity and, hence, may be useful for treatment of neuroblastoma. The strategy presented here should be applicable to optimize the ADCC activity of other therapeutic IgGs.

...read moreread less

1,034 citations

Journal Article•DOI•

Epigenetic Codes for Heterochromatin Formation and Silencing: Rounding up the Usual Suspects

[...]

Eric J. Richards¹, Sarah C. R. Elgin¹•Institutions (1)

University of Washington¹

22 Feb 2002-Cell

TL;DR: A self-reinforcing network of interactions among the three best-characterized covalent modifications that mark heterochromatin suggest a mechanistic basis for spreading of heterochromaatin over large domains and for stable epigenetic inheritance of the silent state.

...read moreread less

879 citations

Journal Article•DOI•

How introns influence and enhance eukaryotic gene expression.

[...]

Hervé Le Hir¹, Ajit Nott¹, Melissa J. Moore¹•Institutions (1)

Howard Hughes Medical Institute¹

01 Apr 2003-Trends in Biochemical Sciences

TL;DR: Recent progress is summarized in understanding of how introns and the act of their removal by the spliceosome can influence and enhance almost every step of mRNA metabolism.

...read moreread less

709 citations

"Production of recombinant protein t..." refers background in this paper

...It is known, however, that efficient cytoplasmic transport and translation of the mRNA in eukaryotic cells depends on splicin...
[...]

Journal Article•DOI•

High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker.

[...]

Christopher R. Bebbington, G Renner, S Thomson, David J. King, D Abrams, Geoffrey Yarranton - Show less +2 more

01 Feb 1992-Nature Biotechnology

TL;DR: A method for introducing a glutamine synthetase (GS) selectable marker into myeloma cells in which transfectants are selected by growth in a glutamines-free medium is reported.

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Abstract: We report a method for introducing a glutamine synthetase (GS) selectable marker into myeloma cells in which transfectants are selected by growth in a glutamine-free medium. Vector amplification can subsequently be selected using the specific inhibitor of GS, methionine sulphoximine (MSX). Using this system, DNA sequences encoding a chimeric B72.3 IgG4 antibody were expressed from hCMV-MIE promoters in NSO myeloma cells. A cell line was isolated after a single round of selection for vector amplification which contains approximately 4 copies of the vector, secretes 10-15 pg/cell/day cB72.3 antibody during exponential growth and can accumulate 560 mg/l antibody in a fed-batch air-lift fermentation system. Productivity is stable in the absence of MSX selection.

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544 citations

"Production of recombinant protein t..." refers background in this paper

...At a concentration of 10–100 μM MSX, resistant clones can be identified in selected NS0 cell populations that have amplified the transgene complex containing the GS gene and the desired gene(s) of interes...
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