WO1996007730A2 - Chemical process for promoting the proliferation of animal cells - Google Patents

Chemical process for promoting the proliferation of animal cells 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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Prior art keywords
cells
serum
suramin
culture
free
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PCT/CH1995/000191
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German (de)
French (fr)
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WO1996007730A3 (en
Inventor
Wolfgang A. Renner
Hans M. Eppenberger
James Edwin Bailey
Original Assignee
Renner Wolfgang A
Eppenberger Hans M
James Edwin Bailey
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Application filed by Renner Wolfgang A, Eppenberger Hans M, James Edwin Bailey filed Critical Renner Wolfgang A
Priority to EP95928931A priority Critical patent/EP0733100A1/en
Publication of WO1996007730A2 publication Critical patent/WO1996007730A2/en
Publication of WO1996007730A3 publication Critical patent/WO1996007730A3/en

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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

The invention relates to means and a process for the serumless and proteinless proliferation of animal cells in cell cultures and the use of suramine-like compounds, especially suramine, as an additive for serumless and proteinless culture media.

Description

Chemisches Verfahren zur Förderung der Proliferation von tierischen Zellen Chemical process to promote the proliferation of animal cells
Die Erfindung betrifft Zellkulturen- insbe- sonders Tierzellkulturen, die aufgrund chemischer Mass- nahmen in serum- und insbesonders serum- und proteinfrei¬ er Umgebung wachsen und sich vermehren können, sowie Me¬ diumzusätze, welche es Tierzellen erlauben in protein- und serumfreier Umgebung zu wachen und sich zu vermehren. Ebenfalls betrifft die Erfindung tierische Zellkulturen, welche die Fähigkeit besitzen in Suspension zu wachsen und sich zu vermehren sowie Mediumzusätze, welche die obgenannten Eigenschaften vermitteln sowie MediumsZusät¬ ze, welche die Zell-Zelladhäsion blockieren und auf diese Weise die Aggregatbildung von Tierzellen in Suspensions¬ kultur verhindern.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.
Kulturen genetisch veränderter Säugetierzel¬ len werden zur Herstellung von pharmazeutischen Substan¬ zen verwendet. Sie besitzen im Unterschied zu Mikroorga- nismen die Fähigkeit, posttranslational modifizierte und richtig gefaltete Proteine herzustellen- Zum Wachstum be¬ nötigen Säugetierzellen nebst der im Grundmedium enthal¬ tenen niedermolekularen Substanzen, Wachstumsfaktoren oder Hormone, die üblicherweise durch Zusatz von fötalen Blutseren beigegeben werden. Die Verwendung dieser Blut¬ seren bringt allerdings etliche schwerwiegende Probleme mit sich, sie stellen die grösste Gefahr von Kontamina¬ tionen des Produktionsprozesses mit Viren, Mycoplasmen und Prionen (Erreger der spongiformen Enzephalitis (BSE)) dar. Während Viren und Mycoplasmen durch leichtes Erhit¬ zen zerstört bzw. durch Filtration entfernt werden kön¬ nen, ist dies bei Prionen wie dem BSE-Erreger nicht mög¬ lich, ohne dabei die wichtigen Peptid-Wachstumsfaktoren zu zerstören. Als weitere gravierende Nachteile des Ge- brauchs von fötalen Seren sind die wesentliche Erschwe¬ rung der Produktreinigung durch den hohen Fremdprotein¬ gehalt (über 90 %) sowie die hohen Kosten zu erwähnen. Es wird befürchtet, dass die im Serum enthaltenen Proteine zu allergischen Reaktionen bei Patienten führen können, wenn sie noch in Spuren im Endprodukt vorhanden sind. Zu¬ dem muss beim Gebrauch von Serum mit variierender Quali- tat gerechnet werden. Alter, Gesundheitszustand und Nah¬ rung der Tiere können die Qualität des Serums stark be¬ einflussen. Dazu kommt, dass die Zusammensetzung des Se¬ rums wegen seiner Komplexität im wesentlichen unbekannt ist, was eine exakte Prozesskontrolle verunmöglicht. Selbstverständlich sprechen auch tierschützerische Aspek¬ te gegen die Verwendung von fötalen Seren, werden doch grosse Mengen in einem Produktionsprozess verwendet (üb¬ lich sind Kulturmedien mit bis zu 10 % fötalem Kälberse¬ rum) . Wachstumsfaktoren regulieren das Wachstum und die Differenzierung von tierischen Zellen. Sie werden von spezifischen Rezeptoren auf der Zelloberfläche erkannt und induzieren damit ein intrazelluläres Signal, das zur DNA-Synthese sowie letztendlich zur Teilung der Zelle führt (Chao, M. V. Cell 68, 995-7 (1992)). Nebst den aktivierenden Wachstumsfaktoren gibt es auch solche, welche das Wachstum von tierischen Zellen inhibieren. Diese Faktoren spielen als Antagonisten eine wesentliche Rolle bei der Regulation des Wachstums im gesamten Orga- nismus. C. Gandor, Diss. ETH Nr. 10087 zeigte, dass ge¬ wisse Zellen in Kultur über einen autokrinen Stimula¬ tionsmechanismus zum Wachstum angeregt werden. Dabei wur¬ de das Augenmerk auf positiv wirkende Faktoren gelegt.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)). During viruses and mycoplasmas due to slight heating zen destroyed or removed by filtration, this is not possible with prions such as the BSE pathogen without destroying the important peptide growth factors. Further serious disadvantages of the use of fetal sera are the substantial complication of product cleaning due to the high foreign protein content (over 90%) and the high costs. It it is feared that the proteins contained in the serum can lead to allergic reactions in patients if they are still present in traces in the end product. In addition, varying quality must be expected when using serum. The age, state of health and diet of the animals can have a strong influence on the quality of the serum. In addition, the composition of the serum is essentially unknown due to its complexity, which makes exact process control impossible. Of course, animal protection aspects also speak against the use of fetal sera, since large amounts are used in a production process (culture media with up to 10% fetal calf serum are common). Growth factors regulate the growth and differentiation of animal cells. They are recognized by specific receptors on the cell surface and thus induce an intracellular signal, which leads to DNA synthesis and ultimately to cell division (Chao, MV Cell 68, 995-7 (1992)). In addition to the activating growth factors, there are also those that inhibit the growth of animal cells. As antagonists, these factors play an essential role in regulating growth in the entire organism. C. Gandor, Diss. ETH No. 10087 showed that certain cells in culture are stimulated to grow via an autocrine stimulation mechanism. The focus was on positive factors.
Es wurde gezeigt, dass CHO-Zellen durch Muta- genese mit Ethylmethansulfonat an eine serumfreie Umge¬ bung angepasst werden können und es sind auch Protokolle bekannt, welche die spontane Mutation ausnutzen, um nach monatelanger Selektion eine serumfrei wachsende Zellinie zu erhalten (C. Gandor, Diss. ETH Nr. 10087). Diese Me- thoden haben den Nachteil, dass sich die Zelle in beiden Fällen durch Mutationen in unbekannter Weise ändert. Soll beispielsweise ein bestehender Produktionsprozess auf se- rumfreies Medium umgestellt werden, ist keineswegs si¬ chergestellt, dass bei dem mehrere Monate dauernden Prozess die erwünschten Eigenschaften der Produzenten¬ zelle erhalten bleiben. Mutationen im Produktgen oder in einem Gen, das das Produkt posttranslational modifiziert, könnten verheerende Folgen haben.It has been shown that 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). 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.
Es konnte ebenfalls gezeigt werden, dass durch die Ueberexpression von zellzyklusregulatorischen Proteinen (Cycline, Transkriptionsfaktoren) das Wachstum in serum- und proteinfreier Umgebung sowie in Suspension ermöglicht wird (Bailey; Vortrag, "Cell Culture Engineer¬ ing", San Diego, 8.3.1994). Dabei wurde in den aktivierenden Teil der Wachstumsregulation eingegriffen, was zur Proliferation der Zellen führte. Vorteil dieser Methode ist, dass durch dieses genau definierte Verfahren innert kurzer Zeit eine protein- und serumfrei wachsende Zellinie erhalten wird.It was also possible to show that the overexpression of cell cycle regulatory proteins (cyclins, transcription factors) enables growth in a serum- and protein-free environment and in suspension (Bailey; lecture, "Cell Culture Engineering", San Diego, 8.3.1994 ). This involved intervening in the activating part of the growth regulation, which led to the proliferation of the cells. The advantage of this method is that this well-defined procedure quickly produces a protein and serum-free cell line.
Viele Zellen benötigen zum Wachstum und zur Vermehrung nebst Wachstumsfaktoren eine Oberfläche auf der sie adhärieren können. Diese muss dazu speziell be¬ schichtet sein. Beim Scale up eines biotechnologischen Prozesses stellt diese Oberflächenabhängigkeit ein gros- ses Problem dar. Roller bottles, an denen die Zellen ad¬ härieren, eignen sich nicht zur Massstabsvergrösserung, üblicherweise werden die Zellen zur Produktion in gross- volumigen Reaktoren auf Microcarriern gezüchtet. Im Ku¬ bikmetermassstab stellt die Verwendung solcher Micro- carrier einen wesentlichen Kostenfaktor dar, völlig sus¬ pendiertes Wachstum ist daher von grossem Vorteil. Auch ist die unvermeidliche Inhomogenität der Suspension bei der Verwendung von Microcarriern problematisch. Einige Zelltypen zeigen als Reaktion auf den Entzug von entspre¬ chenden Oberflächen starke Aggregatbildung. Dabei sterben durch die fehlende Vaskualisierung die im Inneren eines Aggregates liegenden Zellen ab; der Anteil lebender Zel¬ len in einer solchen Kultur ist entsprechend gering. Of¬ fenbar existieren mehrere Mechanismen, die zur Aggregat- bildung führen können. Es konnte gezeigt werden, dass die aus toten Zellen austretende chromosomale DNA Nachbarzel¬ len verklebt und so zur Ausbildung grosser Aggregate füh¬ ren kann (W. Renner et al., Biotechnology and Bioengi- neering 41, 188-193 (1993)). Durch Zugabe von DNase wird diese Art der Verklumpung verhindert resp. rückgängig ge¬ macht. Offensichtlich existieren auch Arten der Verklum¬ pung, die auf anderen Mechanismen beruhen. Diese Aggre¬ gate entstehen auch ohne das Vorhandensein toter Zellen, es handelt sich dabei um einen Adhäsionsvorgang der Zel¬ len aufeinander. Während die erstgenannte Art der Aggre¬ gatbildung durch eine schonende Züchtung (mit entspre¬ chend geringem Anteil toter Zellen) verhindert werden kann, bedarf es zur Verhinderung der zweitgenannten Art der Aggregation einer aktiven Blockierung der entspre¬ chenden biologischen Abläufe.In addition to growth factors, many cells require a surface on which to adhere in order to grow and multiply. For this purpose, this must be specially coated. When scaling up a biotechnological process, this surface dependency is a major problem. Roller bottles to which the cells adhere are not suitable for scale-up; the cells are usually grown on microcarriers for production in large-volume reactors. The use of such microcarriers represents a significant cost factor on a cubic meter scale, so completely suspended growth is of great advantage. The inevitable inhomogeneity of the suspension is also problematic when using microcarriers. Some cell types show strong aggregation in response to the withdrawal of the corresponding surfaces. The lack of vascularization causes the cells inside an aggregate to die; the proportion of living cells in such a culture is correspondingly low. Apparently, there are several mechanisms that can lead education. It could be shown that the chromosomal DNA emerging from dead cells adheres to neighboring cells and can thus lead to the formation of large aggregates (W. Renner et al., Biotechnology and Bioengineering 41, 188-193 (1993)). This type of clumping is prevented or added by adding DNase. undone. Obviously there are also types of clumping which are based on other mechanisms. These aggregates are formed even without the presence of dead cells; this is an adhesion process of the cells to one another. While the first-mentioned type of aggregate formation can be prevented by gentle cultivation (with a correspondingly small proportion of dead cells), active blocking of the corresponding biological processes is required to prevent the second-mentioned type of aggregation.
Obwohl tierische Zellen zur Produktion von bereits kommerziell vertriebenen pharmazeutischen Wirk¬ stoffen verwendet werden, stellt die Verwendung von fö- talen Blutseren als Zusatz im Kulturmedium ein in jeder Hinsicht unbefriedigendes System dar. Die hohe Gefahr von Kontaminationen (Viren, Mycoplasmen, Prionen sowie aller- gen wirkende Serumproteine), die Erschwerung der Produk¬ tereinigung, variirende Qualität des Serums, die hohen Kosten sowie ethische und tierschützerische Aspekte spre¬ chen klar gegen eine weitere Verwendung dieser Mediumzu¬ sätze. Die durch die Oberflächengebundenheit der Zellen auftretenden Probleme in biotechnologischen Prozessen können zwar durch den Einsatz von Microcarriern gelöst werden, die hohen Kosten der Carrier beeinflussen die Wirtschaftlichkeit des Prozesses jedoch enorm.Although animal cells are used for the production of commercially active pharmaceutical ingredients, the use of fetal blood sera as an additive in the culture medium represents an unsatisfactory system in every respect. The high risk of contamination (viruses, mycoplasma, prions and all gene active serum proteins), the difficulty in cleaning the product, the varying quality of the serum, the high costs and ethical and animal protection aspects speak clearly against further use of these medium additives. The problems that arise in biotechnological processes due to the surface bond of the cells can be solved by using microcarriers, but the high costs of the carriers have an enormous impact on the economics of the process.
Es besteht deshalb ein Bedürfnis für ein serum- und proteinfreies Wachstumsmedium, in dem Zellen ohne spezielle Mutagenese oder gentechnologische Verände- rung proliferieren können. Die Bereitstellung eines Medi¬ ums, welches tierischen Zellen durch extrazelluläre Mass- nahmen suspendiertes Wachstum ermöglicht, war Aufgabe der vorliegenden Erfindung.There is therefore a need for a serum- and protein-free growth medium in which cells can proliferate without special mutagenesis or genetic engineering changes. The provision of a medium which animal cells by extracellular mass took suspended growth enabled was the object of the present invention.
Es ist bekannt, dass Suramin Natrium die Bin¬ dung von verschiedenen Wachstumsfaktoren an den entspre- chenden Rezeptoren hemmt, wodurch auch die Proliferation dieser Zellen gehemmt wird. Voogd, T.E. et al. Pharmaco- logical Reviews, 45, 177-203 (1993). Unter den Wachstums¬ faktoren, bei welchen der erwähnte Effekt von Suramin Na¬ trium beschrieben wurde, waren unter anderem bFGF, IGF I und II, PDGF, TGF beta etc. Selbst der mitogene Effekt von fötalem Kälberserum konnte auf diese Art unterdrückt werden. Die Vielfalt der Effekte von Suramin Natrium in bezug auf Wachstumsfaktorregulation lässt auf einen eher generellen Mechanismus schliessen. Die favorisierte Hypo- these geht von einer Maskierung des Wachstumsfaktors mit Suramin Natrium aus. In all den bisher beschriebenen Fäl¬ len hatte Suramin Natrium eine wachstumsinhibierende Wir¬ kung, es wurde daher schon versuchsweise als Chemothera- peutikum gegen Krebs eingesetzt. Ueberraschenderweise konnte nun gezeigt wer¬ de, dass tierischen Zellen durch Zugabe von suraminähnli¬ chen Verbindungen, insbesonders Suraminsalzen wie Suramin Natrium, zum Kulturmedium suspendiertes Wachstum in serum- und/oder proteinfreier Umgebung ermöglicht werden kann. Unter suraminähnlichen Verbindungen sind Stoffe zu verstehen, die mindestens teilweise die räumliche Struk¬ tur des Suramins aufweisen, z.B. die Symmetrie, die ioni¬ sche Gruppen wie Sulfono- oder Phosphonogruppen, sowie aromatische Funktionen enthalten, insbesonders zumindest teilweise wasserlösliche Salze eines sulfonierten, gege¬ benenfalls alkylsubstituierten aromatischen Oligoamid- harnstoffs der allgemeinen Formel I *n Hn It is known that suramin sodium inhibits the binding of various growth factors to the corresponding receptors, which also inhibits the proliferation of these cells. Voogd, TE et al. Pharmacological Reviews, 45, 177-203 (1993). 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. In all the cases described so far, suramin sodium had a growth-inhibiting effect, and it has therefore already been used on a trial basis as a chemotherapeutic agent against cancer. Surprisingly, it has now been shown that 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. Compounds which are similar to suramin are to be understood as meaning substances which at least partially have the spatial structure of suramin, for example the symmetry which contains ionic groups such as sulfono- or phosphono groups, and aromatic functions, in particular at least partially water-soluble salts of a sulfonated counter-product optionally alkyl-substituted aromatic oligoamide urea of the general formula I * n H n
(Ar - X ) -Ar l V ι(Ar - X) -Ar l V ι
R'm R'm R ' m R ' m
Ar = unabhängig voneinander Phenyl oder Naphthyl ist,Ar = independently of one another is phenyl or naphthyl,
R = unabhängig voneinander Wasserstoff oder eine niedere Alkylgruppe mit 1 bis 4 C-R = independently of one another hydrogen or a lower alkyl group with 1 to 4 C-
Atomen bedeutet,Atoms means
R' = -SO3Y ist, wobei Y unabhängig voneinander ein Aequivalent eines Kations ist, insbesonders Na, K,R '= -SO3Y, where Y is independently an equivalent of a cation, in particular Na, K,
Li 0Li 0
1111
X = unabhängig voneinander -NH-C- oderX = independently of one another -NH-C- or
0 f|0 f |
-NH-C-NH- bedeuten, n und m unabhängig voneinander ganze Zahlen von 0 bis 7 bedeuten, deren Summe pro Ar nicht grösser als 7 ist und wobei pro Gesamtmolekül mindestens ein Sub- stituent R' vorhanden sein musε, und p =1 bis 20 ist.-NH-C-NH-, n and m independently of one another are integers from 0 to 7, the sum of which per Ar is not greater than 7 and where at least one substituent R 'must be present per total molecule, and p = 1 is up to 20.
Bevorzugte Oligoamidhamstoffe sind in den abhängigen Ansprüchen 3 bis 6 beschrieben. Durch Zugabe von z.B. Suramin Natrium zumPreferred oligoamide ureas are described in dependent claims 3 to 6. By adding e.g. Suramin sodium for
Nährmedium lassen sich Zellinien, welche zuvor serum-, protein- und/oder oberflächenabhängig gewachsen sind, serum- und proteinfrei sowie als Einzelzellsuspension mit sehr geringer Aggregatbildung züchten. Erfindungsgemäss gezüchtete Zellkulturen sind in den Bildern und Figuren veranschaulicht.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.
Bild 1 zeigt CHO Kl Zellen 4 Tage nach Trans¬ fer in unsupplementiertes FMX 8 Medium,Figure 1 shows CHO Kl cells 4 days after transfer into unsupplemented FMX 8 medium,
Bild 2 zeigt CHO Kl Zellen 4 Tage nach Trans- fer in FMX 8 Medium supplementiert mit 0,5 mg/mnl Suramin Natrium,Figure 2 shows CHO Kl cells 4 days after transfer in FMX 8 medium supplemented with 0.5 mg / ml suramin sodium,
Figur 3 zeigt das Wachstum von CHO Kl Zellen in Spinnerkultur, Figur 4 zeigt das Wachstum von CHO Kl:cycE Zellen im COLOR Bioreaktor,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,
Figur 5 zeigt das Wachstum von tPA produzie¬ renden CHO Zellen (CHO 1-15500; ATCC Nr. CRL 9606), Bild 6 zeigt die Morphologie von BHK 21 Zel¬ len in Adhärenzkultur in unsupplementiertem FMX 8 Medium,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,
Bild 7 zeigt die Morphologie von BHK 21 Zel¬ len in Suspensionskultur in unsupplementiertem FMX 8 Me¬ dium, Bild 8 zeigt die Morphologie von BHK 21 Zel¬ len in FMX 8 Medium supplementiert mit 1 mg/ml Suramin Natrium,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,
Figur 9 zeigt das UV-DifferenzSpektrum einer 1 mg/ml BSA Lösung, welcher 99,9 % des Suramin Natrium (0,5 mg/ml) durch Ultrafiltration in 1 M NaCl Lösung ent¬ zogen wurden gegen eine reine BSA Lösung, undFIG. 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
Figur 10 zeigt das UV-Differenzspektrum einer 1 mg/ml BSA Lösung, welche 1/1000 der Mediumskonzentra¬ tion von Suramin Natrium (0,5 μg/ml) enthält gegen eine reine BSA Lösung.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 μg / ml) against a pure BSA solution.
Es wurde bereits früher beobachtet, dass sich CHO Zellen nach dem Transferieren in ein neues Kulturge- fäss ein bis zweimal teilen und erst dann in einen ruhen¬ den Zustand übergehen. Es ist möglich, dass dies mit der Produktion von Faktoren zusammenhängt, die die Polifera- tion inhibieren und dass solche inhibierend wirkenden Faktoren beim Zusatz von fötalen Blutseren durch die in hoher Konzentration vorhandenen aktivierenden Faktoren kompensiert oder überspielt werden. Offenbar wird eine gewisse Zeit für die Produktion resp. Demaskierung der die Proliferation hindernden Faktoren, beispielsweise in¬ hibierende Faktoren, benötigt, um eine aktive Schwellen¬ konzentration zu erreichen. Durch Zugabe von Suramin Natrium resp. diesem ähnliche Verbindungen ist es nun möglich, serum- und proteinfrei wachsende CHO Zellen ohne Adaptationsphase in einem völlig serum- und proteinfreien Medium (z.B. dem FMX-8 Medium der Firma Messi Cell Culture Technologies, Zürich) zu züchten. Es wird ange¬ nommen, dass die Wirkung der suraminähnlichen Substanzen primär extrazellulär ist, intrazelluläre Wirkungen sind als unwahrscheinlich einzustufen. Die sechs Sulfonatgrup- pen des Suramins selbst lassen einen Durchgang durch die Zellmembran als unwahrscheinlich erscheinen.It has already been observed earlier that CHO cells divide once or twice after transfer into a new culture vessel and only then go into a resting state. It is possible that this is related to the production of factors that inhibit the poliferation and that such inhibiting factors when the fetal blood sera are added are compensated for or overplayed by the activating factors present in high concentration. Apparently there is a certain amount of time for production or Unmasking the factors preventing proliferation, for example inhibiting factors, is required to achieve an active threshold concentration. By adding suramin sodium resp. It is now possible to use these similar compounds to grow serum and protein-free CHO cells without an adaptation phase in a completely serum and protein-free medium (eg the FMX-8 medium from Messi Cell Culture Technologies, Zurich). It is assumed that the effect of the suramin-like substances is primarily extracellular, intracellular effects can be classified as unlikely. The six sulfonate groups of suramin themselves make passage through the cell membrane seem unlikely.
Die Wachstumsraten in suraminhaltigem Medium variieren leicht von Zelllinie zu Zellinie etwa in dem Masse, wie dies schon in der serumhaltigen ursprünglichen Kultur der Fall ist. Wöchentliche Verdünnungsraten im se¬ rum- und proteinfreien FMX-8 Medium liegen im Bereich von 1/25 bis 1/250 und sind somit sehr geeignet für den Ein¬ satz in einem Produktionsprozess. Diese günstigen Effekte konnten bei mehreren Produktionszellinien beobachtet wer- den, so z.B. bei CHO Produzentenzellen für tPA oder der ursprünglichen CHO Kl und DUKX Zelllinien.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. These beneficial effects could be observed in several production cell lines, e.g. at CHO producer cells for tPA or the original CHO Kl and DUKX cell lines.
Suramin Natrium hatte selbst im Fall von be¬ reits serum- und proteinfrei wachsenden CHO Zellen eine überaus positive Wirkung. Diese Zellen waren vorgängig mit einem Expressionsvektor für Cyclin E transfiziert worden. Nach Zugabe von Suramin Natrium konnte schnelle¬ res Wachstum, ein höherer Lebendzellanteil sowie eine deutlich höhere Endzellkonzentration beobachtet werden. Offensichtlich ist bei diesen Zellen weiterhin ein extra- zellulärer Inhibitionsmechanismus aktiv, welcher durch Suramin unterdrückt wird.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 Zellen (BHK 21) werden ebenfalls zur Produktion von pharmazeutisch wirksamen Substanzen eingesetzt. Dabei werden Medien verwendet, welche entweder fötales Kälberserum als Zusatz enthalten oder Wachstumsfaktoren und Proteine wie Transferrin und Insulin. Ueberraschenderweise ist es den Erfindern gelun¬ gen BHK 21 Zellen in einem ursprünglich für CHO Zellen entwickelten Medium zu züchten (FMX 8 Medium der Firma Messi Cell Culture Technologies Zürich, F. Messi, Diss. ETH Nr. 9559(1991)). Dabei wurde ein sehr schnelles Wachstum und ein hoher Lebendzellanteil beobachtet. Wöchentliche Verdünnungsraten von 1/100 konnten über min¬ destens drei Monate lang aufrecht erhalten werden. Die Morphologie der Zellen war dabei, wie in Bild 6 zu er¬ kennen, ausgebreitet und adhärent. BHK 21 Zellen können jedoch alternativ auch als suspendierte Einzelzellkultur gezüchtet werden mit extrem geringer Aggregatbildung. Dabei ist ein weiterer positiver wie überraschender Effekt von Suramin Natrium, dass es Zeil-Zeil und Zeil-Substrat Adhäsionsprozesse blockiert. Oberflächengebunden wachsende CHO oder BHK 21 Zellen wachsen nach Zugabe von 0,5 - 1 mg/ml Suramin Na¬ trium zum Kulturmedium in Suspension als Einzelzellkul¬ tur. In all den oben beschriebenen Fällen konnte ein vollständiger Uebergang zu abgerundeter Morphologie und suspendiertem Wachstum beobachtet werden (siehe Bild 8).Baby hamster kidney cells (BHK 21) 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. Surprisingly, 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)). 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. Alternatively, BHK 21 cells can also be grown as a suspended single cell culture with extremely low aggregate formation. Another positive and surprising effect of suramin sodium is that it 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 Zellen bilden in Suspen¬ sionskultur (z.B. Spinnerkultur) in Medien mit oder ohne Serum und Proteinen sphärische Aggregate aus, welche da¬ durch gekennzeichnet sind, dass sich die Zellen aufeinan- der und auf schon bestehenden Aggregaten ausbreiten (sie¬ he Bild 7). Diese Art der Aggregation unterscheidet sich deutlich von jener der CHO Zellen. Die Adhäsion von BHK 21 Zellen aneinander ist ein aktiver Prozess. Dieser Ad- häsionsprozess kann durch Zugabe von Suramin Natrium ins Nährmedium verhindert werden. BHK 21 Zellen wachsen so als Einzelzellsuspension, es kann keine Ausbreitung der Zellen aufeinander beobachtet werden (siehe Bild 8). Da¬ mit erhöht sich der Lebendzellanteil von BHK Zellkulturen enorm. Die NährstoffVersorgung ist nicht mehr limitiert wie dies im Inneren von Zellaggregaten der Fall ist. In biotechnologischen Prozessen mit tierischen Zellen ist das vollkommen suspendierte Wachstum ein grosser Vorteil. Die Vermeidung teurer Microcarrier vereinfacht und ver¬ billigt den Prozess enorm. Das Einphasensystem erlaubt zudem eine homogenere Prozessführung und -kontrolle.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.
Suraminähnliche Verbindungen werden dem Zell- kulturmedium in Mengen von 8*10~5 bis lO-*-* molar zuge- geben, Suramin Natrium vorzugsweise in Mengen von 0,2 - 1 mg/ml. In diesem Konzentrationsbereich werden keinerlei toxische Effekte beobachtet (LD^Q in Maus = 620 mg/kg Körpergewicht i.V.). Die Verwendung von Suramin Natrium als solches in Zellkulturprozessen ist ebenfalls unbe¬ denklich, da die Substanz selbst als therapeutisches Pro¬ dukt für Menschen zugelassen ist und somit sämtliche kli¬ nischen Tests zur Ermittlung eventueller Toxizität er¬ folgreich durchlaufen hat. Suramin Natrium ist kommerziell erhältlich und relativ günstig, so dass es, obschon es dem Medium in relativ hoher Menge zugegeben wird, nur einen minimen Kostenfaktor darstellt, der - verglichen mit den Kosten eines Zellkulturprozesses - absolut vernachlässigbar ist. Es wird angenommen, dass die Wirkung vonSuramin-like compounds are added to the cell culture medium in amounts of 8 * 10 ~ 5 to 10 - * - * molar give Suramin sodium preferably in amounts of 0.2 - 1 mg / ml. No toxic effects were observed in this concentration range (LD ^ Q in mouse = 620 mg / kg body weight IV). 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 Natrium durch relativ unspezifische Wechselwir¬ kungen mit Proteinen zustandekommt. Somit muss auch davon ausgegangen werden, dass eine Wechselwirkung zwischen Suramin Natrium und dem gewünschten Endprodukt besteht, eventuell sogar eine Bindung auftritt. Es ist daher von grosser Wichtigkeit, Methoden zur Hand zu haben, mit wel¬ chen sich Suramin Natrium aus dem Endprodukt entfernen sowie nachweisen lässt. Eine günstige Methode zur Entfer¬ nung ist Ultrafiltration nach der Neutralisierung von ionischen Wechselwirkungen. So konnten in einem Schritt 99.9 % der ursprünglich vorhandenen Menge Suramin Natrium aus einer Proteinlösung entfernt werden. Der Restgehalte entsprach 1 Molekül Suramin Natrium pro 5 Molekülen Rin¬ der Serum Albumin, einem Protein, das wegen seiner hohen Adsorptionsfähigkeit als Modellprotein gewählt wurde. Zum Nachweis von Suramin Natrium wurde in der vorliegenden Arbeit UV Spektroskopie gewählt. Durch die aromatischen Gruppen im Molekül kann ein charakteristisches Absorp¬ tionsmaximum bei 310 n zur Detektion herangezogen wer- den. Die in Proteinen enthaltenen Aromaten (Phe, Tyr, Trp) absorbieren allesamt bei niedrigeren Wellenlängen. Beispiel 1Suramin 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
Serum- und proteinfreies Wachstum von CHO Kl ZellenSerum and protein free growth of CHO Kl cells
Zum Uebergang von serum- und oberflächenab¬ hängigem Wachstum zu serum- und proteinfreiem Wachstum in Suspension wird keinerlei Adaptations- oder Selektions¬ phase benötigt. Die Zellen einer konfluenten T75 Flasche werden durch Trypsinisierung (Life Technologies) abgelöst und zur Inaktivierung des Trypsins in 10 ml einer 1 mg/ml Soybean Trypsin Inhibitor (Sigma) Lösung in Medium aufge¬ nommen. 0,2 ml dieser Zellsuspension werden in 25 ml- FMX 8 Medium aufgenommen, welches Suramin Natrium (Bayer AG) in einer Konzentration von 0,5 mg/ml enthält. DieserNo transition or selection phase is required for the transition from serum and surface-dependent growth to serum and protein-free growth in suspension. The cells of a confluent T75 bottle are detached by trypsinization (Life Technologies) and taken up in 10 ml of a 1 mg / ml Soybean trypsin inhibitor (Sigma) solution in medium to inactivate the trypsin. 0.2 ml of this cell suspension is taken up in 25 ml FMX 8 medium which contains suramin sodium (Bayer AG) in a concentration of 0.5 mg / ml. This
Zellkultur wird nach 4 Tagen 25 ml des gleichen suramin- haltigen Mediums zugegeben. Nach einer Woche kann die Kultur durch einfaches Verdünnen 1/50 gesplittet werden. (1 ml Kultur in 24ml frisches Medium plus refeed nach 4 Tagen etc.). Auf diese Weise wurden CHO Kl Zellen während drei Monaten stabil in Kultur gehalten. CHO Kl Zellen sind vier Tage nach dem Serumentzug in Medium ohne Sura¬ min Natrium auf Bild 1 sowie in Medium mit Suramin Natri¬ um auf Bild 2 dargestellt. CHO Kl Zellen wurden zur Be- Stimmung der Wachstumsparameter in 0,5 1 Spinnerflaschen (Technomara) in FMX 8 Medium mit 0,5 g/1 Suramin Natrium Zusatz gezüchtet. Wachstumskurven dieser Kultur sind Fi¬ gur 3 zu entnehmen. Zelldichten wurden nach Trypanblau- färbung im Hämacytometer bestimmt. Glukosekonzentrationen wurden mit Hilfe eines YSI Glucoseanalysers bestimmt. Beispiel 2After 4 days, 25 ml of the same suramin-containing medium are added to cell culture. After a week, the culture can be split by simply diluting 1/50. (1 ml culture in 24 ml fresh medium plus refeed after 4 days etc.). In this way, 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. To determine the growth parameters, CHO Kl cells were grown in 0.5 1 spinner bottles (Technomara) in FMX 8 medium with 0.5 g / 1 suramin sodium additive. 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. Example 2
Serum- und proteinfreies Wachstum von CHO KlcycE Zellen Suramin Natrium hat ebenfalls einen günstigenSerum and protein free growth of CHO KlcycE cells suramin sodium also has a favorable one
Einfluss auf das Wachstum von CHO Zellen, welchen durch andere Methoden das Wachstum in serum- und proteinfreiemInfluence on the growth of CHO cells, which by other methods the growth in serum and protein free
Medium ermöglicht wurde. Dazu wurden Vergleichskulturen angesetzt, welche unter sonst identischen Bedingungen in Medien mit resp. ohne Suramin Natrium-Zusatz kultiviert wurden.Medium was made possible. For this purpose, comparative cultures were set up which, under otherwise identical conditions, in the media with resp. were grown without suramin sodium addition.
Herstellung von Kl cvc E-Zellen Die cDNA des humanen Cyclin E Gens kann durchProduction of Kl cvc E cells The cDNA of the human cyclin E gene can be by
Standard-Hybridisierungsverfahren aus einer HeLa cDNA- Bank isoliert werden. Sämtliche folgenden Methoden sind Standard Labor Technik und wurden nach Sambrook et al. ausgeführt. HeLa mRNA wurde mit Hilfe eines RNA Extrak- tionskits der Firma Pharmacia isoliert. Nach cDNA Syn¬ these und Einbau in den Phagen lambda entsprechend Her¬ stellerangaben (Stratagene) wurde mittels Standard Hy- bridisierungstechniken die cDNA des humanen Cyclin E Gens isoliert (Sambrook,J. , Fritsch, E.F. und Maniatis, T. Molecular cloning Cold Spring Harbor Laboratory PressStandard 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
(1989)). Nach in vivo Excision ("Zappen") der cDNA-Bank entsprechend Herstellerangaben lag die cDNA im Plasmid pBluescript vor. Nach Restriktionsverdau mit Eco RI liess sich ein Fragment mit einer Grosse von 2.5 kb aus einem 0.8 % low melt-Agarose Gel isolieren. Nach dem Lineari- sieren des Vektors pRc/CMV (Invitrogen) mit dem Restrik¬ tionsenzym Bst XI wurden Fragment und Vektor mit dem Klenowenzy aufgefüllt. Nach Ligation und Transformation in den E.coli Stamm DH5alpha und Identifizierung eines Konstruktes in Sinn-Orientierung wurden grössere Mengen des Expressionsvektors mit dem FlexiPrep Kit (Pharmacia) hergestellt. CHO Kl Zellen wurden so in eine Kulturschale einer "Six-well-plate" (TPP) eingesät, dass am Tag der Transfektion 50-70 % Konfluenz erreicht wurde. Dabei be¬ fanden sich die Zellen in Medium das 10 % fötales Kälber- serum enthält (z.B. Ham's F12, Gibco BRL) .(1989)). After in vivo excision ("zapping") of the cDNA bank in accordance with the manufacturer's instructions, 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. The cells were found in medium containing 10% fetal calf serum (eg Ham's F12, Gibco BRL).
Zur Lipofektion wurden als erstes zwei Scha¬ len einer 24 well-plate jeweils mit 100 μl FMX-8 Medium gefüllt. In eine dieser Schalen wurden 10 μl LipofektAmin (Gibco) und in die andere 1-2 μg DNA des pRC Cyclin E Ex- pressionsvektors gegeben. Nach Mischung der beiden Lösun¬ gen wurde 30 min bei Raumtemperatur inkubiert. In der Zwischenzeit wurden die CHO Kl Zellen dreimal mit serum¬ freiem FMX-8 Medium gewaschen. Nach Zugabe von 800 μl Me¬ dium zu den 200 μl LipofektAmin/DNA Mischung wurde dieses Reagenz zu den gewaschenen Zellen gegeben. Nach 6h Inku¬ bation bei 37°C und 5% C02 wurden weitere 1.5 ml FMX 8 Medium zugegeben.For lipofection, two shells of a 24-well plate were first filled with 100 μl FMX-8 medium. 10 μl LipofektAmin (Gibco) were placed in one of these dishes and 1-2 μg DNA of the pRC Cyclin E expression vector in the other. After the two solutions had been mixed, they were incubated at room temperature for 30 min. In the meantime, the CHO Kl cells were washed three times with serum-free FMX-8 medium. After adding 800 μl of medium to the 200 μl lipofectamine / DNA mixture, this reagent was added to the washed cells. After 6 hours of incubation at 37 ° C. and 5% CO 2, a further 1.5 ml of FMX 8 medium were added.
24 h nach Beginn der Lipofektion wurden die Zellen durch Trypsinisierung abgelöst. Dazu wurde das Medium entfernt, 1 ml Trypsinlösung (Gibco BRL) zuge¬ geben, etwa 1 min bei leichtem Schütteln gewartet, das Trypsin abgesogen und etwa 10 min bei 37°C und 5% C02 inkubiert. Die abgelösten Zellen wurden in 2 ml FMX 8 Medium aufgenommen, das 1/1000 (w/vol) Trypsininhibitor (Sigma) enthielt.24 hours after the start of lipofection, the cells were detached by trypsinization. For this purpose, 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).
Die Kulturflaschen wurden während der ersten drei Wochen mit Fibronectin (Boehringer Mannheim) be¬ schichtet (lμg/cm***), um in der Uebergangsphase den Zellen die Adhäsion zu erleichtern. 1 ml sowie 0.5 ml der abgelösten Zellen wur¬ den nun in 5 ml FMX 8 Medium in T-25 Kulturflaschen aufgenommen und inkubiert. Da die Effizienz der Lipofek¬ tion, sowie der Anteil an überlebenden Zellen nach der Lipofektion variieren können, ist es ratsam beim Subkul- tivieren verschiedene Zellkonzentrationen einzusetzen um das Ueberleben der Kulturen zu sichern. Nach einigen Tagen Proliferation, die auf transiente Expression des Cyclin E Gens zurückzuführen ist, wird üblicherweise nach einer Woche ein vorüberge¬ hender Rückgang der Proliferation beobachtet, bis die Zellen, die den Vektor stabil eingebaut haben, die Kultur überwachsen haben. Daher muss von Fall zu Fall, die Ver¬ dünnungsrate eingestellt werden. Trotzdem ist es ratsam, jede Woche zu subkultivieren, da offenbar Rückstände to¬ ter Zellen am Plastik inhibierend wirken. Nach einer Woche wurde die Kultur in eine be¬ schichtete T-75 Flasche transferiert (Verdünnungsrate 1/2 bis 1/5). Nach etwa drei Wochen weiterer Kultivierung mit Verdünnungsraten zwischen 1/2 und 1/20, Hessen sich- die Zellen mit wöchentlichen Verdünnungsraten von 1/40 in un- beschichteten Kulturflaschen züchten. Diese Zellen können in serum- und proteinfreiem FMX-8 Medium während langer Zeit in Kultur gehalten werden.The culture bottles were coated with fibronectin (Boehringer Mannheim) during the first three weeks (1 μg / 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. After one week, the culture was transferred to a coated T-75 bottle (dilution rate 1/2 to 1/5). After about three weeks of further cultivation with dilution rates between 1/2 and 1/20, Hessen, 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.
Einfluss von Suramin Natrium auf das Wachstum von CHO Kl cvc E ZellenInfluence of suramin sodium on the growth of CHO Kl cvc E cells
In T-Flaschen zeigt sich der Unterschied durch eine deutlich höhere Endzelldichte sowie durch die grössere wöchentliche Verdünnungsrate von 1/250, mit wel- eher diese Zellen gezüchtet werden können, im Vergleich zu 1/50 ohne Suramin Zusatz.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.
Figur 4 zeigt die Wachstumsparameter von CHO KlcycE Zellen mit Zusatz von 0,5 mg/ml Suramin Natrium im compact loop Bioreaktor (Bioengineering). Die physikali- sehen Parameter wurden wie folgt eingestellt: Arbeitsvo¬ lumen 2,3 1; Temperatur 37°C, pH 7,3; p02 50% Luftsauer¬ stoffSättigung; Rührerdrehzahl 580 rpm. Zelldichten wur¬ den nach Trypanblaufärbung im Hämacytometer gezählt. Glu¬ kosekonzentrationen wurden mit Hilfe eines YSI Glucose- analysers bestimmt.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.
Ein Vergleich der Wachstumsparameter von CHO Kl cyc E-Zellen mit und ohne Suramin Natrium Zusatz ist der Tabelle zu entnehmen. Tabelle ohne Suramin mit SuraminThe table shows a comparison of the growth parameters of CHO Kl cyc E cells with and without suramin sodium. Table without suramin with suramin
Zelldichte pro ml 800O00 2'700'000 Wachstumsrate (pro Tag) 0.8 1.05 Verdoppelungszeit (Std. ) 21 15.8Cell density per ml 800,000 2,700,000 growth rate (per day) 0.8 1.05 Doubling time (hours) 21 15.8
Beispiel 3Example 3
Serum- und proteinfreies Wachstum von tPA produzierenden CHO Zellen (CHO 1-15500; ATCC Nr. CRL-9606.Serum and protein free growth of tPA producing CHO cells (CHO 1-15500; ATCC No. CRL-9606.
Der Uebergang dieser Zellinie in serum- und proteinfreies Medium wird wie unter Beispiel 1 vollzogen. CHO tPA Zellen werden in FMX 8 Medium mit 0,5 mg/ml Zu¬ satz Suramin Natrium gezüchtet. Die dabei zu erreichenden wöchentlichen Verdünnungsraten liegen ebenfalls bei 1/50. Die Morphologie dieser Zellen änderte sich in selbem Mas¬ se wie in Beispiel 1 für CHO Kl Zellen beschrieben. Die Wachstumsparameter der CHO tPA Zellen sind in Figur 5 dargestellt.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.
Beispiel 4Example 4
Serum- und proteinfreies adhärentes Wachstum von BHK 21 ZellenAdherent growth of BHK 21 cells free of serum and protein
BHK 21 Zellen können im serum- und protein¬ freien FMX 8 Medium der Firma Messi Cell Culture Techno¬ logies adhärent gezüchtet werden. Zum Uebergang von se¬ rumabhängigem Wachstum zu serum- und proteinfreiem Wachs¬ tum in Suspension wird keinerlei Adaptations- oder Selek¬ tionsphase benötigt. Die Zellen einer konfluenten T75 Flasche (TTP) werden durch Trypsinisierung abgelöst und zur Inaktivierung des Trypsins in 10 ml einer 1 mg/ml Soybean Trypsin Inhibitor (Sig a) Lösung in Medium aufge¬ nommen. 0,1 ml dieser Zellsuspension werden in 25 ml FMX 8 Medium aufgenommen. Nach 4 Tagen werden 25 ml FMX 8 Me¬ dium zugegeben und nach 1 Woche trypsinisiert wie oben beschrieben. BHK 21 Zellen konnten auf diese Weise minde¬ stens drei Monate lang mit wöchentlichen Verdünnungsraten von 1/100 gezüchtet werden. Die Morphologie der Zellen in Adhärenzkultur ist auf Bild 6 abgebildet.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.
Beispiel 5Example 5
Einzelzell-Suspensionskulturen von BHK 21Single cell suspension cultures from BHK 21
ZellenCells
BHK 21 Zellen können alternativ auch in Sus¬ pension gezüchtet werden. Ohne Zugabe von Suramin Natrium bilden sich in Suspensionskultur sehr grosse Aggregate (siehe Bild 7). Diese entstehen in gerührter (Spinner oder Bioreaktor) wie in ungerührter Suspensionskultur in BSA (Rinder Serum Albumin) beschichteten T-Flaschen. Die¬ se Aggregation wird durch Zugabe von 1 mg/ml Suramin Na- trium zum Kulturmedium gänzlich verhindert. Die Morpholo¬ gieänderung nach Suramin Zugabe ist in Bild 8 darge¬ stellt. Die Wachstumsparameter einer Spinnerkultur von BHK 21 Zellen in suraminhaltigem Medium sind in Figur 9 dargestellt. Die Eigenschaften dieser Zellkulturen eignen sich besonders für den Einsatz in Produktionsprozessen, besonders hinsichtlich einfacher Handhabung, geringer Einsaatdichten sowie schnellen Wachstums zu hohen Zell¬ dichten bei gleichzeitiger Verhinderung von Aggregaten.Alternatively, 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.
Beispiel 6Example 6
Entfernung von Suramin Natrium aus ProteinRemoval of suramin sodium from protein
Lösungen Zur Entfernung von Suramin Natrium aus Prote¬ inlösungen (z.B. Zellkulturüberständen) wurde die Methode der Ultrafiltration gewählt. Als Modellprotein wurde Rinder Serum Albumin (BSA) gewählt. Dieses Protein zeich¬ net sich durch seine hohe Adsorptionsfähigkeit aus. Einer 1 mg/ml BSA Lösung wurde Suramin Natrium in Mengen wie sie im Zellkulturmedium vorhanden sind (500 μg/ml) zuge- geben. 100 ml dieser Lösung wurde NaCl in einer Konzen¬ tration von IM zugegeben. Diese Lösung wurde durch eine Membran mit einer Porengrösse von 10'000 Dalton ultrafil¬ triert. Das Filtrat wurde in 100 ml einer IM Kochsalz¬ lösung aufgenommen und filtriert; dieser Vorgang wurde im ganzen dreimal durchgeführt. Das so gereinigte Filtrat wurde in Wasser aufgenommen, so dass eine 1 mg/ml BSA Lö¬ sung resultierte. Der Restgehalt an Suramin Natrium wurde mittels UV-Spektroskopie ermittelt. Dabei konnte durch Vergleich mit Referenzspektren von Suramin/BSA Masslösun- gen eine Restkonzentration von 0,5 μg/ml ermittelt werden (Figuren 9 und 10). Bezogen auf die Anfangskonzentration wurden somit 99,9 % des Suramin Natriums der Proteinlö¬ sung entzogen, d.h. nach der Reinigung befand sich pro 5 Moleküle BSA nur ein Molekül Suramin Natrium in der Lö- sung. Solutions The method of ultrafiltration was chosen to remove suramin sodium from protein solutions (eg cell culture supernatants). As a model protein 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 μg / 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 μg / 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.

Claims

Patentansprüche claims
1. Serumfreies und insbesonders serum- und proteinfreies Kulturmedium, dadurch gekennzeichnet, dass es mindestens eine suraminähnliche Verbindung enthält.1. Serum-free and in particular serum and protein-free culture medium, characterized in that it contains at least one suramin-like compound.
2. Kulturmedium, dadurch gekennzeichnet, dass die suraminähnliche Verbindung ein zumindest teilweise wasserlösliches Salz eines sulfonierten, gegebenenfalls Alkyl-substituierten aromatischen Oligoamidharnstoff enthält, der der allgemeinen Formel I2. Culture medium, characterized in that the suramin-like compound contains an at least partially water-soluble salt of a sulfonated, optionally alkyl-substituted aromatic oligoamide urea, which has the general formula I
(Ar - X ) -Ar I(Ar - X) -Ar I
R m κ m R m κ m
entspricht, in dercorresponds in which
Ar = unabhängig voneinander Phenyl oderAr = independently of one another phenyl or
Naphthyl ist,Is naphthyl,
R = unabhängig voneinander Wasserstoff oder eine niedere Alkylgruppe mit 1 bis 4 C- Atomen bedeutet,R = independently of one another is hydrogen or a lower alkyl group having 1 to 4 carbon atoms,
R' = -SO3Y ist, wobeiR '= -SO3Y, where
Y = unabhängig voneinander ein Aequivalent eines Kations ist, insbesonders Na, K,Y = independently of one another is a cation equivalent, in particular Na, K,
Li 0Li 0
// X unabhängig voneinander -NH-C- oder// X independently of one another -NH-C- or
OO
Ü -NH-C-NH- bedeuten,Ü -NH-C-NH- mean
n und m unabhängig voneinander ganze Zahlen von 0 bis 7 bedeuten, deren Summe pro Ar nicht grösser als 7 ist und wobei im Gesamtmolekül mindestens ein Substitu- ent R' vorhanden sein muss, und p = 1 bis 20 ist.n and m independently of one another are integers from 0 to 7, the sum of which per Ar is not greater than 7 and where at least one substituent R 'must be present in the total molecule, and p = 1 to 20.
3. Kulturmedium gemäss Anspruch 2, dadurch gekennzeichnet, dass p = 5 ist. 3. Culture medium according to claim 2, characterized in that p = 5.
4. Kulturmedium gemäss Anspruch 2 oder 3, dadurch gekennzeichnet, dass R unabhängig voneinander -H oder -CH3 und R' = -Sθ3Na und die Summe über alle m im Molekül >.2 ist.4. Culture medium according to claim 2 or 3, characterized in that R independently of one another is -H or -CH3 and R '= -Sθ3Na and the sum over all m in the molecule is> .2.
5. Kulturmedium gemäss Anspruch 4, dadurch gekennzeichnet, dass die suraminähnliche Verbindung die folgende Strukturformel II5. Culture medium according to claim 4, characterized in that the suramin-like compound has the following structural formula II
Figure imgf000021_0001
Figure imgf000021_0001
besitzt.owns.
6 . Kulturmedium gemäss einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, dass es eine suramin- ähnliche Verbindung in einer Konzentration von 8-10~5 bis 10~3 molar enthält.6. Culture medium according to one of claims 1 to 5, characterized in that it contains a suramin-like compound in a concentration of 8-10 ~ 5 to 10 ~ 3 molar.
7. Tierzellkultur mit suspendierten, im we¬ sentlichen nicht agglomerierten Tierzellen in einem se¬ rumfreien Medium, dadurch gekennzeichnet, dass das Medium ein Medium gemäss einem der Ansprüche 1 bis 6 ist.7. Animal cell culture with suspended, essentially non-agglomerated animal cells in a serum-free medium, characterized in that the medium is a medium according to one of claims 1 to 6.
8. Tierzellkultur gemäss Anspruch 7, dadurch gekennzeichnet, dass die Tierzellen Säugetierzellen sind.8. Animal cell culture according to claim 7, characterized in that the animal cells are mammalian cells.
9. Tierzellkultur gemäss Anspruch 8, dadurch gekennzeichnet, dass die Tierzellen Chinese Hamster Ovary oder Baby Hamster Kidney Zellen sind.9. Animal cell culture according to claim 8, characterized in that the animal cells are Chinese Hamster Ovary or Baby Hamster Kidney cells.
10. Verwendung von suraminähnlichen Verbin¬ dungen als Zusatz zu serumfreiem, insbesondere serum- und proteinfreiem Kulturmedium.10. Use of suramin-like compounds as an additive to serum-free, in particular serum and protein-free culture medium.
11. Verwendung gemäss Anspruch 10, dadurch gekennzeichnet, dass die suraminähnliche Verbindung eine11. Use according to claim 10, characterized in that the suramin-like compound
Verbindung entsprechend der allgemeinen Formel I gemäss Anspruch 2 enthält. Contains compound according to general formula I according to claim 2.
12. Verwendung gemäss Anspruch 11, dadurch gekennzeichnet, dass p = 5 ist.12. Use according to claim 11, characterized in that p = 5.
13. Verwendung gemäss Anspruch 11 oder 12, dadurch gekennzeichnet, dass R unabhängig voneinander Wasserstoff oder -CH3 bedeuten und R'= -SÜ3Na und die Summe aller m in Molekül __ 2 ist.13. Use according to claim 11 or 12, characterized in that R is independently hydrogen or -CH3 and R '= -SÜ3Na and the sum of all m in molecule __ 2.
14. Verwendung gemäss Anspruch 13, dadurch gekennzeichnet, dass als suraminähnliche Verbindung eine Verbindung der Strukturformel II gemäss Anspruch 5 ver- wendet wird.14. Use according to claim 13, characterized in that a compound of structural formula II according to claim 5 is used as the suramin-like compound.
15. Verwendung gemäss einem der Ansprüche 9 bis 14, dadurch gekennzeichnet, dass das Kulturmedium FMX 8 ist.15. Use according to one of claims 9 to 14, characterized in that the culture medium is FMX 8.
16. Verfahren zum Züchten von Tierzellen in serumfreiem und insbesonders in serum- und proteinfreiem16. Method for growing animal cells in serum-free and especially in serum and protein-free
Medium, dadurch gekennzeichnet, dass die Zellen in ein Kulturgefäss transferiert werden, welches ein Kulturme¬ dium gemäss einem der Ansprüche 1 bis 6 enthält, und dass sie bei nachfolgenden Verdünnungsstufen ebenfalls in Kul- turgefässe, enthaltend ein Kulturmedium gemäss einem der Ansprüche 1 bis 6, weitertransferiert werden.Medium, characterized in that the cells are transferred into a culture vessel which contains a culture medium according to one of claims 1 to 6, and that, in the subsequent dilution stages, they are also transferred into culture vessels containing a culture medium according to one of claims 1 to 6 , to be transferred further.
17. Verwendung von FMX 8 zur adhärenten Kul¬ tivierung von Baby Hamster Kidney Zellen. 17. Use of FMX 8 for the adherent cultivation of baby hamster kidney cells.
PCT/CH1995/000191 1994-09-09 1995-09-05 Chemical process for promoting the proliferation of animal cells WO1996007730A2 (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
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US9758568B2 (en) 2006-01-04 2017-09-12 Baxalta GmbH Oligopeptide-free cell culture media

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WO1998026084A1 (en) * 1996-12-09 1998-06-18 Wolfgang Andreas Renner Expression of active interferon beta 1 using recombinant rna replicons
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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