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WO1996007730A2 - Procede chimique favorisant la proliferation de cellules animales - Google Patents

Procede chimique favorisant la proliferation de cellules animales Download PDF

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Publication number
WO1996007730A2
WO1996007730A2 PCT/CH1995/000191 CH9500191W WO9607730A2 WO 1996007730 A2 WO1996007730 A2 WO 1996007730A2 CH 9500191 W CH9500191 W CH 9500191W WO 9607730 A2 WO9607730 A2 WO 9607730A2
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WO
WIPO (PCT)
Prior art keywords
cells
serum
suramin
culture
free
Prior art date
Application number
PCT/CH1995/000191
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German (de)
English (en)
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WO1996007730A3 (fr
Inventor
Wolfgang A. Renner
Hans M. Eppenberger
James Edwin Bailey
Original Assignee
Renner Wolfgang A
Eppenberger Hans M
James Edwin Bailey
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Renner Wolfgang A, Eppenberger Hans M, James Edwin Bailey filed Critical Renner Wolfgang A
Priority to EP95928931A priority Critical patent/EP0733100A1/fr
Publication of WO1996007730A2 publication Critical patent/WO1996007730A2/fr
Publication of WO1996007730A3 publication Critical patent/WO1996007730A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/005—Protein-free medium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the invention relates to cell cultures, in particular animal cell cultures, which can grow and multiply as a result of chemical measures in a serum- and in particular serum- and protein-free environment, and medium additives which allow animal cells in a protein- and serum-free environment to watch and multiply.
  • the invention also relates to animal cell cultures which have the ability to grow and multiply in suspension, as well as medium additives which impart the abovementioned properties and medium additives which block cell-cell adhesion and in this way the formation of aggregates of animal cells in suspension culture prevent.
  • Cultures of genetically modified mammalian cells are used for the production of pharmaceutical substances. In contrast to microorganisms, they have the ability to produce post-translationally modified and correctly folded proteins. Mammalian cells required for growth in addition to the low-molecular substances, growth factors or hormones contained in the basic medium, which are usually added by adding fetal blood sera. However, the use of these blood sera brings with it a number of serious problems; they represent the greatest risk of contamination of the production process with viruses, mycoplasmas and prions (pathogens of spongiform encephalitis (BSE)).
  • BSE pathogens of spongiform encephalitis
  • CHO cells can be adapted to a serum-free environment by mutagenesis with ethyl methanesulfonate and protocols are also known which use the spontaneous mutation to obtain a serum-free cell line after months of selection (C. Gandor , Diss. ETH No. 10087).
  • C. Gandor Diss. ETH No. 10087.
  • These methods have the disadvantage that in both cases the cell changes in an unknown way due to mutations. For example, should an existing production process be rum-free medium is by no means ensured that the desired properties of the producer cell are retained in the process lasting several months. Mutations in the product gene or in a gene that modifies the product post-translationally could have devastating consequences.
  • suramin sodium inhibits the binding of various growth factors to the corresponding receptors, which also inhibits the proliferation of these cells.
  • Among the growth factors in which the above-mentioned effect of suramin sodium was described were, inter alia, bFGF, IGF I and II, PDGF, TGF beta etc. Even the mitogenic effect of fetal calf serum could be suppressed in this way.
  • the variety of effects of suramin sodium in relation to growth factor regulation suggests a more general mechanism. The preferred hypothesis assumes masking the growth factor with suramin sodium.
  • suramin sodium had a growth-inhibiting effect, and it has therefore already been used on a trial basis as a chemotherapeutic agent against cancer.
  • animal cells can be allowed to grow in a serum and / or protein-free environment by adding suramin-like compounds, in particular suramin salts such as suramin sodium, to the culture medium.
  • Ar independently of one another is phenyl or naphthyl
  • R independently of one another hydrogen or a lower alkyl group with 1 to 4 C-
  • R ' -SO3Y, where Y is independently an equivalent of a cation, in particular Na, K,
  • X independently of one another -NH-C- or
  • Preferred oligoamide ureas are described in dependent claims 3 to 6.
  • Cell lines which have previously grown serum, protein and / or surface-dependent can be grown in culture medium, serum and protein-free and as a single cell suspension with very little aggregate formation. Cell cultures grown according to the invention are illustrated in the figures and figures.
  • Figure 1 shows CHO Kl cells 4 days after transfer into unsupplemented FMX 8 medium
  • Figure 2 shows CHO Kl cells 4 days after transfer in FMX 8 medium supplemented with 0.5 mg / ml suramin sodium
  • FIG. 3 shows the growth of CHO Kl cells in spinner culture
  • FIG. 4 shows the growth of CHO Kl: cycE cells in the COLOR bioreactor
  • FIG. 5 shows the growth of tPA-producing CHO cells (CHO 1-15500; ATCC No. CRL 9606)
  • FIG. 6 shows the morphology of BHK 21 cells in adherence culture in unsupplemented FMX 8 medium
  • Figure 7 shows the morphology of BHK 21 cells in suspension culture in unsupplemented FMX 8 medium
  • Figure 8 shows the morphology of BHK 21 cells in FMX 8 medium supplemented with 1 mg / ml suramin sodium
  • FIG. 9 shows the UV difference spectrum of a 1 mg / ml BSA solution, from which 99.9% of the suramin sodium (0.5 mg / ml) were removed by ultrafiltration in 1 M NaCl solution against a pure BSA solution, and
  • FIG. 10 shows the UV difference spectrum of a 1 mg / ml BSA solution which contains 1/1000 of the medium concentration of suramin sodium (0.5 ⁇ g / ml) against a pure BSA solution.
  • the growth rates in suramin-containing medium vary slightly from cell line to cell line to the extent that this is already the case in the original culture containing serum.
  • Weekly dilution rates in the serum- and protein-free FMX-8 medium are in the range of 1/25 to 1/250 and are therefore very suitable for use in a production process.
  • Suramin sodium had an extremely positive effect even in the case of CHO cells that were already free of serum and protein. These cells had previously been transfected with an expression vector for cyclin E. After the addition of suramin sodium, rapid growth, a higher proportion of living cells and a significantly higher end cell concentration were observed. Obviously, an extra cellular inhibition mechanism is still active in these cells, which is suppressed by suramin.
  • Baby hamster kidney cells are also used to produce pharmaceutically active substances.
  • Media are used which either contain fetal calf serum as an additive or growth factors and proteins such as transferrin and insulin.
  • the inventors succeeded in growing BHK 21 cells in a medium originally developed for CHO cells (FMX 8 medium from Messi Cell Culture Technologies Zurich, F. Messi, Diss. ETH No. 9559 (1991)).
  • FMX 8 medium from Messi Cell Culture Technologies Zurich, F. Messi, Diss. ETH No. 9559 (1991)
  • a very rapid growth and a high proportion of living cells were observed.
  • Weekly dilution rates of 1/100 could be maintained for at least three months.
  • the morphology of the cells as can be seen in Figure 6, was widespread and adherent.
  • BHK 21 cells can also be grown as a suspended single cell culture with extremely low aggregate formation.
  • suramin sodium blocks cell-line and cell-substrate adhesion processes.
  • Surface-growing CHO or BHK 21 cells grow after the addition of 0.5-1 mg / ml suramin sodium to the culture medium in suspension as a single cell culture. In all of the cases described above, a complete transition to rounded morphology and suspended growth was observed (see Figure 8).
  • Baby hamster kidney cells form spherical aggregates in suspension culture (eg spinner culture) in media with or without serum and proteins, which are characterized in that the cells spread out on one another and on already existing aggregates (see picture 7).
  • This type of aggregation differs significantly from that of the CHO cells.
  • the adhesion of BHK 21 cells to each other is an active process. This adhesion process can be prevented by adding suramin sodium to the nutrient medium.
  • BHK 21 cells grow in this way as a single cell suspension; no spreading of the cells towards one another can be observed (see Fig. 8). This increases the living cell proportion of BHK cell cultures enormously.
  • the nutrient supply is no longer limited, as is the case inside cell aggregates. In biotechnological processes with animal cells, the completely suspended growth is a great advantage. The avoidance of expensive microcarriers simplifies and cheapens the process enormously.
  • the single-phase system also allows more homogeneous process control and control.
  • the use of suramin sodium as such in cell culture processes is likewise harmless, since the substance itself is approved as a therapeutic product for humans and has therefore successfully passed all clinical tests to determine any toxicity.
  • Suramin sodium is commercially available and relatively inexpensive, so that although it is added to the medium in a relatively large amount, it represents only a minimal cost factor which, compared to the costs of a cell culture process, is absolutely negligible. It is believed that the effect of
  • Suramin sodium comes about through relatively unspecific interactions with proteins. It must therefore also be assumed that there is an interaction between suramin sodium and the desired end product, and that there may even be a bond. It is therefore of great importance to have methods at hand with which suramin sodium can be removed from the end product and can be detected. A cheap method of removal is ultrafiltration after the neutralization of ionic interactions. In one step, 99.9% of the originally available amount of suramin sodium could be removed from a protein solution. The residual contents corresponded to 1 molecule of suramin sodium per 5 molecules of Rin ⁇ serum albumin, a protein which was chosen as the model protein because of its high adsorption capacity. For the detection of suramin sodium, UV spectroscopy was chosen in the present work. Due to the aromatic groups in the molecule, a characteristic absorption maximum at 310 n can be used for detection. The aromatics (Phe, Tyr, Trp) contained in proteins all absorb at lower wavelengths. example 1
  • CHO Kl cells were kept stable in culture for three months. CHO Kl cells are shown four days after the serum withdrawal in medium without suramin sodium on picture 1 and in medium with suramin sodium on picture 2.
  • Figure 3 shows growth curves of this culture. Cell densities were determined after trypan blue staining in the hemacytometer. Glucose concentrations were determined using a YSI glucose analyzer.
  • Serum and protein free growth of CHO KlcycE cells suramin sodium also has a favorable one
  • the cDNA of the human cyclin E gene can be by
  • Standard hybridization methods can be isolated from a HeLa cDNA library. All of the following methods are standard laboratory technology and were developed according to Sambrook et al. executed. HeLa mRNA was isolated using an RNA extraction kit from Pharmacia. After cDNA synthesis and incorporation into the phage lambda according to the manufacturer's instructions (Stratagene), the cDNA of the human cyclin E gene was isolated using standard hybridization techniques (Sambrook, J., Fritsch, EF and Maniatis, T. Molecular cloning Cold Spring Harbor Laboratory Press
  • the cDNA was present in the plasmid pBluescript. After restriction digestion with Eco RI, a fragment with a size of 2.5 kb could be isolated from a 0.8% low melt agarose gel. After linearizing the vector pRc / CMV (Invitrogen) with the restriction enzyme Bst XI, the fragment and the vector were filled in with the Klenowenzy. After ligation and transformation into the E.coli strain DH5alpha and identification of a construct in sense orientation, larger amounts of the expression vector were produced with the FlexiPrep Kit (Pharmacia).
  • CHO Kl cells were sown in a culture dish of a "six-well plate” (TPP) in such a way that 50-70% confluency was reached on the day of the transfection.
  • TPP ix-well plate
  • the cells were found in medium containing 10% fetal calf serum (eg Ham's F12, Gibco BRL).
  • the cells were detached by trypsinization.
  • the medium was removed, 1 ml of trypsin solution (Gibco BRL) was added, the mixture was waited for about 1 min with gentle shaking, the trypsin was sucked off and incubated for about 10 min at 37 ° C. and 5% CO 2.
  • the detached cells were taken up in 2 ml of FMX 8 medium containing 1/1000 (w / vol) trypsin inhibitor (Sigma).
  • the culture bottles were coated with fibronectin (Boehringer Mannheim) during the first three weeks (1 ⁇ g / cm ***) in order to facilitate the adhesion of the cells in the transition phase. 1 ml and 0.5 ml of the detached cells were then taken up in 5 ml FMX 8 medium in T-25 culture bottles and incubated. Since the efficiency of lipofection and the proportion of surviving cells can vary after lipofection, it is advisable to use different cell concentrations when subculturing to ensure the survival of the cultures. After a few days of proliferation, which can be attributed to transient expression of the cyclin E gene, a temporary decrease in proliferation is usually observed after one week until the cells which have incorporated the vector stably have overgrown the culture.
  • the dilution rate must therefore be set from case to case. Nevertheless, it is advisable to subculture every week, since residues of dead cells on the plastic obviously have an inhibiting effect.
  • the culture was transferred to a coated T-75 bottle (dilution rate 1/2 to 1/5).
  • the cells grow at weekly dilution rates of 1/40 in uncoated culture bottles. These cells can be kept in culture in serum and protein free FMX-8 medium for a long time.
  • T bottles The difference in T bottles is shown by a significantly higher end cell density and the larger weekly dilution rate of 1/250, with which these cells can be grown, compared to 1/50 without suramin additive.
  • FIG. 4 shows the growth parameters of CHO KlcycE cells with the addition of 0.5 mg / ml suramin sodium in the compact loop bioreactor (bioengineering).
  • the physical parameters were set as follows: working volume 2.3 1; Temperature 37 ° C, pH 7.3; p0 2 50% atmospheric oxygen saturation; Stirrer speed 580 rpm. Cell densities were counted after trypan blue staining in the hemacytometer. Glucose concentrations were determined using a YSI glucose analyzer.
  • the table shows a comparison of the growth parameters of CHO Kl cyc E cells with and without suramin sodium. Table without suramin with suramin
  • Example 1 The transition of this cell line into serum and protein-free medium is carried out as in Example 1.
  • CHO tPA cells are grown in FMX 8 medium with 0.5 mg / ml addition of suramin sodium. The weekly dilution rates to be achieved are also 1/50. The morphology of these cells changed to the same extent as described in Example 1 for CHO Kl cells.
  • the growth parameters of the CHO tPA cells are shown in FIG. 5.
  • BHK 21 cells can be grown adherently in the serum and protein free FMX 8 medium from Messi Cell Culture Technologies. No transition or selection phase is required for the transition from serum-dependent growth to serum- and protein-free growth in suspension.
  • the cells of a confluent T75 bottle (TTP) are detached by trypsinization and taken up in 10 ml of a 1 mg / ml Soybean trypsin inhibitor (Sig a) solution in medium to inactivate the trypsin.
  • 0.1 ml of this cell suspension are in 25 ml FMX 8 medium added. After 4 days, 25 ml of FMX 8 medium are added and after 1 week trypsinized as described above. In this way, BHK 21 cells could be grown for at least three months at weekly dilution rates of 1/100.
  • the morphology of the cells in the adherence culture is shown in Figure 6.
  • BHK 21 cells can also be grown in suspension. Without the addition of suramin sodium, very large aggregates form in suspension culture (see Figure 7). These arise in stirred (spinner or bioreactor) as well as in non-stirred suspension culture in BSA (cattle serum albumin) coated T-bottles. This aggregation is completely prevented by adding 1 mg / ml suramin sodium to the culture medium.
  • the change in morphology after the addition of Suramin is shown in Figure 8.
  • the growth parameters of a spinner culture of BHK 21 cells in a medium containing suramin are shown in FIG. 9. The properties of these cell cultures are particularly suitable for use in production processes, particularly with regard to simple handling, low seed densities and rapid growth to high cell densities while at the same time preventing aggregates.
  • the method of ultrafiltration was chosen to remove suramin sodium from protein solutions (eg cell culture supernatants).
  • protein solutions eg cell culture supernatants.
  • Cattle Serum Albumin (BSA) selected. This protein is characterized by its high adsorption capacity.
  • Suramin sodium was added to a 1 mg / ml BSA solution in amounts as present in the cell culture medium (500 ⁇ g / ml). 100 ml of this solution was added to NaCl in a concentration of IM.
  • This solution was ultrafiltered through a membrane with a pore size of 10,000 daltons. The filtrate was taken up in 100 ml of an 1M saline solution and filtered; this was done three times in all.
  • the filtrate purified in this way was taken up in water, so that a 1 mg / ml BSA solution resulted.
  • the residual suramin sodium content was determined by means of UV spectroscopy. By comparison with reference spectra of suramin / BSA standard solutions, a residual concentration of 0.5 ⁇ g / ml could be determined (FIGS. 9 and 10). Based on the initial concentration, 99.9% of the suramin sodium was thus withdrawn from the protein solution, ie after the purification there was only one molecule of suramin sodium per 5 molecules of BSA in the solution.

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Abstract

L'invention concerne des moyens et un procédé permettant de faire proliférer, en l'absence de sérum et de protéines, des cellules animales dans des cultures cellulaires, ainsi que l'utilisation de composés semblables à la suramine comme additif pour des milieux de culture exempts de sérum et de protéines.
PCT/CH1995/000191 1994-09-09 1995-09-05 Procede chimique favorisant la proliferation de cellules animales WO1996007730A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP95928931A EP0733100A1 (fr) 1994-09-09 1995-09-05 Procede chimique favorisant la proliferation de cellules animales

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CH2763/94-9 1994-09-09
CH276394 1994-09-09

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WO1996007730A2 true WO1996007730A2 (fr) 1996-03-14
WO1996007730A3 WO1996007730A3 (fr) 1996-04-18

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998026084A1 (fr) * 1996-12-09 1998-06-18 Wolfgang Andreas Renner EXPRESSION DE L'INTERFERON β1 ACTIF AU MOYEN DE REPLICONS D'ARN RECOMBINANT
US7955833B2 (en) 2002-07-09 2011-06-07 Baxter International Inc. Animal protein free media for cultivation of cells
US8021881B2 (en) 1999-09-28 2011-09-20 Baxter Innovations Gmbh Medium for the protein-free and serum-free cultivation of cells
US8080414B2 (en) 1997-06-20 2011-12-20 Baxter Innovations Gmbh Recombinant cell clones having increased stability and methods of making and using the same
US8440408B2 (en) 2004-10-29 2013-05-14 Baxter International Inc. Animal protein-free media for cultivation of cells
US9758568B2 (en) 2006-01-04 2017-09-12 Baxalta GmbH Oligopeptide-free cell culture media

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF ENDOCRINOLOGY, Bd. 134, 1992 Seiten 505-511, K. TRIEB ET AL. 'SURAMIN AFFECTS DIFFERENTIATED AND UNDIFFERENTIATED HUMAN THYROID EPITHELIAL CELLS IN VITRO.' *
SURGERY TODAY, Bd. 24, Nr. 3, 1994 Seiten 234-240, HIDENORI MUKAIDA ET AL. 'THE INHIBITORY EFFECT CAUSED BY SURAMIN ON THE PARACRINE GROWTH OF HUMAN CANCER CELLS AND FIBROBLASTS.' *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998026084A1 (fr) * 1996-12-09 1998-06-18 Wolfgang Andreas Renner EXPRESSION DE L'INTERFERON β1 ACTIF AU MOYEN DE REPLICONS D'ARN RECOMBINANT
US8329465B2 (en) 1997-06-20 2012-12-11 Baxter Innovations Gmbh Recombinant cell clones having increased stability and methods of making and using the same
US8080414B2 (en) 1997-06-20 2011-12-20 Baxter Innovations Gmbh Recombinant cell clones having increased stability and methods of making and using the same
US8084252B2 (en) 1997-06-20 2011-12-27 Baxter Innovations Gmbh Recombinant cell clones having increased stability and methods of making and using the same
US8084251B2 (en) 1997-06-20 2011-12-27 Baxter Innovations Gmbh Recombinant cell clones having increased stability and methods of making and using the same
USRE46745E1 (en) 1997-06-20 2018-03-06 Baxalta Incorporated Recombinant cell clones having increased stability and methods of making and using the same
USRE46897E1 (en) 1997-06-20 2018-06-19 Baxalta Incorporated Recombinant cell clones having increased stability and methods of making and using the same
USRE46860E1 (en) 1997-06-20 2018-05-22 Baxalta Incorporated Recombinant cell clones having increased stability and methods of making and using the same
US9982286B2 (en) 1999-09-28 2018-05-29 Baxalta Incorporated Medium for the protein-free and serum-free cultivation of cells
US8021881B2 (en) 1999-09-28 2011-09-20 Baxter Innovations Gmbh Medium for the protein-free and serum-free cultivation of cells
US8722406B2 (en) 1999-09-28 2014-05-13 Baxter Innovations Gmbh Medium for the protein-free and serum-free cultivation of cells
US9441203B2 (en) 1999-09-28 2016-09-13 Baxalta Innovations Gmbh Medium for the protein-free and serum-free cultivation of cells
US8524497B2 (en) 2002-07-09 2013-09-03 Baxter International Inc. Animal protein free media for cultivation of cells
US9163211B2 (en) 2002-07-09 2015-10-20 Baxter International Inc. Animal protein free media for cultivation of cells
US7955833B2 (en) 2002-07-09 2011-06-07 Baxter International Inc. Animal protein free media for cultivation of cells
US8748156B2 (en) 2004-10-29 2014-06-10 Baxter International Inc. Animal protein-free media for cultivation of cells
US9809796B2 (en) 2004-10-29 2017-11-07 Baxalta GmbH Animal protein-free media for cultivation of cells
US9714411B2 (en) 2004-10-29 2017-07-25 Baxalta GmbH Animal protein-free media for cultivation of cells
US9222075B2 (en) 2004-10-29 2015-12-29 Baxalta Incorporated Animal protein-free media for cultivation of cells
US8440408B2 (en) 2004-10-29 2013-05-14 Baxter International Inc. Animal protein-free media for cultivation of cells
US10138461B2 (en) 2004-10-29 2018-11-27 Baxalta GmbH Animal protein-free media for cultivation of cells
US10655099B2 (en) 2004-10-29 2020-05-19 Baxalta Incorporated Animal protein-free media for cultivation of cells
US9758568B2 (en) 2006-01-04 2017-09-12 Baxalta GmbH Oligopeptide-free cell culture media

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EP0733100A1 (fr) 1996-09-25

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