WO1993005144A1 - Process for cultivating mammal cells in a fluidised bed reactor - Google Patents

Process for cultivating mammal cells in a fluidised bed reactor Download PDF

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Publication number
WO1993005144A1
WO1993005144A1 PCT/EP1992/001918 EP9201918W WO9305144A1 WO 1993005144 A1 WO1993005144 A1 WO 1993005144A1 EP 9201918 W EP9201918 W EP 9201918W WO 9305144 A1 WO9305144 A1 WO 9305144A1
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cells
cell
bed reactor
mammalian cells
cultivation
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PCT/EP1992/001918
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German (de)
French (fr)
Inventor
Peter Reuschenbach
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Basf Aktiengesellschaft
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Publication of WO1993005144A1 publication Critical patent/WO1993005144A1/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
    • C12N5/0068—General culture methods using substrates
    • C12N5/0075—General culture methods using substrates using microcarriers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/16—Particles; Beads; Granular material; Encapsulation
    • C12M25/20—Fluidized bed
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/10—Mineral substrates
    • C12N2533/12—Glass
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides

Definitions

  • the cultivation of mammalian cells is the basis for a large number of biotechnological production processes, in particular the production of pharmaceutical proteins.
  • Carrier bodies made of glass are reusable and autoclavable, however, the cell yield is with these
  • EP 303 262 discloses a support body made of glass or ceramic with a content-containing surface layer; however, the use of these carrier bodies for mammalian cell culture is not described.
  • the object of the present invention was to provide a method for the cultivation of mammalian cells immobilized on support bodies, in which the support bodies ensure a high cell yield, are autoclavable, easy to clean and reusable.
  • the method for cultivating mammalian cells in a fluidized bed reactor is particularly advantageous if the mammalian cells are immobilized on porous glass support bodies, the surface layer of which contains amino groups bonded to a support. It has also been found that a surface layer containing diethylaminoethyl dextran (DEAE dextran) is particularly suitable. Furthermore, it was found that the mouse fibroblast cell line C-127 (ATCC CRL 1616) can be cultivated particularly well with this method.
  • the porous glass support body can be in any shape; they are preferably used in a spherical shape.
  • the size of the carrier body is expediently from 0.4 to 5 mm, preferably 1 to 2 mm.
  • the pore size of the porous support body is generally 20 to 500 ⁇ m, preferably 60 to 300 ⁇ m.
  • micropores of 1 to 10 ⁇ m can also be present in the carrier bodies.
  • Open-pore sintered glass carrier bodies e.g. of the Siran® brand (Schott, Mainz).
  • the carrier bodies can consist of soda-lime glass or borosilicate glass.
  • Sintered glass carrier bodies made of borosilicate glass are particularly preferred.
  • Suitable amino groups are dialkylamino groups, in particular dialkylaminoalkyl groups, the alkyl groups advantageously having 1 to 4 carbon atoms.
  • Amino groups bound to a carrier include amino groups which are linked to a polymer, e.g. are covalently linked to a polysaccharide. Amino groups which are bonded to glucans, in particular dextran, are preferred. Diethyla inoethyl-dextran (DEAE-dextran) is particularly preferably used.
  • the glass support bodies containing amino groups bonded to the surface of the support are commercially available (Schott, Mainz, Product Information No. 6196d).
  • Suitable mammalian cells for the method according to the invention are both adherent and suspension cells growing in suspension. Permanent cell lines or primary cultures can be used. Genetically modified (recombinant) cell lines can also be used. Hybridoma cells and fibroblasts, in particular the mouse fibroblast cell line C-127 (ATCC CRL 1616) are preferably used. The cell line C-127 secreting the tissue plasminogen activator (t-PA), which transforms with a bovine papomomirus expression vector, is particularly preferred had been used. The production of this cell line is by Reddy et al. (J. Cell. Biochem., Supplement 10 D, 154, 1986).
  • tissue plasminogen activator t-PA
  • bioreactors are suitable as fluidized bed reactors for the process according to the invention, such as those e.g. by Lobby and Griffiths (Cytotechnology 1, 339-346, 1988; Advances in Ani.mal Cell Biology and Technology for Bioprocesses, Eds. Spier et al., Butterworths, Guilford, 336-344, 1989).
  • the volume of the support body in the unexpanded state is usually 10 to 80%, preferably 20 to 60% of the reactor volume.
  • All conventional cell culture media can be used as nutrient media for cultivating the mammalian cells.
  • Serum-free culture media can also be used. Whether serum needs to be added depends on the mammalian cell used.
  • the immobilization of the mammalian cells on the carrier bodies expediently takes place in that the mammalian cells are pre-cultivated by conventional cell culture techniques and then introduced into the fluidized bed reactor containing the carrier bodies, so that a cell density in the reactor of 5-10 '+ to 1-106, preferably from 1 to 8-105 cells / ml.
  • the cultivation of the mammalian cells according to the method according to the invention can be carried out continuously or batchwise. However, work is preferably carried out continuously.
  • a large number of cells is expediently generated by cell multiplication and then in a second phase (the production phase) these cells form the valuable substance.
  • the method according to the invention makes it possible to cultivate mammalian cells to a high density on porous support bodies.
  • the carrier bodies have excellent mechanical properties, so that in the fluidized bed reactor e.g. there is no abrasion which reduces the cell yield.
  • these carrier bodies can be autoclaved by superheated steam; you can e.g. be sterilized in the reactor before cultivation begins. This also makes it possible to reuse the carrier bodies after cleaning and sterilization.
  • the mammalian cells were first grown in commercially available cell culture bottles. For this purpose, cell culture bottles were inoculated with 0 r 5 - 1 r 0 x 105 cells.
  • DMEM medium 4.5 g glucose per liter, 584 mg glutamine per liter
  • fetal calf serum 10%)
  • polyalkylene glycol were used as growth medium
  • the fluidized bed reactor consisted of a 3.2 cm x 28 cm long glass cylinder provided with a double jacket and conically converging at the lower end and a 2.8 x 13 cm conditioning vessel also provided with a double jacket.
  • the total volume of the unit including the connecting hoses was 380 ml.
  • the medium was conveyed with the help of a peristaltic pump between the conditioning vessel and the fluidized bed reactor at a speed of 20 to 60 cm per minute.
  • Fresh medium was fed into the conditioning vessel via a peristaltic pump and used medium was removed from the conditioning vessel via an overflow.
  • the media exchange was started on the 3rd day with an exchange rate of 0.5 reactor volume per day and gradually increased in the production phase to an average value of 4.3 reactor volume per day.
  • the system was tempered via the double jackets of the reactor and the conditioning vessel using a water bath.
  • the incubation temperature was 37 ° C in the first 6 days, then 35 ° C.
  • the oxygen partial pressure was set to 30% (air saturation). This partial pressure was maintained with the aid of a measuring and control unit by automatically introducing air into the head space or oxygen into the liquid of the conditioning vessel.
  • the pH setpoint was regulated by automatic metering of C0 2 into the head space of the conditioning vessel between pH 6.9 and 7.2.
  • porous sintered glass carrier bodies with a DEAE dextran surface layer and having a diameter of 1 to 2 mm and a pore size between 60 and 300 ⁇ m served as carrier material for the C-127 cells.
  • the reactor was inoculated with 7 x 105 cells / ml.
  • DMEM medium 4.5 g glucose per liter, 584 mg glutamine per liter
  • fetal calf serum 10%
  • Pluronic F-68® (0.157o) were used as culture medium in the first 6 days.
  • a medium consisting of cell culture medium (IGl®, Genzyme), glutamine (292 mg / 1), insulin (5 mg / 1), polyalkylene glycol (Pluronic F-68®) (0.15%), bovine serum albumin (150 mg / l) and fetal calf serum.
  • the serum concentration in the medium was gradually reduced from 10% at the beginning of the fermentation to 0% on the 16th day.
  • the amidolytic activity of the secreted protein was determined using the COA set (S 2251, Kabi vitrum).
  • the cell number was derived from the oxygen consumption.
  • Table I Cultivation of C-127 cells on Siran® carrier bodies containing DEAE dextran surface layer. The data were determined in the production phase (day 16-106, serum-free culture medium).
  • the production phase lasted 49 days.
  • Table 2 Cultivation of C-127 cells on collagen carriers (Verax VX-100®). The data were determined in the production phase (16th-65th day, serum-free culture medium)

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Abstract

The present invention relates to a novel process for cultivating mammal cells in a fluidised bed reactor.

Description

Verfahren zur Kultivierung von Säugerzellen im FließbettreaktorProcess for the cultivation of mammalian cells in a fluidized bed reactor
Beschreibungdescription
Die Kultivierung von Säugerzellen ist die Grundlage für eine Vielzahl von biotechnologischen Produktionsverfahren, insbe¬ sondere der Herstellung von Pharmaproteinen.The cultivation of mammalian cells is the basis for a large number of biotechnological production processes, in particular the production of pharmaceutical proteins.
Die Kultivierung von Säugerzellen im Fließbettreaktor unter Verwendung von Trägerkörpern ist bekannt. Von Looby und Griffiths werden in einem Übersichtsartikel (TIBTECH August 1990, 204-209) verschiedene Trägerkörper für die Kultivierung von Zellen im Fließbettreaktor verglichen. Besonders hohe Zellausbeuten werden mit Trägerkörpern erhalten, die aus Kollagen (Verax®) bestehen.The cultivation of mammalian cells in a fluidized bed reactor using carrier bodies is known. An overview article (TIBTECH August 1990, 204-209) by Looby and Griffiths compares different carrier bodies for the cultivation of cells in a fluidized bed reactor. Particularly high cell yields are obtained with carrier bodies that consist of collagen (Verax®).
Jedoch können diese Trägerkörper nicht autoklaviert und auch nicht wiederverwendet werden.However, these carrier bodies cannot be autoclaved and cannot be reused.
Trägerkörper aus Glas (z.B. Siran®) sind wiederverwendbar und autoklavierbar, jedoch ist die Zellausbeute bei diesenCarrier bodies made of glass (e.g. Siran®) are reusable and autoclavable, however, the cell yield is with these
Trägerkörpern deutlich geringer als bei Verax® (TIBTECH August 1990, 204-209).Carrier bodies significantly smaller than with Verax® (TIBTECH August 1990, 204-209).
Aus der Offenlegungsschri ft EP 303 262 ist ein Trägerkörper aus Glas oder Keramik mit a inhaltiger Oberflächenschicht bekannt; die Verwendung dieser Trägerkörper für die Säugerzellkultur wird jedoch nicht beschrieben.EP 303 262 discloses a support body made of glass or ceramic with a content-containing surface layer; however, the use of these carrier bodies for mammalian cell culture is not described.
Aufgabe der vorliegenden Erfindung war es, ein Verfahren zur Kultivierung von auf Trägerkörpern immobilisierten Säugerzellen bereitzustellen, bei dem die Trägerkörper eine hohe Zellausbeute gewährleisten, autoklavierbar, leicht reinigbar und wiederver¬ wendbar sind.The object of the present invention was to provide a method for the cultivation of mammalian cells immobilized on support bodies, in which the support bodies ensure a high cell yield, are autoclavable, easy to clean and reusable.
Demgemäß wurde gefunden, daß das Verfahren zur Kultivierung von Säugerzellen im Fließbettreaktor besonders vorteilhaft ist, wenn die Säugerzellen auf porösen Trägerkörpern aus Glas, deren Ober¬ flächenschicht an einen Träger gebundene Aminogruppen enthält, immobilisiert werden. Außerdem wurde gefunden, daß eine Diethyl- aminoethyl-Dextran (DEAE-Dextran) enthaltende Oberflächenschicht besonders gut geeignet ist. Weiterhin wurde gefunden, daß sich mit diesem Verfahren besonders gut die Maus-Fibroblastenzell inie C-127 (ATCC CRL 1616) kultivieren läßt. Die porösen Trägerkörper aus Glas können in beliebiger Gestalt vorliegen; bevorzugt werden sie in einer kugelförmigen Gestalt verwendet.Accordingly, it has been found that the method for cultivating mammalian cells in a fluidized bed reactor is particularly advantageous if the mammalian cells are immobilized on porous glass support bodies, the surface layer of which contains amino groups bonded to a support. It has also been found that a surface layer containing diethylaminoethyl dextran (DEAE dextran) is particularly suitable. Furthermore, it was found that the mouse fibroblast cell line C-127 (ATCC CRL 1616) can be cultivated particularly well with this method. The porous glass support body can be in any shape; they are preferably used in a spherical shape.
Die Größe der Trägerkörper beträgt zweckmäßig von 0,4 bis 5 mm, bevorzugt 1 bis 2 mm.The size of the carrier body is expediently from 0.4 to 5 mm, preferably 1 to 2 mm.
Die Porengröße der porösen Trägerkörper beträgt im allgemeinen 20 bis 500 μm, bevorzugt 60 bis 300 μm.The pore size of the porous support body is generally 20 to 500 μm, preferably 60 to 300 μm.
Darüber hinaus können in den Trägerkörpern auch sogenannte Mikroporen von 1 bis 10 μm vorhanden sein.In addition, so-called micropores of 1 to 10 μm can also be present in the carrier bodies.
Bevorzugt werden offenporige Sinterglas-Trägerkörper, z.B. der Marke Siran® (Schott, Mainz) verwendet. Die Trägerkörper können aus Kalk-Natron-Glas oder aus Borosilikatglas bestehen. Besonders bevorzugt werden Sinterglas-Trägerkörper aus Borosilikatglas.Open-pore sintered glass carrier bodies, e.g. of the Siran® brand (Schott, Mainz). The carrier bodies can consist of soda-lime glass or borosilicate glass. Sintered glass carrier bodies made of borosilicate glass are particularly preferred.
Die Beschichtung der gläsernen Trägerkörper mit Träger gebundenen Aminogruppen, insbesondere DEAE-Dextran-Gruppen ist in der Offen- legungsschrift EP 303 262 beschrieben. Wie dort erwähnt, kann die Beschichtung sowohl adsorptiv als auch kovalent erfolgen. Als Aminogruppen eignen sich Dialkylaminogruppen, insbesondere Dialkylaminoalkylgruppen, wobei die Alkylgruppen zweckmäßig 1 bis 4 C-Atome aufweisen. Als an Träger gebundene Aminogruppen kommen Aminogruppen, die mit einem Polymer, z.B. einem Polysaccharid kovalent verknüpft sind, in Frage. Bevorzugt werden Aminogruppen, die an Glucane, insbesondere Dextran gebunden sind. Besonders bevorzugt wird Diethyla inoethyl-Dextran (DEAE-Dextran) verwendet.The coating of the glass support bodies with support-bound amino groups, in particular DEAE-dextran groups, is described in the published patent application EP 303 262. As mentioned there, the coating can take place both adsorptively and covalently. Suitable amino groups are dialkylamino groups, in particular dialkylaminoalkyl groups, the alkyl groups advantageously having 1 to 4 carbon atoms. Amino groups bound to a carrier include amino groups which are linked to a polymer, e.g. are covalently linked to a polysaccharide. Amino groups which are bonded to glucans, in particular dextran, are preferred. Diethyla inoethyl-dextran (DEAE-dextran) is particularly preferably used.
Die in ihrer Oberflächenschicht Träger gebundene Aminogruppen enthaltenden gläsernen Trägerkörper sind kommerziell erhältlich (Schott, Mainz, Produktinformation Nr. 6196d).The glass support bodies containing amino groups bonded to the surface of the support are commercially available (Schott, Mainz, Product Information No. 6196d).
Als Säugerzellen eignen sich für das erfindungsgemäße verfahren sowohl adhärente als auch in Suspension wachsende Säugerzellen. Es können permanente Zellinien oder Primärkulturen verwendet werden. Auch gentechnisch veränderte (reko binante) Zellinien können eingesetzt werden. Bevorzugt werden Hybridomzellen und Fibroblasten, insbesondere die Maus-Fibroblasten Zellinie C-127 verwendet (ATCC CRL 1616). Besonders bevorzugt wird die den Gewebeplasminogenaktivator (t-PA) sekretierende Zellinie C-127, die mit einem Rinderpap llomvirus-Expressionsvektor transformiert worden war, verwendet. Die Herstellung dieser Zellinie ist von Reddy et al. (J. Cell. Biochem., Supplement 10 D, 154, 1986) beschrieben worden.Suitable mammalian cells for the method according to the invention are both adherent and suspension cells growing in suspension. Permanent cell lines or primary cultures can be used. Genetically modified (recombinant) cell lines can also be used. Hybridoma cells and fibroblasts, in particular the mouse fibroblast cell line C-127 (ATCC CRL 1616) are preferably used. The cell line C-127 secreting the tissue plasminogen activator (t-PA), which transforms with a bovine papomomirus expression vector, is particularly preferred had been used. The production of this cell line is by Reddy et al. (J. Cell. Biochem., Supplement 10 D, 154, 1986).
Als Fließbettreaktoren für das erfindungsgemäße Verfahren sind die üblichen Bioreaktoren geeignet, wie sie z.B. von Lobby und Griffiths (Cytotechnology 1, 339-346, 1988; Advances in Ani.mal Cell Biology and Technology for Bioprocesses, Eds. Spier et al., Butterworths, Guilford, 336-344, 1989) beschrieben werden.The usual bioreactors are suitable as fluidized bed reactors for the process according to the invention, such as those e.g. by Lobby and Griffiths (Cytotechnology 1, 339-346, 1988; Advances in Ani.mal Cell Biology and Technology for Bioprocesses, Eds. Spier et al., Butterworths, Guilford, 336-344, 1989).
Das Volumen der Trägerkörper im nicht-expandierten Zustand beträgt üblicherweise 10 bis 80 %, bevorzugt 20 bis 60 % des Reaktorvolumens.The volume of the support body in the unexpanded state is usually 10 to 80%, preferably 20 to 60% of the reactor volume.
Als Nährmedien zur Kultivierung der Säugerzellen können alle üblichen Zellkulturmedien verwendet werden. Es können auch Serum-freie Nährmedien verwendet werden. Ob Serum zugesetzt werden muß, hängt von der verwendeten Säugerzelle ab.All conventional cell culture media can be used as nutrient media for cultivating the mammalian cells. Serum-free culture media can also be used. Whether serum needs to be added depends on the mammalian cell used.
Die Immobilisierung der Säugerzellen auf den Trägerkörpern geschieht zweckmäßigerweise dadurch, daß die Säugerzellen durch konventionelle Zellkulturtechniken vorgezüchtet werden und an¬ schließend in den die Trägerkörper enthaltenden Fließbettreaktor eingebracht werden, so daß im Reaktor eine Zelldichte von 5-10'+ bis 1-106, bevorzugt von 1 bis 8-105 zellen/ml vorliegt.The immobilization of the mammalian cells on the carrier bodies expediently takes place in that the mammalian cells are pre-cultivated by conventional cell culture techniques and then introduced into the fluidized bed reactor containing the carrier bodies, so that a cell density in the reactor of 5-10 '+ to 1-106, preferably from 1 to 8-105 cells / ml.
Die Kultivierung der Säugerzellen nach dem erfindungsgemäßen Verfahren kann kontinuierlich oder diskontinuierlich betrieben werden. Bevorzugt wird jedoch kontinuierlich gearbeitet.The cultivation of the mammalian cells according to the method according to the invention can be carried out continuously or batchwise. However, work is preferably carried out continuously.
Als erfindungsgemäße Kultivierung kommen sowohl die Zellver¬ mehrung als auch die Nährstoffversorgung von Zellen ohne Zell¬ vermehrung in Betracht.Both the cell multiplication and the nutrient supply of cells without cell multiplication come into consideration as cultivation according to the invention.
Beide Kultivierungsverfahren werden insbesondere bei der Her¬ stellung von Proteinen aus Zellen mit gutem Erfolg angewendet.Both cultivation methods are used with particular success in the production of proteins from cells.
Zweckmäßigerweise wird dabei in einer ersten Phase (der Wachs¬ tumsphase) durch Zellvermehrung eine große Zellzahl erzeugt und anschließend in einer zweiten Phase (der Produktionsphase) von diesen Zellen der Wertstoff gebildet.In a first phase (the growth phase), a large number of cells is expediently generated by cell multiplication and then in a second phase (the production phase) these cells form the valuable substance.
Das erfindungsgemäße Verfahren erlaubt es, Säugerzellen bis zu einer hohen Dichte auf porösen Trägerkörpern zu kultivieren. Die Trägerkörper besitzen ausgezeichnete mechanische Eigenschaften, so daß im Fl eßbettreaktor z.B. kein die Zellausbeute verringernder Abrieb entsteht. Weiterhin sind diese Trägerkörper durch Heißdampf autoklavierbar; sie können z.B. im Reaktor vor Beginn der Kultivierung sterilisiert werden. Dadurch ist es auch möglich, die Trägerkörper nach Reinigung und Sterilisation wieder zu verwenden.The method according to the invention makes it possible to cultivate mammalian cells to a high density on porous support bodies. The carrier bodies have excellent mechanical properties, so that in the fluidized bed reactor e.g. there is no abrasion which reduces the cell yield. Furthermore, these carrier bodies can be autoclaved by superheated steam; you can e.g. be sterilized in the reactor before cultivation begins. This also makes it possible to reuse the carrier bodies after cleaning and sterilization.
Die Erfindung wird durch folgende Beispiele veranschaulicht:The invention is illustrated by the following examples:
Beispiel 1example 1
Kultivierung einer t-PA-sekretierenden C-127 Zellinie auf DEAE-Dextran-beschichteten Siran®-TrägerkörpernCultivation of a t-PA-secreting C-127 cell line on DEAE-dextran-coated Siran® carrier bodies
Die Experimente wurden mit einer adhärenten, t-PA sekretierenden C-127 Mausfibroblastenzellinie, die mit einem Rindei— apillom- virus-Expressionsvektor transformiert waren, durchgeführt (J. Cell. Biochem., Supplement 10 D, 154, 1986).The experiments were carried out with an adherent, t-PA secreting C-127 mouse fibroblast cell line which had been transformed with a bark apillomavirus expression vector (J. Cell. Biochem., Supplement 10 D, 154, 1986).
Die Anzucht der Säugerzellen erfolgte zunächst in handelsüblichen Zellkulturflaschen. Dazu wurden Zellkulturflaschen mit 0r5 - lr0 x 105 Zellen beimpft. Als Wachstumsmedium wurde DMEM- Mediu (4,5 g Glucose pro Liter, 584 mg Glutamin pro Liter), foetales Kälberserum (10 %) und PolyalkylenglykolThe mammalian cells were first grown in commercially available cell culture bottles. For this purpose, cell culture bottles were inoculated with 0 r 5 - 1 r 0 x 105 cells. DMEM medium (4.5 g glucose per liter, 584 mg glutamine per liter), fetal calf serum (10%) and polyalkylene glycol were used as growth medium
(Pluronic F-68®) (0,15 %) eingesetzt. Die Inkubation erfolgte in einem für Zellkulturen geeigneten Brutschrank bei 37°C. Beatmet wurden die Kulturen mit einem Gasgemisch bestehend aus Luft und CO2 (7 %). Nach 2 bis 3 Tagen wurde das verbrauchte Medium entfernt und die am Boden haftenden Zellen mit einem Gemisch aus Trypsin (0-125 %) und Ethylendiamintetraessigsäure (EDTA) (0,02 %)- gelöst in PBS-Puffer (137 mM NaCl, 2,7 M KC1, 10 mM Phosphatpuffer) gelöst. Der Ablösevorgang wurde durch DMEM (4,5 g Glucose pro Liter. 584 mg Glutamin pro Liter), foetalem Kälberserum (10 %) und Polyalkylenglykol (Pluronic F-68®)(Pluronic F-68®) (0.15%). Incubation was carried out in an incubator suitable for cell cultures at 37 ° C. The cultures were ventilated with a gas mixture consisting of air and CO2 (7%). After 2 to 3 days, the used medium was removed and the cells adhering to the bottom with a mixture of trypsin (0-125%) and ethylenediaminetetraacetic acid (EDTA) (0.02%) - dissolved in PBS buffer (137 mM NaCl, 2 , 7 M KC1, 10 mM phosphate buffer). The detachment was carried out by DMEM (4.5 g glucose per liter. 584 mg glutamine per liter), fetal calf serum (10%) and polyalkylene glycol (Pluronic F-68®)
(0,15 %) gestoppt, nachdem der überwiegende Teil der Zellen in Suspension gegangen war. Die Zellzahl wurde mit Hilfe eines Hämocytometers ermittelt und die Zellen anschließend in einen Fließbett-Reaktor überführt. Der Fließbett-Reaktor bestand aus einem mit einem Doppelmantel versehenen, 3,2 cm x 28 cm langen, am unteren Ende konisch zusam¬ menlaufenden Glaszylinder und einem ebenfalls mit einem Doppel¬ mantel versehenen, 2,8 x 13 cm großen Konditionierungsgefäß. Das Gesamtvolumen der Einheit inklusive der Verbindungsschläuche betrug 380 ml. Das Medium wurde mit Hilfe einer Schlauchpumpe zwischen Konditionierungsgefäß und Fließbett-Reaktor mit einer Geschwindigkeit von 20 bis 60 cm pro Minute gefördert. Eine Edelstahlkugelschüttung (3 mm Durchmesser, 25 ml Volumen) im konisch zusammenlaufenden Teil des Reaktors bewirkte eine gleich¬ mäßige Verteilung der Flüssigkeit und der porösen Trägerkörper im Reaktor. Frisches Medium wurde über eine Schlauchpumpe ins Kondi¬ tionierungsgefäß geleitet und verbrauchtes Medium via Überlauf aus dem Konditionierungsgefäß entfernt.(0.15%) stopped after the majority of the cells went into suspension. The cell number was determined using a hemocytometer and the cells were then transferred to a fluidized bed reactor. The fluidized bed reactor consisted of a 3.2 cm x 28 cm long glass cylinder provided with a double jacket and conically converging at the lower end and a 2.8 x 13 cm conditioning vessel also provided with a double jacket. The total volume of the unit including the connecting hoses was 380 ml. The medium was conveyed with the help of a peristaltic pump between the conditioning vessel and the fluidized bed reactor at a speed of 20 to 60 cm per minute. A stainless steel ball bed (3 mm diameter, 25 ml volume) in the conically converging part of the reactor brought about a uniform distribution of the liquid and the porous support bodies in the reactor. Fresh medium was fed into the conditioning vessel via a peristaltic pump and used medium was removed from the conditioning vessel via an overflow.
Der Medienaustausch wurde am 3. Tag mit einer Austauschrate von 0,5 Reaktorvolumen pro Tag begonnen und in der Produktionsphase schrittweise auf einen mittleren Wert von 4,3 Reaktorvolumen pro Tag erhöht.The media exchange was started on the 3rd day with an exchange rate of 0.5 reactor volume per day and gradually increased in the production phase to an average value of 4.3 reactor volume per day.
Temperiert wurde das System über die Doppelmäntel des Reaktors und des Konditionierungsgefäßes mit Hilfe eines Wasserbades. Die Inkubationstemperatur betrug in den ersten 6 Tagen 37°C, danach 35°C. Der Sauerstoffpartialdruck wurde auf 30 % (Luftsättigung) eingestellt. Dieser Partialdruck wurde mit Hilfe einer Meß- und Regeleinheit durch automatische Einleitung von Luft in den Kopf¬ raum bzw. Sauerstoff in die Flüssigkeit des Konditionsgefäßes aufrechterhalten. Der pH-Sollwert wurde durch automatische Zu- dosierung von C02 in den Kopfraum des Konditionsierungsgefäßes zwischen pH 6,9 und 7,2 geregelt.The system was tempered via the double jackets of the reactor and the conditioning vessel using a water bath. The incubation temperature was 37 ° C in the first 6 days, then 35 ° C. The oxygen partial pressure was set to 30% (air saturation). This partial pressure was maintained with the aid of a measuring and control unit by automatically introducing air into the head space or oxygen into the liquid of the conditioning vessel. The pH setpoint was regulated by automatic metering of C0 2 into the head space of the conditioning vessel between pH 6.9 and 7.2.
120 ml poröse, mit einer DEAE-Dextran-Oberflächenschicht ver¬ sehene Sinterglas-Trägerkörper (Siran®) mit einem Durchmesser von 1 bis 2 mm und einer Porengröße zwischen 60 bis 300 μm dienten als Trägermaterial für die C-127 Zellen.120 ml porous sintered glass carrier bodies (Siran®) with a DEAE dextran surface layer and having a diameter of 1 to 2 mm and a pore size between 60 and 300 μm served as carrier material for the C-127 cells.
Der Reaktor wurde mit 7 x 105 Zellen/ml beimpft. Als Kulturmedium wurde in den ersten 6 Tagen DMEM-Medium (4,5 g Glucose pro Liter, 584 mg Glutamin pro Liter), foetales Kälberserum (10 %) und Pluronic F-68® (0,157o) verwendet. Nach 6 Tagen wurde auf ein Medium bestehend aus Zellkulturmedium (IGl®, Genzyme), Glutamin (292 mg/1), Insulin (5 mg/1), Polyalkylenglykol (Pluronic F-68®) (0,15 %), Rinderserumalbumin (150 mg/l) und foetalem Kälberserum umgestellt. Die Serumkonzentration im Medium wurde stufenweise von 10 % zu Beginn der Fermentation auf 0 % am 16. Tag reduziert.The reactor was inoculated with 7 x 105 cells / ml. DMEM medium (4.5 g glucose per liter, 584 mg glutamine per liter), fetal calf serum (10%) and Pluronic F-68® (0.157o) were used as culture medium in the first 6 days. After 6 days, a medium consisting of cell culture medium (IGl®, Genzyme), glutamine (292 mg / 1), insulin (5 mg / 1), polyalkylene glycol (Pluronic F-68®) (0.15%), bovine serum albumin (150 mg / l) and fetal calf serum. The serum concentration in the medium was gradually reduced from 10% at the beginning of the fermentation to 0% on the 16th day.
Mit der Umstellung auf serü freies Medium am 16. Tag war die Wachstumsphase abgeschlossen und die Produktionsphase begann. Die Produktionsphase dauerte 90 Tage.With the changeover to serü-free medium on the 16th day, the growth phase was completed and the production phase began. The production phase lasted 90 days.
Die amidolytische Aktivität des sekretierten Proteins wurde mit Hilfe des COA-Set (S 2251, Kabi vitrum) bestimmt. Die Zellzahl wurde aus dem Sauerstoffverbrauch abgeleitet.The amidolytic activity of the secreted protein was determined using the COA set (S 2251, Kabi vitrum). The cell number was derived from the oxygen consumption.
Das Ergebnis ist in Tabelle 1 zusammengefaßt.The result is summarized in Table 1.
Tabelle I: Züchtung von C-127 Zellen auf DEAE-Dextran-Ober- flächenschicht enthaltenden Siran®-Trägerkörpern. Die Daten wurden in der Produktionsphase (16.-106. Tag, serumfreies Kulturmedium) ermittelt.Table I: Cultivation of C-127 cells on Siran® carrier bodies containing DEAE dextran surface layer. The data were determined in the production phase (day 16-106, serum-free culture medium).
Prozeßparameter DEAE-Gruppen enthaltende Siran®- TrägerkörperProcess parameters Siran® carrier bodies containing DEAE groups
Zelldichte 2,6 x 107 (Zellen/ml) t-PA Konzentration 38,3 (mg/l) t-PA Produktivität 163 (mg/l x Tag) spezifische Produktivität 6,4 (pg/Zelle x Tag)Cell density 2.6 x 107 (cells / ml) t-PA concentration 38.3 (mg / l) t-PA productivity 163 (mg / l x day) specific productivity 6.4 (pg / cell x day)
Perfusionsrate 4,3 (pro Tag)Perfusion rate 4.3 (per day)
Beispiel 2 (Vergleichsbeispiel)Example 2 (comparative example)
Kultivierung einer t-PA-sekretierenden C-127 Zellinie auf Kollagen-Trägerkörpern (Verax®) Die gleiche Zellinie wurde im gleichen Reaktor, wie in Beispiel 1 beschrieben, kultiviert.Cultivation of a t-PA-secreting C-127 cell line on collagen carriers (Verax®) The same cell line was cultivated in the same reactor as described in Example 1.
Anstelle von 120 ml DEAE-Dextran-Oberflächenschicht enthaltenden Siran®-Trägerkörpern wurden 90 ml poröse Kollagen-Trägerkörper der Firma Verax Corporation, Lebanon, USA (Verax VX-100®) verwendet.Instead of 120 ml of Siran® carrier bodies containing DEAE dextran surface layer, 90 ml of porous collagen carrier bodies from Verax Corporation, Lebanon, USA (Verax VX-100®) were used.
Die Produktionsphase dauerte 49 Tage.The production phase lasted 49 days.
Das Ergebnis ist in Tabelle 2 zusammengefaßt.The result is summarized in Table 2.
Tabelle 2: Züchtung von C-127 Zellen auf Kollagen-Trägerkörpern (Verax VX-100®) . Die Daten wurden in der Produktionsphase (16.-65. Tag, serumfreies Kulturmedium) ermitteltTable 2: Cultivation of C-127 cells on collagen carriers (Verax VX-100®). The data were determined in the production phase (16th-65th day, serum-free culture medium)
Prozeßparameter Kollagen-Trägerkörper (Verax VX-100®)Process parameters collagen carrier body (Verax VX-100®)
Zelldichte 2,1 x 107Cell density 2.1 x 107
•(Zellen/ml) t-PA Konzentration 37,7 • (cells / ml) t-PA concentration 37.7
(mg/l) t-PA Produktivität 160(mg / l) t-PA productivity 160
(mg/l x Tag) spezifische Produktivität 7,8(mg / l x day) specific productivity 7.8
(pg/Zelle x Tag)(pg / cell x day)
Perfusionsrate 4,2 (pro Tag) Perfusion rate 4.2 (per day)

Claims

Patentansprüche Claims
1. Verfahren zur Kultivierung von Säugerzellen im Fließbett¬ reaktor, dadurch gekennzeichnet, daß die Säugerzellen auf porösen Trägerkörpern aus Glas, deren Oberflächenschicht an einen Träger gebundene Aminogruppen enthält, immobilisiert werden.1. A process for the cultivation of mammalian cells in the fluidized bed reactor, characterized in that the mammalian cells are immobilized on porous glass support bodies, the surface layer of which contains amino groups bonded to a support.
2. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet- daß eine Diethylaminoethyl-Dextran enthaltende Oberflächenschicht verwendet wird.2. The method according to claim 1, characterized in that a surface layer containing diethylaminoethyl dextran is used.
3. Verfahren gemäß Anspruch 1 oder 2, dadurch gekennzeichnet, daß als Säugerzellen die Maus-Fibroblastenzellinie C-127 verwendet wird.3. The method according to claim 1 or 2, characterized in that the mouse fibroblast cell line C-127 is used as mammalian cells.
4. Verfahren gemäß Anspruch 3, dadurch gekennzeichnet, daß eine Gewebeplasminogenaktivator sekretierende rekombinante Zellinie C-127 verwendet wird. 4. The method according to claim 3, characterized in that a tissue plasminogen activator secreting recombinant cell line C-127 is used.
PCT/EP1992/001918 1991-08-30 1992-08-21 Process for cultivating mammal cells in a fluidised bed reactor WO1993005144A1 (en)

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