EP0848751A1 - Method for the continuous preparation of recombinant proteins from cell cultures - Google Patents

Method for the continuous preparation of recombinant proteins from cell cultures

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Publication number
EP0848751A1
EP0848751A1 EP96909159A EP96909159A EP0848751A1 EP 0848751 A1 EP0848751 A1 EP 0848751A1 EP 96909159 A EP96909159 A EP 96909159A EP 96909159 A EP96909159 A EP 96909159A EP 0848751 A1 EP0848751 A1 EP 0848751A1
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Prior art keywords
cells
spin filter
cell
cell cultures
recombinant proteins
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EP96909159A
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German (de)
French (fr)
Inventor
Jürgen VORLOP
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GSK Vaccines GmbH
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Chiron Behring GmbH and Co KG
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Publication of EP0848751A1 publication Critical patent/EP0848751A1/en
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    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis

Definitions

  • the present invention relates to a process for the continuous production of recombinant proteins from cell cultures which are grown in a continuous fermentation.
  • the disclosed method allows the stable expression of recombinant proteins with a constant quality of the recombinant protein over a fermentation period of several months.
  • Recombinant proteins can be produced using genetically modified bacteria or using cell cultures.
  • Cells from higher organisms are used for cell cultures.
  • Cell lines derived from humans, animals, plants or insects are used particularly frequently. Examples of such cell lines are mouse fibroblast cells, CHO cells (chinese hamster ovary), plant cells or insect cells.
  • the recombinant proteins are often produced from cell cultures by batch fermentation.
  • recombinant tPA is currently being produced from CHO cells on an industrial scale.
  • a continuous process has several advantages, but the optimal fermentation management is difficult.
  • recombinant coagulation factor VIII is currently being produced from BHK cells.
  • the advantage of the continuous process over the batch process is the high space-time product yield. This advantage can be achieved in particular if the cells can be separated from the outflowing cell culture supernatant in a suitable manner and returned to the fermentation process.
  • a spin filter essentially consists of a mesh sieve shaped into a cylinder that rotates about its longitudinal axis.
  • WO 92/05242 describes a perfusion bioreactor with spin filter for cell culture.
  • screen cylinders are used, which preferably have a mesh size of 5 ⁇ m, and the spin filters should preferably rotate at speeds between 100 and 150 rpm.
  • EPA 88.118999.7 describes a continuous cell cultivation system which is particularly suitable for mammalian cells. It should be noted that the spin filters tend to block and then do not allow the medium to pass through. For example, at a spin filter speed of 150 rpm, the filter was blocked after 284 hours. The separation of the cells from the culture fluid is therefore achieved according to the teaching of EPA 88.118999.7 by an external cell separation.
  • spin filter for continuous cell culture BioTec, No. 2, March 1993, pp. 46-511
  • spin filters that can be used in continuous fermentation.
  • the relationships between the type of cells used (cells on microcarriers, aggregate-forming cells and suspension cells), pore size of the spin filter and number of revolutions of the spin filter are discussed. However, it is not shown what influence the process parameters have on the quality and yield of the products formed.
  • the object of the present invention is to provide the recombinant products, in particular proteins, in a good quality of the product with a consistently high yield over a long period of time.
  • the object is achieved by the process according to the invention for the continuous production of recombinant proteins from cell cultures comprising a continuous fermentation in which the cells are retained in the fermenter system by at least one spin filter, the speed of the Spin filter during the fermentation is between about 30 min -1 and about 50 min -1 .
  • the speed of the spin filter is between about 40 and about 49 min -1 .
  • a speed of 45 min -1 is very particularly preferred.
  • cells are preferably removed daily from the culture medium in the continuous fermentation. The amount removed can be between 10% and 30% of the cells per day, based on the total number of cells. In a preferred manner, however, the daily cell extraction only starts on the 10th day, calculated from the start of the continuous fermentation.
  • the cell cultures used are preferably animal cells.
  • the process according to the invention is preferably carried out at a temperature of about 37 ° C. and the pH is preferably adjusted to the range between 6.7 and 7.2, very preferably to about 7.1.
  • the p ⁇ 2 is preferably between 10% and 80% and is very particularly preferably around 50%.
  • the flow rate is preferably 2 d "1 .
  • the spin filters used according to the invention preferably consist of fine-mesh stainless steel wire mesh and are constructed from a robust filter body with an approximately cylindrical shape.
  • the bottom is preferably conical and, in a particularly preferred embodiment, the spin filter has an overflow which is open at the top.
  • the spin filter used in the method according to the invention preferably has a pore size of approximately 20 ⁇ m.
  • the recombinant product can be of a consistently good quality with a relatively high yield, which remains uniform at a relatively high level.
  • recombinant human interleukin-4 receptor (rhu IL-4R) from CHO cells was chosen as the model system in the examples.
  • the cells were used in a 10 1 fermenter (Biostat E, B. Braun Biotec International, Melsungen, Germany), on the agitator shaft of which a spin filter was attached.
  • the spin filter was purchased from B. Braun Biotec International, Melsungen, Germany and had a diameter of 105 mm and a height of 260 mm. The pore size was 20 ⁇ m.
  • Two inclined blade stirrers were attached below the spin filter cone, which were offset by 90 ° to each other.
  • the membrane gassing basket of the fermenter served as a guide tube, so that a directed overturning flow could be realized in the fermenter.
  • Serum-free Ultra-CHO medium which was purchased from Bio Whittaker, USA, was used as the medium.
  • Figure 1 shows the cell density and the product concentration (rhu IL-4 receptor) over a period of about four months.
  • the 10 1 fermenter was provided with a spin filter, which was operated at a speed of about 50 rpm.
  • Figure 2 shows the cell density and the product concentration over a period of about two months.
  • the cells were grown in a 10 liter fermenter.
  • the spin filter was operated at a speed of 150 rpm and no cells were removed.
  • the product concentration was observed to drop below 10 mg / l from day 55.
  • Figure 3 shows the cell density and product concentration over a period of approximately three months.
  • the cells were grown in a 2 liter fermenter, the spin filter being operated at a speed of 200 rpm. The following amounts of cells were removed: from day 38 10% from day 52 20% from day 63 30% from day 70 10%

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Abstract

The method disclosed for the continuous preparation of recombinant proteins from cell cultures comprises a continuous fermentation in which the cells in the fermenter are retained by at least one rotating filter, the rotational speed of the filter during the fermentation lying between 30 and 50 min<-1>.

Description

Verfahren zur kontinuierlichen Herstellung rekombinanter Process for the continuous production of recombinant
Proteine aus ZellkulturenProteins from cell cultures
Die vorliegende Erfindung betrifft ein Verfahren zur kontinuierlichen Herstellung rekombinanter Proteine aus Zellkulturen, die in einer kontinuierlichen Fermentation angezogen werden. Das offenbarte Verfahren erlaubt die stabile Expression rekombinanter Proteine mit einer gleich¬ bleibenden Qualität des rekombinanten Proteins über einen mehrmonatigen Fermentationszeitraum.The present invention relates to a process for the continuous production of recombinant proteins from cell cultures which are grown in a continuous fermentation. The disclosed method allows the stable expression of recombinant proteins with a constant quality of the recombinant protein over a fermentation period of several months.
Rekombinante Proteine können mit Hilfe von genetisch veränderten Bakterien oder mit Hilfe von Zellkulturen hergestellt werden. Für die Zellkulturen werden Zellen von höheren Organismen eingesetzt. Besonders häufig finden Zellinien Verwendung, die von Menschen, Tieren, Pflanzen oder Insekten abstammen. Beispiele derartiger Zellinien sind Mausfibroblastenzellen, CHO-Zellen (chinese hamster ovary) , Pflanzenzellen oder Insektenzellen.Recombinant proteins can be produced using genetically modified bacteria or using cell cultures. Cells from higher organisms are used for cell cultures. Cell lines derived from humans, animals, plants or insects are used particularly frequently. Examples of such cell lines are mouse fibroblast cells, CHO cells (chinese hamster ovary), plant cells or insect cells.
Die Herstellung der rekombinanten Proteine aus Zellkulturen erfolgt häufig durch eine ansatzweise Fermentation (batch fermentation) . So wird beispielsweise derzeit auch im industriellen Maße rekombinantes tPA aus CHO-Zellen hergestellt. Demgegenüber weist ein kontinuierliches Verfahren verschiedene Vorteile auf, jedoch stellt sich die optimale Fermentationsführung als schwierig dar. Im industriellen Maßstab wird derzeit beispielsweise der rekombinante Blutgerinnungsfaktor VIII aus BHK-Zellen hergestellt. Vorteil bei den kontinuierlichen Verfahren gegenüber dem ansatzweisen Verfahren ist die hohe Raum-Zeit- Produktausbeute. Dieser Vorteil kann insbesondere dann erzielt werden, wenn es gelingt, die Zellen auf geeignete Weise aus dem abströmenden Zellkulturüberstand abzutrennen und wieder dem Fermentationsprozeß zuzuführen.The recombinant proteins are often produced from cell cultures by batch fermentation. For example, recombinant tPA is currently being produced from CHO cells on an industrial scale. In contrast, a continuous process has several advantages, but the optimal fermentation management is difficult. On an industrial scale, for example, recombinant coagulation factor VIII is currently being produced from BHK cells. The advantage of the continuous process over the batch process is the high space-time product yield. This advantage can be achieved in particular if the cells can be separated from the outflowing cell culture supernatant in a suitable manner and returned to the fermentation process.
Bei der Zeilrückhaltung wird zwischen externer und interner Zeilrückhaltung unterschieden. Bei der externen Zell- rückhaltung wird der Zellkulturüberstand aus dem Fermenter abgezogen. Die zeilhaltige Flüssigkeit durchströmt dann eine Vorrichtung, die eine teilweise oder vollständige Abtrennung der Zellen von der Kulturflüssigkeit ermöglicht . Anschließend werden die abgetrennten Zellen dem Fermenter über eine Rückleitung wieder zugeführt. Hier ist zwar eine Reihe von Verfahren beschrieben, ein wesentlicher Nachteil dieses Verfahrens ist jedoch die Belastung der Zellen bei der Rückführung, da mechanische Belastungen (beispielsweise durch Pumpen) und physiologische Belastungen durch unkontrollierte pH-Werte und pθ2-Werte nicht kontrollierbar sind.With line retention, a distinction is made between external and internal line retention. With external cell retention, the cell culture supernatant is withdrawn from the fermenter. The cell-containing liquid then flows through a device which enables a partial or complete separation of the cells from the culture liquid. The separated cells are then returned to the fermenter via a return line. Although a number of methods are described here, a major disadvantage of this method is the load on the cells during the return, since mechanical loads (for example due to pumps) and physiological loads due to uncontrolled pH values and pθ2 values cannot be controlled.
Bei der internen Zeilrückhaltung wird bereits im Fermenter eine teilweise oder vollständige Zurückhaltung der Zellen angestrebt. Dadurch können die Nachteile, die bei der externen Zellrückhaltung auftreten, bei der internen Zell-rückhaltung nicht stören. Eine der Möglichkeiten der internen Zellrückhaltung ist der Einsatz von Spinfiltern. Diese Technik wurde zum ersten Mal von Himmelfarb et al . beschrieben (Himmelfarb et al . , Science 164, S. 555-557 (1969) ; Spin filter culture: The propagation of mammalian cells in Suspension) .With internal cell retention, partial or complete cell retention is already sought in the fermenter. As a result, the disadvantages that occur with external cell retention cannot interfere with internal cell retention. One of the options for internal cell retention is the use of spin filters. This technique was first developed by Himmelfarb et al. (Himmelfarb et al., Science 164, pp. 555-557 (1969); Spin filter culture: The propagation of mammalian cells in suspension).
Ein Spinfilter besteht im wesentlichen aus einem zu einem Zylinder geformten Maschensieb, das um seine Längsachse rotiert . Die WO 92/05242 beschreibt einen Perfusionsbioreaktor mit Spinfilter zur Zellkultur. In dieser Literaturstelle werden Siebzylinder verwendet, die bevorzugt eine Maschenweite von 5 μm haben, und die Spinfilter sollen bevorzugt mit Geschwindigkeiten zwischen 100 und 150 Upm rotieren.A spin filter essentially consists of a mesh sieve shaped into a cylinder that rotates about its longitudinal axis. WO 92/05242 describes a perfusion bioreactor with spin filter for cell culture. In this reference, screen cylinders are used, which preferably have a mesh size of 5 μm, and the spin filters should preferably rotate at speeds between 100 and 150 rpm.
In der EPA 88.118999.7 wird ein kontinuierliches Zeilkulti¬ vierungssystem beschrieben, das vor allem für Säugerzellen geeignet ist. Es wird darauf hingewiesen, daß die Spinfilter zum Verblocken neigen und danach das Medium nicht mehr durchtreten lassen. So wurde beispielsweise bei einer Drehzahl des Spinfilters von 150 Upm eine Verblockung des Filters nach 284 Stunden festgestellt. Die Abtrennung der Zellen von der Kulturflüssigkeit wird daher gemäß der Lehre der EPA 88.118999.7 durch eine externe Zellabtrennung gelöst.EPA 88.118999.7 describes a continuous cell cultivation system which is particularly suitable for mammalian cells. It should be noted that the spin filters tend to block and then do not allow the medium to pass through. For example, at a spin filter speed of 150 rpm, the filter was blocked after 284 hours. The separation of the cells from the culture fluid is therefore achieved according to the teaching of EPA 88.118999.7 by an external cell separation.
Fenge et al. beschreiben in dem Aufsatz "Spinfilter für kontinuierliche Zellkultur" (BioTec, Nr. 2, März 1993, S. 46- 51) Spinfilter, die bei der kontinuierlichen Fermentation Verwendung finden können. Die Beziehungen zwischen Art der verwendeten Zellen (Zellen auf Microcarriern, aggregat¬ bildende Zellen und Suspensionszellen) , Porenweite des Spinfilters und Umdrehungszahl des Spinfilters werden erörtert. Nicht dargestellt wird jedoch, welchen Einfluß die Verfahrensparameter auf die Qualität und Ausbeute der gebildeten Produkte haben.Fenge et al. describe in the article "Spin filter for continuous cell culture" (BioTec, No. 2, March 1993, pp. 46-51) spin filters that can be used in continuous fermentation. The relationships between the type of cells used (cells on microcarriers, aggregate-forming cells and suspension cells), pore size of the spin filter and number of revolutions of the spin filter are discussed. However, it is not shown what influence the process parameters have on the quality and yield of the products formed.
Aufgabe der vorliegenden Erfindung ist es, die rekombinanten Produkte, insbesondere Proteine in einer guten Qualität des Produktes mit einer gleichbleibend hohen Ausbeute über einen langen Zeitraum bereitzustellen.The object of the present invention is to provide the recombinant products, in particular proteins, in a good quality of the product with a consistently high yield over a long period of time.
Die Aufgabe wird gelöst durch das erfindungsgemäße Verfahren zur kontinuierlichen Herstellung rekombinanter Proteine aus Zellkulturen umfassend eine kontinuierliche Fermentation, bei der die Zellen in dem Fermentersystem durch wenigstens einen Spinfilter zurückgehalten werden, wobei die Drehzahl des Spinfilters während der Fermentation zwischen etwa 30 min-1 und etwa 50 min-1 beträgt.The object is achieved by the process according to the invention for the continuous production of recombinant proteins from cell cultures comprising a continuous fermentation in which the cells are retained in the fermenter system by at least one spin filter, the speed of the Spin filter during the fermentation is between about 30 min -1 and about 50 min -1 .
In einer bevorzugten Ausführungsform liegt die Drehzahl des Spinfilters zwischen etwa 40 und etwa 49 min-1. Ganz besonders bevorzugt ist eine Drehzahl von 45 min-1. Bevorzugterweise werden darüber hinaus bei der kontinuierlichen Fermentation täglich Zellen aus dem Kulturmedium entnommen. Die entnommene Menge kann dabei täglich zwischen 10% und 30% der Zellen, bezogen auf die Gesamtzellzahl, betragen. In bevorzugter Weise wird jedoch mit der täglichen Zellentnahme erst ab dem 10. Tag, gerechnet ab Beginn der kontinuierlichen Fermentation, begonnen.In a preferred embodiment, the speed of the spin filter is between about 40 and about 49 min -1 . A speed of 45 min -1 is very particularly preferred. In addition, cells are preferably removed daily from the culture medium in the continuous fermentation. The amount removed can be between 10% and 30% of the cells per day, based on the total number of cells. In a preferred manner, however, the daily cell extraction only starts on the 10th day, calculated from the start of the continuous fermentation.
Auch wenn bei dem erfindungsgemäßen Verfahren die verschie¬ densten eukaryontischen Zellen verwendet werden können, handelt es sich doch bevorzugt bei den eingesetzten Zellkulturen um tierische Zellen.Even if the most varied eukaryotic cells can be used in the method according to the invention, the cell cultures used are preferably animal cells.
Das erfindungsgemäße Verfahren wird vorzugsweise bei einer Temperatur von etwa 37°C durchgeführt und der pH-Wert wird bevorzugt auf dem Bereich zwischen 6,7 und 7,2, ganz bevorzugt auf etwa 7,1 eingestellt. Der pθ2 beträgt Vorzugs-weise zwischen 10% und 80% und liegt ganz besonders bevorzugt bei etwa 50%. Die Durchflußrate beträgt vorzugsweise 2 d"1.The process according to the invention is preferably carried out at a temperature of about 37 ° C. and the pH is preferably adjusted to the range between 6.7 and 7.2, very preferably to about 7.1. The pθ2 is preferably between 10% and 80% and is very particularly preferably around 50%. The flow rate is preferably 2 d "1 .
Die erfindungsgemäß verwendeten Spinfilter bestehen vorzugsweise aus feinmaschigem Edelstahldrahtgewebe und sind aus einem robusten Filterkörper mit etwa zylindrischer Gestalt aufgebaut. Der Boden ist bevorzugt konisch und in besonders bevorzugter Ausführungsform weist der Spinfilter einen oben offenen Überlauf auf. Der bei dem erfindungs-gemäßen Verfahren verwendete Spinfilter weist bevorzugt eine Porenweite von etwa 20 μm auf.The spin filters used according to the invention preferably consist of fine-mesh stainless steel wire mesh and are constructed from a robust filter body with an approximately cylindrical shape. The bottom is preferably conical and, in a particularly preferred embodiment, the spin filter has an overflow which is open at the top. The spin filter used in the method according to the invention preferably has a pore size of approximately 20 μm.
Durch das erfindungsgemäße Verfahren kann das rekombinante Produkt in gleichbleibend guter Qualität mit einer verhältnismäßig hohen Ausbeute, die gleichmäßig auf relativ hohem Niveau bleibt, hergestellt werden.By means of the method according to the invention, the recombinant product can be of a consistently good quality with a relatively high yield, which remains uniform at a relatively high level.
Überraschenderweise wurde trotz der erfindungsgemäß verwendeten niedrigen Drehzahl keine Verblockung des Spinfilters über einen Zeitraum von mehr als vier Monaten beobachtet . Bei der bevorzugten Entnahme der Zellen konnte eine Beibehaltung der hohen Vitalität der Zellen beobachtet werden und darüber hinaus konnte eine homogene Verteilung der Zellaggregate erzielt werden.Surprisingly, despite the low speed used according to the invention, no blocking of the spin filter was observed over a period of more than four months. With the preferred removal of the cells, a maintenance of the high vitality of the cells was observed and, moreover, a homogeneous distribution of the cell aggregates could be achieved.
Die nachfolgenden Beispiele belegen die Vorteile des erfindungsgemäßen Verfahrens.The following examples demonstrate the advantages of the method according to the invention.
Als Modellsystem in den Beispielen wurde die Herstellung von rekombinantem humanem Interleukin-4 Rezeptor (rhu IL-4R) aus CHO-Zellen gewählt. Die Zellen wurden in einem 10 1 Fermenter (Biostat E, B. Braun Biotec International, Melsungen, BRD) verwendet, auf dessen Rührwelle ein Spinfilter befestigt war. Der Spinfilter wurde von der Firma B. Braun Biotec International, Melsungen, BRD, bezogen und wies einen Durchmesser von 105 mm bei einer Höhe von 260 mm auf. Die Porenweite betrug 20 μm. Unterhalb des Spinfilterkonus waren zwei Schrägblattrührer befestigt, die um 90° gegeneinander versetzt waren. Der Membranbegasungskorb des Fermenters diente als Leitrohr, so daß eine gerichtete UmwurfStrömung im Fermenter realisiert werden konnte. Als Medium wurde serumfreies Ultra-CHO Medium, das käuflich von Bio Whittaker, USA, erworben wurde, eingesetzt.The production of recombinant human interleukin-4 receptor (rhu IL-4R) from CHO cells was chosen as the model system in the examples. The cells were used in a 10 1 fermenter (Biostat E, B. Braun Biotec International, Melsungen, Germany), on the agitator shaft of which a spin filter was attached. The spin filter was purchased from B. Braun Biotec International, Melsungen, Germany and had a diameter of 105 mm and a height of 260 mm. The pore size was 20 μm. Two inclined blade stirrers were attached below the spin filter cone, which were offset by 90 ° to each other. The membrane gassing basket of the fermenter served as a guide tube, so that a directed overturning flow could be realized in the fermenter. Serum-free Ultra-CHO medium, which was purchased from Bio Whittaker, USA, was used as the medium.
Im Rahmen der vorliegenden Erfindung wurde gefunden, daß die Produktion von rhu IL-4R über einen langen Zeitraum ständig zurückging, wenn der Spinfilter bei hoher Drehzahl bewegt wurde und keine Zellen entnommen wurden. Durch den Einsatz einer niedrigen Drehzahl konnte die Produktbildung stabilisiert werden und weiterhin durch eine Zellentnahme verbessert werden. Durch die erfindungsgemäße Verfahrensführung wurde erst nach sehr langen Zeiträumen (etwa vier Monaten) eine Verblockung des Siebes beobachtet. Durch die bevorzugte Konstruktion, nämlich den oben offenen Überlauf des Siebfilters, war es dennoch möglich, die Zelldichte auf einem hohen Niveau zu stabilisieren.In the context of the present invention it was found that the production of rhu IL-4R decreased continuously over a long period of time when the spin filter was moved at high speed and no cells were removed. By using a low speed, the product formation could be stabilized and further improved by removing cells. The process according to the invention only observed blocking of the sieve after very long periods of time (approximately four months). Thanks to the preferred construction, namely the open overflow of the sieve filter, it was still possible to stabilize the cell density at a high level.
Beispiel 1example 1
In Abbildung 1 ist die Zelldichte und die Produkt- konzentration (rhu IL-4 Rezeptor) über einen Zeitraum von etwa vier Monaten angegeben. Der 10 1 Fermenter wurde mit einem Spinfilter versehen, der mit einer Drehzahl von etwa 50 Upm betrieben wurde.Figure 1 shows the cell density and the product concentration (rhu IL-4 receptor) over a period of about four months. The 10 1 fermenter was provided with a spin filter, which was operated at a speed of about 50 rpm.
Ab dem 15. Tag wurden etwa 10% der Zellen entnommen. Die Zellentnahme wurde auf etwa 20% ab dem 84. Tag erhöht. Es wurde beobachtet, daß sich die Produktkonzentration stabil auf einem Wert zwischen 20 und 60 mg/1 einstellte.From day 15, about 10% of the cells were removed. The cell withdrawal was increased to about 20% from the 84th day. It was observed that the product concentration was stable between 20 and 60 mg / l.
Beispiel 2 (Vergleichsbeispiel)Example 2 (comparative example)
Abbildung 2 zeigt die Zelldichte und die Produktkonzentration über einen Zeitraum von etwa zwei Monaten.Figure 2 shows the cell density and the product concentration over a period of about two months.
Die Zellen wurden in einem 10 1 Fermenter angezogen. Der Spinfilter wurde mit einer Drehzahl von 150 Upm betrieben und es fand keine Zellentnahme statt. Es wurde beobachtet, daß die Produktkonzentration ab dem 55. Tag unter 10 mg/1 absank.The cells were grown in a 10 liter fermenter. The spin filter was operated at a speed of 150 rpm and no cells were removed. The product concentration was observed to drop below 10 mg / l from day 55.
Beispiel 3 (Vergleichsbeispiel)Example 3 (comparative example)
In Abbildung 3 ist die Zelldichte und Produktkonzentration über einen Zeitraum von etwa drei Monaten dargestellt.Figure 3 shows the cell density and product concentration over a period of approximately three months.
Die Zellen wurden in einem 2 1 Fermenter angezogen, wobei der Spinfilter bei einer Drehzahl von 200 Upm betrieben wurde. Es wurden folgende Mengen an Zellen entnommen: ab Tag 38 10% ab Tag 52 20% ab Tag 63 30% ab Tag 70 10%The cells were grown in a 2 liter fermenter, the spin filter being operated at a speed of 200 rpm. The following amounts of cells were removed: from day 38 10% from day 52 20% from day 63 30% from day 70 10%
Es wurde festgestellt, daß die Produktkonzentration am Tag 70 unter 10 mg/1 fiel trotz Zellentnahme bei hoher Drehzahl. Durch eine Verminderung der Zellentnahme konnte nur kurzfristig eine höhere Produktkonzentration erzielt werden. Am Tag 90 fiel die Produktkonzentration abermals auf 10 mg/1. It was found that the product concentration fell below 10 mg / l on day 70 despite cell removal at high speed. By reducing the cell removal, a higher product concentration could only be achieved for a short time. On day 90 the product concentration dropped again to 10 mg / 1.

Claims

Patentansprüche claims
1. Verfahren zur kontinuierlichen Herstellung rekombinanter Proteine aus Zellkulturen umfassend eine kontinuierliche Fermentation, bei der die Zellen in dem Fermentersystem durch wenigstens einen Spinfilter zurückgehalten werden, dadurch gekennzeichnet, daß die Drehzahl des Spinfilters während der Fermentation zwischen 30 min-1 und 50 min-1 beträgt.1. A process for the continuous production of recombinant proteins from cell cultures comprising a continuous fermentation, in which the cells are retained in the fermenter system by at least one spin filter, characterized in that the speed of the spin filter during the fermentation between 30 min -1 and 50 min -1 is.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß die Drehzahl des Spinfilters 40 - 49 min-1 beträgt.2. The method according to claim 1, characterized in that the speed of the spin filter is 40 - 49 min -1 .
3. Verfahren nach Anspruch 1 oder 2, dadurch gekenn¬ zeichnet, daß bei der kontinuierlichen Fermentation täglich Zellen aus dem Kulturmedium entnommen werden.3. The method according to claim 1 or 2, characterized gekenn¬ characterized in that cells are removed from the culture medium daily in the continuous fermentation.
4. Verfahren nach Anspruch 3, dadurch gekennzeichnet, daß täglich zwischen 10% und 30% der Zellen, bezogen auf die Gesamtzellzahl, dem Kulturmedium entnommen werden.4. The method according to claim 3, characterized in that daily between 10% and 30% of the cells, based on the total number of cells, are removed from the culture medium.
5. Verfahren nach den Ansprüchen 3 und 4, dadurch gekenn¬ zeichnet, daß die tägliche Zellentnahme ab dem 10. Tag, gerechnet ab Beginn der kontinuierlichen Fermentation, erfolgt.5. The method according to claims 3 and 4, characterized gekenn¬ characterized in that the daily cell removal from the 10th day, calculated from the beginning of the continuous fermentation takes place.
6. Verfahren nach einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, daß die Porenweite des eingesetzten Spin¬ filters 20 μm beträgt.6. The method according to any one of claims 1 to 5, characterized in that the pore size of the spin filter used is 20 microns.
7. Verfahren nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, daß der eingesetzte Spinfilter einen oben offenen Überlauf aufweist.7. The method according to any one of claims 1 to 6, characterized in that the spin filter used has an open overflow.
8. Verfahren nach einem der Ansprüche 1 bis 7, dadurch gekennzeichnet, daß es sich bei den eingesetzten Zellkulturen um tierische Zellen handelt. 8. The method according to any one of claims 1 to 7, characterized in that the cell cultures used are animal cells.
EP96909159A 1995-09-09 1996-04-01 Method for the continuous preparation of recombinant proteins from cell cultures Withdrawn EP0848751A1 (en)

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DE19533460 1995-09-09
DE19533460 1995-09-09
PCT/EP1996/001425 WO1997009416A1 (en) 1995-09-09 1996-04-01 Method for the continuous preparation of recombinant proteins from cell cultures

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DE10116888B4 (en) * 2001-04-04 2005-10-20 Fraunhofer Ges Forschung Dynamic filter system

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US5126269A (en) * 1990-09-13 1992-06-30 Life Techologies, Inc. Spin filter perfusion bioreactor (sfpb) cell culture apparatus

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