Production of Recombinant Proteins in Chinese Hamster Ovary Cells Using A Protein-Free Cell Culture Medium

Zang, Michael A.; Trautmann, Helmut; Gandor, Christine R.; Messi, F.; Asselbergs, Fred A.M.; Leist, C.; Fiechter, Armin; Reiser, Jakob

doi:10.1038/nbt0495-389

Cited by 65 publications

(49 citation statements)

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“…Experiments similar to those described above for the serumfree medium formulation revealed that generation times are significantly increased in the protein-free formulation for cell lines CHO DUKXB11, CHO-A11-A2, and CHO-A11-A279 when compared to the serum-free formulation (Table 5). However, they are comparable with generation times reported for CHO SSF3 cells producing urokinase-type plasminogen activator (about 27 h, Zang et al, 1995) or a humanized immunoglobin G kappa light chain (about 15 h, Zang et al, 1995). The increased generation times in the protein-free medium are probably due to a lack of insulin and indicate a partial block in progression through the cell cycle, presumably a prolongation of the G1-phase.…”

Section: Growth Characteristics and Atiii Secretion In Protein-free Msupporting

confidence: 62%

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Serum- and protein-free media formulations for the Chinese hamster ovary cell line DUKXB11

Schröder

Matischak

Friedl

2004

Journal of Biotechnology

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Citation for published item:hr¤ oderD wF nd w tis h kD uF nd priedlD F @PHHRA 9 erumE nd proteinEfree medi formul tions for the ghinese h mster ov ry ell line h u fIIF9D tourn l of iote hnologyFD IHV @QAF ppF PUWEPWPF Further information on publisher's website: httpXGGdxFdoiForgGIHFIHITGjFj iote FPHHQFIPFHHS Publisher's copyright statement:Additional information: Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-pro t purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. AbstractThe production of therapeutic proteins in mammalian cell lines is of outstanding importance. The maintenance of most mammalian cell lines in culture requires the addition of serum to the culture medium.The elimination of serum from mammalian cell culture is desirable since serum is expensive and a source of contaminants, e.g. viruses, mycoplasma or prions. Here we describe the composition of serum-and protein-free media for the Chinese hamster ovary (CHO) cell line DUKXB11. The serum-free formulation supports excellent growth of CHO DUKXB11 cells at low (23 cells/cm 2 ) and high (2·10 4 cells/cm 2 ) seeding densities characterized by a generation time of 10 -12 h, and, after addition of 0.2% pluronic F-68, the growth of a recombinant suspension cell line derived from DUKXB11. In addition, this formulation also allowed us to adapt recombinant cell lines expressing various amounts of human antithrombin ATIII (ATIII)to serum-free conditions. Secretion of ATIII was readily observed in the serum-free medium. Minor changes to the serum-free formulation resulted in a protein free formulation that supported growth of CHO DUKXB11 cells, growth of recombinant CHO cells expressing ATIII, and production of ATIII. Key wordsChinese hamster ovary cells, serum-free medium, protein-free medium, recombinant protein production,

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Section: Growth Characteristics and Atiii Secretion In Protein-free Msupporting

confidence: 62%

“…CHO SSF3, were selected from DUKXB11 cells by replacing part of the medium every week . A protein-free medium for CHO SSF3 cells was described (Zang et al, 1995). In addition, a cell line preadapted to serum-free conditions, derived from DUKXB11 cells was described (Sinacore et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Serum- and protein-free media formulations for the Chinese hamster ovary cell line DUKXB11

Schröder

Matischak

Friedl

2004

Journal of Biotechnology

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“…It was not necessary to use protease inhibitors in the medium or to include an additional purification step to eliminate the enzyrnatically active form. The highest levels of production of this and other recombinant proteins obtained with stably transfected SSF3-derived cell lines were, like observed with the parental CHO(dhfr-)-derived cells, around 20 50/zg per 10 6 cells day -1 or 2-5 10 t4 molecules cell -1 day ~ [23]. The availability of procedures to manipulate mammalian cells genetically in the absence of serum also opens the possibility of using these cells for other experiments than production of recombinant proteins, where serum-containing medium would interfere, for example in assays of signal transduction by hormones naturally present in serum.…”

Section: Prospects For Animal Cell Culture In Protein-free Mediamentioning

confidence: 61%

Amplification and expression of recombinant genes in serum‐independent Chinese hamster ovary cells

1995

FEBS Letters

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CHO SSF3 cells grow as a suspension culture in unmodified commercial medium with only low-molecular weight ingredients. Continuous serum-free culture unexpectedly induced expression of a low dihydrofolate reductase activity in the originally dhfr-CHO cells. Nevertheless, it was possible with methotrexate to induce amplification of a gene coding for the hybrid plasminogen activator K2tu-PA cotransfected with a dhfr gene.Expression of K2tu-PA expression was proportionally increased to that of dh)9, which was measured with fluorescent methotrexate. Because no serum proteases were present, secreted K2tu-PA was not converted to the enzymatically active form, but was exclusively recovered in proenzyme form.

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“…However, secretion of uPA by yeast was hampered due to the formation of large aggregates in the endoplasmic reticulum (Agaphonov et al 2002). Zang and colleagues have previously explored the use of a mammalian expression system to avoid complications observed with prokaryotic or yeast systems (Zang et al 1995). Using normal, wild type CHO cells, they were able to generate reasonable quantities of recombinant uPA, however it was not reported in this study if the recombinant uPA was proteolytically active or structurally comparable to native uPA.…”

Section: Discussionmentioning

confidence: 92%

“…Expression of recombinant human uPA has been attempted using bacterial Lenich et al 1992;Wang et al 2000), yeast (Agaphonov et al 2002), and mammalian (Zang et al 1995) expression systems with mixed results. In many cases, recombinant protein yields are low and/or post-translational modifications are missing or aberrant.…”

Section: Introductionmentioning

confidence: 99%

Development of a Mammalian Suspension Culture for Expression of Active Recombinant Human Urokinase-type Plasminogen Activator

et al. 2005

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The development of specific catalytic inhibitors for the serine protease urokinase-type plasminogen activator (uPA) has been hindered due to difficulties in producing sufficient amounts of active recombinant uPA that is catalytically equivalent to native uPA. The purpose of this study was to develop an efficient system for the expression of recombinant human uPA that exhibits comparable proteolytic activity to that of the native protein. Since post-translational modifications (e.g. glycosylations) of uPA are necessary for efficient proteolytic activity, we have used a mammalian cell line [Chinese hamster ovary (CHO)-S] to express recombinant human uPA. CHO-S cells were selected to stably express full-length recombinant human uPA containing a hexahistidine tag at its C-terminus to permit purification by nickel-based affinity chromatography. Secretion of recombinant uPA into the culture media was confirmed by immunoblotting and the presence of an N-linked glycosylation was confirmed by PNGase sensitivity. Enzymatic activity of purified recombinant uPA was demonstrated using zymography and quantitatively compared to native uPA by kinetic analysis using an uPA-specific substrate. Native uPA and the recombinant uPA demonstrated comparable K m values (55.7 and 39 lM, respectively). Furthermore, inhibition studies using benzamidine resulted in a K i of 195 lM for native uPA, while recombinant uPA had a K i of 112 lM. These data indicate that recombinant human uPA expressed by CHO-S cells is functionally comparable to native uPA.Abbreviations: CHO-S -Chinese hamster ovary

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Production of Recombinant Proteins in Chinese Hamster Ovary Cells Using A Protein-Free Cell Culture Medium

Cited by 65 publications

References 28 publications

Serum- and protein-free media formulations for the Chinese hamster ovary cell line DUKXB11

Serum- and protein-free media formulations for the Chinese hamster ovary cell line DUKXB11

Amplification and expression of recombinant genes in serum‐independent Chinese hamster ovary cells

Development of a Mammalian Suspension Culture for Expression of Active Recombinant Human Urokinase-type Plasminogen Activator

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