EP0357738A1 - Process and device for producing viruses and viral antigens - Google Patents

Process and device for producing viruses and viral antigens

Info

Publication number
EP0357738A1
EP0357738A1 EP19890903142 EP89903142A EP0357738A1 EP 0357738 A1 EP0357738 A1 EP 0357738A1 EP 19890903142 EP19890903142 EP 19890903142 EP 89903142 A EP89903142 A EP 89903142A EP 0357738 A1 EP0357738 A1 EP 0357738A1
Authority
EP
European Patent Office
Prior art keywords
bioreactor
line
cells
suspension
virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19890903142
Other languages
German (de)
French (fr)
Inventor
Horst S. H. Seifert
Helge BÖHNEL
Frauke Roth
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut fur Angewandte Biotechnologie Der Tropen An Der Georg-August-Universitat
Original Assignee
Institut fur Angewandte Biotechnologie Der Tropen An Der Georg-August-Universitat
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut fur Angewandte Biotechnologie Der Tropen An Der Georg-August-Universitat filed Critical Institut fur Angewandte Biotechnologie Der Tropen An Der Georg-August-Universitat
Publication of EP0357738A1 publication Critical patent/EP0357738A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/58—Reaction vessels connected in series or in parallel
    • 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
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/10—Separation or concentration of fermentation products
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/00051—Methods of production or purification of viral material

Definitions

  • the invention relates to a method for producing virus or viral antigen by culture on animal cells in a sewing medium suspension, and to a device for carrying out this method.
  • a culture in suspension can to date only be carried out in roller bottles or in static fermenter cultures.
  • laborious preparations have to be made in cell cultures in the static fermenter until the culture is successful in large volumes and these cultures have the further disadvantage that after each culture, after each so-called “batch”, the system is cleaned and must be started again.
  • a possible contamination of a large fermenter also means the loss of large quantities of nutrient medium.
  • a further problem in the cultivation of viruses for the production of virus vaccines is that the conventional filtrations for separating the cell residues remove non-low molecular weight components which can lead to severe allergic symptoms, particularly in the case of repeated vaccinations.
  • There was therefore a need for a method for cultivating viruses which avoids the disadvantages of a "batch" mode of operation and which further succeeds in separating low-molecular components from the virus and also minimizes the risk of contamination.
  • the object of the invention was therefore to provide a breeding process for viruses or viral antigens which is easy to handle and in which contamination can be practically ruled out.
  • the object is achieved by a process for the production of virus or viral antigen by culture on animal cells in nutrient medium suspension, which is characterized in that the animal cells are cultivated in suspension in a first bioreactor A with continuous addition of nutrient medium after they have been reached at a certain cell concentration, the cell suspension is continuously transferred to a second bioreactor B and used there as a culture medium for virus cultivation with the continuous addition of a preservation medium, after generating a certain amount of virus in a first filtration arrangement, continuously separating cells and cell residues from the virus-containing culture medium suspension, which Discards retentate, continuously separates virus or viral antigen from the nutrient medium suspension in the filtrate in a second filtration arrangement, concentrating it in the process and discarding the used nutrient medium.
  • the method according to the invention makes it possible to carry out a continuous virus cultivation, in which a cell culture is cultivated, this is used as a wax basis for the viruses to be cultivated, and virus is first extracted from cell lines via two filtration stages. residues, and then can also be separated from low molecular weight components.
  • the growth rate of the cells is controlled in bioreactor A, ie in the cell culture by adding the nutrient medium suspension, which is preferably carried out under computer control. Both this nutrient medium suspension addition and the flow rate of animal cells in nutrient medium suspension into the second bioreactor B are controlled by photometric measurement of the density of the cell suspension during transfer into the bioreactor B. If the cell suspension has not yet reached a sufficient cell density , the flow rate between bioreactor A and B is throttled.
  • the cells are at a concentration of at least
  • bioreactor B 4x10 5 cells and preferably 2x106 cells per ml continuously transferred to bioreactor B. If the cell concentration is below this value, the flow rate from bioreactor A to bioreactor B is accordingly reduced in the preferred embodiment of the invention, so that the cells can grow to a higher density before they are transferred to bioreactor B.
  • virus is then grown which can multiply in the animal cells. After the virus, which has a cytopathic effect, multiplies in these cells, the cell is lysed and the daughter viruses are released. This results in a measurable decrease in cell density and thereby a suspension of the suspension in bioreactor B, which is also measured as a cytopathic effect in a photometer when the cell-virus suspension is passed from bioreactor B into the first filtration arrangement and thereby the flow rate from bioreactor B into the first filtration arrangement can also be optimally controlled.
  • the virus suspension which also contains not yet infected cells and also cell debris from lysed cells, is also transferred continuously into the first filtration arrangement. In a preferred embodiment of the invention, the filtration steps take place, ie cell virus suspension is transferred from bioreactor B into the first filtration arrangement as soon as at least 80% of the cells in bioreactor B are lysed.
  • the cell suspension infected with virus preferably runs continuously from the fermenter B into a collecting vessel. After a time to be determined by preliminary tests (approx. 15 minutes), the ultrasound source is switched on, which destroys all cells according to the time and intensity. Subsequently, the suspension thus obtained is passed into the first filtration arrangement and the collecting vessel is emptied. The time is controlled by the central computer.
  • the residence time in bioreactor B must be determined in preliminary tests. With a continuous inflow from bioreactor A, the residence time in bioreactor B can be varied by changing the working volume in bioreactor B. For virus cultivation, a maintenance medium is continuously added to the bioreactor B to maintain good growth conditions for the cells, so that the growth conditions are optimized.
  • the retentates of the first and / or second filtration arrangement are washed with a washing liquid which is removed as a filtrate in the second filtration arrangement and discarded from the virus product. This results in an increase in the yield of virus in the first filtration arrangement, while in the second filtration arrangement the separation of low molecular weight substances is optimized.
  • the concentration of the cells in the bioreactor A and / or the amount of the lysed cells in the case of cytopathic viruses is determined via the concentration of the still intact cells in the bioreactor B by photometric measurement and the addition from nutrient medium to bioreactor A, the feed from bioreactor A to bioreactor B, the addition of maintenance medium to bioreactor B, the feed from bioreactor B into the first filtration arrangement or / and the addition of liquid to the retentions of the filtration arrangements via a Computer controlled to which the photometers are connected.
  • Hollow fiber bundles are preferably used in the filtration arrangements as filters in the overcurrent process.
  • the nutrient medium is added to bioreactor A
  • the preserving medium is added to bioreactor B
  • the washing liquid is sterile filtered tangentially over hollow fiber bundles in an overflow.
  • the pH and the p0_ in the bioreactors are controlled by gassing through hollow fiber membranes.
  • Hollow fibers of this type for bubble-free fumigation are described, for example, by J. Lehmann et al., BTF-Biotech-Forurr. 2 (1985) 3.
  • the gases used for pH and pO ⁇ regulation are mixed in a computer-controlled manner in a gas mixing vessel and metered in together by only one hollow fiber system.
  • the cultivation of cells and virus is coupled in the method according to the invention, wherein cultivated cells are immediately used further as a basis for the growth of viruses.
  • the entire cultivation of cells and viruses takes place continuously according to the invention, which avoids the disadvantages of a batch of virus cultivation, since it is not necessary to reload and restart the system after a culture cycle has ended.
  • the virus cultivation method according to the invention As soon as the virus cultivation method according to the invention has started, it can be used without interruptions over long periods of time, at least for weeks, without contamination, wherein due to the preferably built-in control computer control continuously no specially trained personnel is necessary and a homogeneous product is obtained, its effectiveness guaranteed i ⁇ t. 1
  • Another object of the invention is a device for carrying out the method for the continuous cultivation of virus or viral antigen by culture on animal cells in nutrient medium suspension, which is characterized in that a bioreactor A, which is connected via a line to nutrient medium storage containers and via a line which is equipped with a photometer, is connected to a bioreactor B, which is connected via a line to preservation medium containers and which is further connected via a line to a first retentate container from which the cell virus suspension is via a Line is passed through a first filtration device, the retentate of which is led back into the first retentate container via a line and is withdrawn from the first retentate container via a line from the system, and the filtrate is fed via a line into a second retentate container is led, which over e a line is connected to a second filtration device, the retentate of which is fed back into the second retentate container via a line and withdrawn from the system via a line and the filtrate
  • the line from bioreactor B into the first filtration arrangement is also equipped with a photometer that is connected to the control system. This enables a new cell concentration measurement, from which one can deduce the amount of virus formed.
  • this line between the bioreactor B and the first filtration arrangement is equipped with a collecting vessel, which contains an ultrasound source, and optionally also with a pump. The use of ultrasound destroys the cells and releases the cultivated viruses when viruses are cultivated which have no lytic effect. From the collection vessel, the suspension is then preferably fed via the pump into the first retentate container.
  • the feed lines from the nutrient medium and maintenance medium storage containers and / or the feed lines to the filtration devices are provided with pumps, which are also preferably controlled by the computer-controlled control system.
  • the bioreactors A and B of the device are provided with hollow fiber membranes, by means of which the pH and p0 2 content of the cell suspension can be regulated by adding gases.
  • the device can also contain a mixing vessel for gas, in which gases are mixed in a computer-controlled manner, and then metered in via the hollow fiber system. It is also possible within the scope of the invention not to use two separate hollow fiber membranes for the two bioreactors, but to use a connected hollow fiber membrane system in both reactors.
  • bioreactors A and B are equipped with magnetic stirrers, which bring about appropriate mixing and stirring in these bioreactors.
  • the lines between bioreactor A and B, between bioreactor B and the first retentate container, between the first retentate container and the second retentate container are the lines for removal the retentates, as well as for the removal of low molecular weight constituents and water and finally the washing liquid feed lines in a preferred embodiment of the invention are equipped with regrowth traps.
  • the bioreactors A and B are preferably equipped with lines with pressure relief valves which open into an exhaust air safety vessel. Differences in pressure and overprints which result from transfer of suspension or from introduction of gases or media can hereby be compensated for.
  • the exhaust air safety vessel is used to clean the exhaust air so that no dangerous virus particles can escape.
  • the retentate containers also have pressure equalization lines which also open into the exhaust air safety vessel. All of these pressure equalization lines are preferably equipped with hollow fiber filters which retain any virus particles that may already be present (no reflux traps in the case of liquid).
  • the retentate containers are each connected to a washing liquid container via a line, which is provided with a pump, through which washing liquid can be introduced into the retentate containers.
  • the line used for this is also provided with regrowth traps.
  • the hollow fiber membrane systems for gassing in bioreactor A and B can also be connected to the exhaust air safety vessel via lines, and preferably also with hollow fiber filter to separate any virus that may be present.
  • the method according to the invention and the device suitable for this purpose make it possible to continuously continuously and continuously grow viru ⁇ or viral antigens which are suitable for vaccination in a simple manner, which method can also be carried out by only little trained personnel and via Leads to a homogeneous product for weeks. Because of the compact devices, the method and the device according to the invention save the construction of large laboratories with the associated infrastructure.
  • the system is also suitable for installation in prefabricated laboratory containers which, when fully equipped and set up at the tropical location, for example, can begin production immediately after being connected to the supply. In this way, vaccines can also be generated ad hoc in problem areas in the event of an outbreak of epidemics from locally specific pathogen strains.
  • the product obtained according to the invention is of high quality and antigenicity, free of harmful by-products (low-molecular substances or cell residues) and can be manufactured with only low costs.
  • the figure shows a preferred device according to the invention with a bioreactor A 2, in which nutrient medium is introduced into the bioreactor A 2 into the nutrient medium storage containers 4 via a line 6, which is equipped with a pump 8.
  • the bioreactor A 2 is further equipped with hollow fiber membranes 58, via which gases for regulating the pH or pH can be introduced.
  • the bioreactor A 2 is connected to the exhaust air safety vessel 70 with a magnetic stirrer 62 and via a line 66 which has a pressure relief valve, the line having a hollow fiber filter 82 for retaining any viruses that may be present.
  • the bioreactor A 2 is connected to the bioreactor B 14 via a line 10, which is equipped with a photometer 12 and a regrowth trap, into which the bioreactor B 14 is connected via a line 18, which is equipped with a pump 20 and a regrowth trap Containers for maintenance medium 16 medium is fed to bioreactor B 14.
  • the bioreactor B 14 is equipped with a hollow fiber membrane 60 which is used for gassing and for regulating the pH and pO_.
  • the bioreactor B 14 has a magnetic stirrer 64 for mixing the ingredients.
  • a line 68 with a pressure relief valve serves to equalize the pressure and leads into the exhaust air safety vessel 70 via a hollow fiber filter 84, in which viruses which may be present are retained.
  • the bioreactor B 14 is connected to the first retentate container 26 of the first filtration arrangement via a line 22 which is equipped with a photometer 24 and a regrowth trap, which in turn is connected via a line 78 with a hollow fiber filter 86 present thereon Exhaust air safety vessel 70 is connected for pressure equalization.
  • Retentate from the first retentate container 26 is withdrawn via line 36, which is equipped with a regrowth trap.
  • a suspension 28 is fed into the first filter 32 via a line 28, which is equipped with a photometer 30, and from this the retentate is returned via line 34 to the first retentate container 26 and the filtrate is passed through the Line 38, which is equipped with a regrowth trap, is introduced into the second retentate container 40, which in turn is provided with a pressure equalization line 80 which opens into the exhaust air safety vessel 70 via a hollow fiber filter 86.
  • the second retentate container 40 is provided with a line 50 which contains a regrowth trap and is drawn off via the virus concentrate.
  • a suspension is fed via a line 42, which is provided with a pump 44, into the filtration device 46, the retentate of which is returned via line 48 to the second retentate container 40, and the filtrate of which is via the line 54, which is equipped with a reflux trap, is withdrawn.
  • Computer-controlled washing lines can also be fed into the retentate containers 26, 40 from the liquid liquid containers 76 via lines 72, which are equipped with reflux traps and provided with a pump 74.
  • the cells BHK 21 clone 13 (ECACC No. 84100501 (to be obtained from Flow Laboratories Meckenheim)) can be grown both in monolayers and in roller bottles. At least inoculation must be done in the fermenter
  • the medium addition (9.538 g / 1 MEM (minimal ess. Media) with Earl's spinner salts, 2.2 g / 1 NaHCO-., 10.0 ml / 1 MEM vitamin solution, 10.0 ml / 1 non-essential amino acids, 10.0% / l fetal calf serum, sterile filtered through hollow fiber bundles, which are directly connected to the bioreactor, was 240 ml per day at the start of fermentation, which corresponds to a dilution of 1: 12.5 and could be proportional to growth up to 1500 ml per day (1: 2).
  • the regulation was carried out via a built-in photometer.
  • the cells grew without a microcarrier.
  • the reaction volume of bioreactor A was 4 liters, the liquid volume 3 liters.
  • the reaction temperature was 37 ° C.
  • the stirring speed was 150 rpm
  • the pH was 7.0
  • the pO was 45% of an air-calibrated solution set.
  • the basic gas flow was 4.5 l / irtin. 40 bar N, 20 mbar 0 "and 30 mbar CO and air without control were used as the upstream pressure. The following were used as devices in the example: 1-
  • module PH BCC electrode pH electrode Ingold autoclavable pH range 0 to 12 type 465-36-90-K9 p0 2 regulation: module P0_ BCC electrode: p0 2 - Ingold electrode autoclavable, type 7455/322756702
  • Pressure reducer Type No. D 142/0 3 Dräger compressed air: continuous air flow
  • Pore size max. 0.54 ⁇ m winding: perpendicular to the liquid level
  • the following medium was used as the cell maintenance medium:
  • MEM vitamin solution 10.0 ml / 1 fetal calf serum 2.0% / l
  • the medium is sterilized by filtration through hollow fiber bundles connected directly to the bioreactor. i ⁇
  • the viru ⁇ ermenter was with a BHK monolayer
  • the reactor volume of the bioreactor B was likewise 4 1, the liquid volume 3 1, the temperature 37 ° C., the stirring speed 150 revolutions per minute, the pH value became 7.0, the p0_ value 40.0% - Set in an air calibrated solution.
  • the gas flow rate was 4.0 l per minute, and 40 mbar N 2 , 20 mbar 0 2 , 30 mbar CO and air were used as the admission pressure without regulating the admission pressure.
  • the medium addition was 2 1 per day and the line additions on average 1.2 x 10 cells per day.
  • the level control built into reactor I opened two solenoid valves with a time delay:
  • Solenoid valve 1 cell flow to reactor II was made possible.
  • Solenoid valve 2 solenoid valve 1 was closed; to flush the supply line of cells, the medium inlet for reactor II was opened; opening time 5 to 10 seconds.
  • Level control and solenoid valves came from the company BCC.
  • the industrial control computer FLS1 with adapted software from the company Münzer and Diehl was used.
  • the host computer system controlled the density of the cell growth in bioreactor A and the virus increase via the cytopathic (lytic effect in bioreactor B and controls the nutrient inflow to bioreactor A and cell delivery from bioreactor B) all material was immediately processed.

Abstract

Afin de produire des virus ou des antigènes viraux par culture sur des cellules animales dans une suspension de milieu de culture, on cultive dans un premier bioréacteur (A) les cellules animales sous un apport constant du milieu de culture en suspension, on transfère en continu dans un deuxième bioréacteur (B) la suspension de cellules, une fois qu'une concentration déterminée de cellules a été atteinte, et on les y utilise comme milieu nutritif pour la culture de virus sous l'apport continu d'un milieu de conservation. On sépare en continu dans un premier agencement de filtrage les cellules et les résidus cellulaires de la suspension de milieu de culture contenant les virus, une fois qu'une quantité déterminée de virus a été obtenue, on jette le rétentat, on sépare le filtrat en continu dans un deuxième agencement de filtrage en virus et antigènes viraux d'une part et en milieu de culture en suspension d'autre part, de façon à les concentrer, et on jette le milieu de culture.In order to produce viruses or viral antigens by culture on animal cells in a suspension of culture medium, the animal cells are cultured in a first bioreactor (A) under a constant supply of the culture medium in suspension, and are continuously transferred in a second bioreactor (B) the suspension of cells, once a determined concentration of cells has been reached, and they are used there as a nutritive medium for the culture of virus under the continuous supply of a preservation medium. The cells and cell residues are continuously separated in a first filtering arrangement from the suspension of culture medium containing the viruses, once a determined quantity of virus has been obtained, the retentate is discarded, the filtrate is separated in continuous in a second arrangement of filtering in virus and viral antigens on the one hand and in culture medium in suspension on the other hand, so as to concentrate them, and the culture medium is discarded.

Description

4. 4th
Verfahren zur Herstellung von Virus und viralem Antigen und Vorrichtung hierzuMethod for producing virus and viral antigen and device therefor
B e s c h r e i b u n gDescription
Die Erfindung betrifft ein Verfahren zur Herstellung von Virus oder viralem Antigen durch Kultur auf tieri¬ schen Zellen in Nähπnedium-Suspension, sowie eine Vor¬ richtung zur Durchführung dieses Verfahrens.The invention relates to a method for producing virus or viral antigen by culture on animal cells in a sewing medium suspension, and to a device for carrying out this method.
Zur Züchtung von Virus- und Chlamydienarten kann bis heute eine Kultur in Suspension nur in Rollerflaschen oder in statischen Fermenterkulturen durchgeführt wer¬ den. Hierzu müssen bei Zellkulturen im statischen Fer¬ menter umständliche Vorbereitungen getroffen werden, bis die Kultur im großen Volumen gelingt und diese Kul¬ turen haben weiter den Nachteil, daß jeweils nach er¬ folgter Kultur, nach jedem sogenannten "batch", das System gereinigt und erneut angefahren werden muß. Eine eventuelle Kontamination eines Großfermenters bedeutet überdies den Verlust von großen Mengen an Nährmedium.For the cultivation of virus and chlamydia species, a culture in suspension can to date only be carried out in roller bottles or in static fermenter cultures. For this, laborious preparations have to be made in cell cultures in the static fermenter until the culture is successful in large volumes and these cultures have the further disadvantage that after each culture, after each so-called “batch”, the system is cleaned and must be started again. A possible contamination of a large fermenter also means the loss of large quantities of nutrient medium.
Kontinuierliche Kulturen tierischer Zellen können bis¬ her nur zur Züchtung von genetisch manipulierten pro- dukt-produzierenden Zellen (Interferon, monoklonale Antikörper) eingesetzt werden.Until now, continuous cultures of animal cells could only be used for the cultivation of genetically manipulated product-producing cells (interferon, monoclonal antibodies).
Ein weiteres Problem bei der Züchtung von Viren zur Herstellung von Virusvakzinen ist, daß die gängigen Filtrationen zur Abtrennung der Zellreεte nicht nieder¬ molekulare Bestandteile beseitigen, die besonders bei Wiederholungεimpfungen zu schweren allergischen Er¬ scheinungen führen können. Es bestand daher ein Bedarf an einem Verfahren zur Züchtung von Viren, das die Nachteile einer "batch"-weisen Betriebsart vermeidet, und durch das es weiter gelingt, niedermolekulare Bestandteile von Virus zu trennen und auch die Gefahr einer Kontamination zu minimieren. Aufgabe der Erfindung war es daher, ein Züchtungsverfah¬ ren für Viren oder virale Antigene bereitzustellen, das leicht zu handhaben ist, und bei dem eine Kontamination praktiεch auεgeschlossen werden kann.A further problem in the cultivation of viruses for the production of virus vaccines is that the conventional filtrations for separating the cell residues remove non-low molecular weight components which can lead to severe allergic symptoms, particularly in the case of repeated vaccinations. There was therefore a need for a method for cultivating viruses which avoids the disadvantages of a "batch" mode of operation and which further succeeds in separating low-molecular components from the virus and also minimizes the risk of contamination. The object of the invention was therefore to provide a breeding process for viruses or viral antigens which is easy to handle and in which contamination can be practically ruled out.
Gelöst wird die Aufgabe durch ein Verfahren zur Herstel¬ lung von Virus oder viralem Antigen durch Kultur auf tierischen Zellen in Nährmediumsuspension, das dadurch gekennzeichnet ist, daß man in einem ersten Bioreaktor A die tierischen Zellen unter kontinuierlicher Nährmedium¬ zugabe in Suspension kultiviert, nach Erreichen einer bestimmten Zellkonzentration die Zellsuspension in einen zweiten Bioreaktor B kontinuierlich überführt und dort als Nährboden für die VirusZüchtung unter kontinuier¬ licher Zugabe eines Erhaltungεmediums verwendet, nach Erzeugung einer beεtimmten Menge an Virus in einer erεten Filtrationsanordnung Zellen und Zellreεte von der virushaltigen Nährmediumsuεpension kontinuierlieh abtrennt, das Retentat verwirft, im Filtrat in einer zweiten Filtrationεanordnung Virus oder virales Antigen von der Nährmediumsuspension kontinuierlich trennt und dabei aufkonzentriert und das verbrauchte Nährmedium verwirft.The object is achieved by a process for the production of virus or viral antigen by culture on animal cells in nutrient medium suspension, which is characterized in that the animal cells are cultivated in suspension in a first bioreactor A with continuous addition of nutrient medium after they have been reached at a certain cell concentration, the cell suspension is continuously transferred to a second bioreactor B and used there as a culture medium for virus cultivation with the continuous addition of a preservation medium, after generating a certain amount of virus in a first filtration arrangement, continuously separating cells and cell residues from the virus-containing culture medium suspension, which Discards retentate, continuously separates virus or viral antigen from the nutrient medium suspension in the filtrate in a second filtration arrangement, concentrating it in the process and discarding the used nutrient medium.
Bevorzugte Ausführungsformen dieses Verfahrens sind in den Unteransprüchen aufgeführt.Preferred embodiments of this method are listed in the subclaims.
Durch das erfindungsgemäße Verfahren wird es ermöglicht, eine kontinuierliche VirusZüchtung durchzuführen, bei der eine Zellkultur gezüchtet wird, diese als Wachs ums- grundlage für die zu züchtenden Viren verwendet wird, und über zwei Filtrationεεtufen Virus zuerst von Zeil- resten, und dann auch von niedermolekularen Bestandteilen getrennt werden kann. Die Steuerung der Wachstumsgeschwin¬ digkeit der Zellen erfolgt im Bioreaktor A, d.h. in der Zellkultur über Zugabe der Nährmediu suspension, was vorzugsweise rechnergesteuert erfolgt. Die Steuerung sowohl dieser Nährmedium-Suspensionszugabe, als auch der Flußgeschwindigkeit von tierischen Zellen in Nähr- mediumsuεpension in den zweiten Bioreaktor B erfolgt über photometrische Messung der Dichte der Zellsuspension bei der Überleitung in den Bioreaktor B. Hat die Zell¬ suspension noch keine ausreichende Zelldichte erreicht, so wird die Durchflußgeεchwindigkeit zwischen dem Bioreaktor A und B gedrosselt.The method according to the invention makes it possible to carry out a continuous virus cultivation, in which a cell culture is cultivated, this is used as a wax basis for the viruses to be cultivated, and virus is first extracted from cell lines via two filtration stages. residues, and then can also be separated from low molecular weight components. The growth rate of the cells is controlled in bioreactor A, ie in the cell culture by adding the nutrient medium suspension, which is preferably carried out under computer control. Both this nutrient medium suspension addition and the flow rate of animal cells in nutrient medium suspension into the second bioreactor B are controlled by photometric measurement of the density of the cell suspension during transfer into the bioreactor B. If the cell suspension has not yet reached a sufficient cell density , the flow rate between bioreactor A and B is throttled.
In einer bevorzugten Ausführungsform der Erfindung wer¬ den die Zellen bei einer Konzentration von mindestensIn a preferred embodiment of the invention, the cells are at a concentration of at least
4x10 5 Zellen und vorzugsweiεe 2x106 Zellen pro ml kontinuierlich in den Bioreaktor B überführt. Liegt die Zellkonzentration unter dieεem Wert, wird daher in der bevorzugten Ausführungsform der Erfindung die Flußge¬ schwindigkeit von Bioreaktor A nach Bioreaktor B ent¬ sprechend verringert, so daß die Zellen zu einer höheren Dichte anwachsen können, bevor sie in den Bioreaktor B überführt werden.4x10 5 cells and preferably 2x106 cells per ml continuously transferred to bioreactor B. If the cell concentration is below this value, the flow rate from bioreactor A to bioreactor B is accordingly reduced in the preferred embodiment of the invention, so that the cells can grow to a higher density before they are transferred to bioreactor B.
Im zweiten Bioreaktor B, der mit einer Viruspräparation angeimpft wird, erfolgt sodann die Züchtung von Virus, das sich in den tierischen Zellen vermehren kann. Nach Vermehrung von Virus, das einen cytopathischen Effekt ausübt, in diesen Zellen, wird die Zelle lysiert und dabei die Tochterviren freigesetzt. Hierdurch wird eine meßbare Abnahme der Zelldichte und dadurch eine Aufhel¬ lung der Suspension im Bioreaktor B bewirkt, die eben¬ falls beim Weiterleiten der Zell-Virus-Suspension aus dem Bioreaktor B in die erste Filtrationsanordnung in einem Photometer als cytopathischer Effekt gemessen und und dadurch die Flußgeschwindigkeit vom Bioreaktor B in die erste Filtrationsanordnung ebenfalls optimal gesteuert werden kann. Die Überführung der VirusSuspension, die auch noch nicht infizierte Zellen und auch Zelltrümmer von lysierten Zellen enthält, in die erste Filtrations¬ anordnung, erfolgt ebenfalls kontinuierlich. In einer bevorzugten Ausführungsform der Erfindung erfolgen die Filtrationsschritte, d.h. wird Zell-Virus-Suεpenεion aus dem Bioreaktor B in die erste Filtrationsanordnung überführt, sobald im Bioreaktor B mindestens 80% der Zellen lysiert sind.In the second bioreactor B, which is inoculated with a virus preparation, virus is then grown which can multiply in the animal cells. After the virus, which has a cytopathic effect, multiplies in these cells, the cell is lysed and the daughter viruses are released. This results in a measurable decrease in cell density and thereby a suspension of the suspension in bioreactor B, which is also measured as a cytopathic effect in a photometer when the cell-virus suspension is passed from bioreactor B into the first filtration arrangement and thereby the flow rate from bioreactor B into the first filtration arrangement can also be optimally controlled. The virus suspension, which also contains not yet infected cells and also cell debris from lysed cells, is also transferred continuously into the first filtration arrangement. In a preferred embodiment of the invention, the filtration steps take place, ie cell virus suspension is transferred from bioreactor B into the first filtration arrangement as soon as at least 80% of the cells in bioreactor B are lysed.
Bei der Züchtung von Viren, die keinen cytopathischen Effekt zeigen, werden die aus dem Bioreaktor B entnom¬ menen Zellen durch Ultraschall zerstört und das darin befindliche Virus freigesetzt.When viruses which do not have a cytopathic effect are grown, the cells removed from the bioreactor B are destroyed by ultrasound and the virus contained therein is released.
Hierzu läuft vorzugsweise aus dem Fermenter B kontinu¬ ierlich die mit Virus infizierte Zellεuspension in ein Sammelgefäß. Nach einer durch Vorversuche zu bestimmen¬ den Zeit (ca. 15 Minuten) wird die Ultraεchallquelle eingeschaltet, die nach Zeit und Intensität regelbar alle Zellen zerεtört. Anεchließend wird die εo erhal¬ tene Suεpenεion in die erste Filtrationsanordnung ge¬ leitet und das Sammelgefäß geleert. Die Zeitsteuerung erfolgt durch den zentralen Rechner.To this end, the cell suspension infected with virus preferably runs continuously from the fermenter B into a collecting vessel. After a time to be determined by preliminary tests (approx. 15 minutes), the ultrasound source is switched on, which destroys all cells according to the time and intensity. Subsequently, the suspension thus obtained is passed into the first filtration arrangement and the collecting vessel is emptied. The time is controlled by the central computer.
Die Verweildauer in Bioreaktor B muß in Vorversuchen ermittelt werden. Bei kontinuierlichem Zufluß aus Bio¬ reaktor A läßt sich die Verweildauer in Bioreaktor B durch Veränderung des in Bioreaktor B befindlichen Arbeitεvolumen variieren. Für die Viruszüchtung wird in den Bioreaktor B zur Aufrechterhaltung guter Wachεtumsbedingungen für die Zellen kontinuierlich ein Erhaltungεmedium zugegeben, daε die Wachεtu εbedingungen optimiert.The residence time in bioreactor B must be determined in preliminary tests. With a continuous inflow from bioreactor A, the residence time in bioreactor B can be varied by changing the working volume in bioreactor B. For virus cultivation, a maintenance medium is continuously added to the bioreactor B to maintain good growth conditions for the cells, so that the growth conditions are optimized.
In einer bevorzugten Ausführungεform der Erfindung werden die Retentate der erεten und/oder zweiten Fil¬ trationsanordnung mit einer Waschflüsεigkeit gewaschen, die als Filtrat in der zweiten Filtrationεanordnung dem Virusprodukt entzogen und verworfen wird. Hierdurch wird in der ersten Filtrationsanordnung eine Erhöhung der Ausbeute an Virus erreicht, in der zweiten Filtra¬ tionsanordnung dagegen die Abtrennung von niedermoleku¬ laren Subεtanzen optimiert.In a preferred embodiment of the invention, the retentates of the first and / or second filtration arrangement are washed with a washing liquid which is removed as a filtrate in the second filtration arrangement and discarded from the virus product. This results in an increase in the yield of virus in the first filtration arrangement, while in the second filtration arrangement the separation of low molecular weight substances is optimized.
In einer besonders bevorzugten Auεführungsform der Erfindung wird die Konzentration der Zellen im Bioreak¬ tor A und/oder die Menge der lyεierten Zellen im Fall von cytopathiεch wirkenden Viren über die Konzentration der noch intakten Zellen im Bioreaktor B durch photo- metriεche Meεεung bestimmt und die Zugabe von Nährmedium zum Bioreaktor A, der Zulauf von Bioreaktor A zu Bio¬ reaktor B, die Zugabe von Erhaltungεmedium zum Bioreaktor B, der Zulauf von Bioreaktor B in die erεte Filtrationε¬ anordnung oder/und die Zugabe von Waεchflüεsigkeit zu den Retentaten der Filtrationεanordnungen über einen Rechner gesteuert, an den die Photometer angeschlossen εind.In a particularly preferred embodiment of the invention, the concentration of the cells in the bioreactor A and / or the amount of the lysed cells in the case of cytopathic viruses is determined via the concentration of the still intact cells in the bioreactor B by photometric measurement and the addition from nutrient medium to bioreactor A, the feed from bioreactor A to bioreactor B, the addition of maintenance medium to bioreactor B, the feed from bioreactor B into the first filtration arrangement or / and the addition of liquid to the retentions of the filtration arrangements via a Computer controlled to which the photometers are connected.
Bevorzugt werden in den Filtrationεanordnungen Hohl¬ faserbündel alε Filter im Uberεtromverfahren verwendet. In weiteren bevorzugten Ausführungεformen der Erfindung wird das Nährmedium vor der Zugabe zu Bioreaktor A, das Erhaltungεmedium vor Zugabe zu Bioreaktor B, sowie die Waεchflüssigkeit vor Zugabe zu den Retentaten der Fil¬ trationsanordnungen tangential über Hohlfaεerbündel im Uberstrom steril filtriert. Hierdurch wird eine Konta¬ mination der Kulturen wirksam vermieden.Hollow fiber bundles are preferably used in the filtration arrangements as filters in the overcurrent process. In further preferred embodiments of the invention, the nutrient medium is added to bioreactor A, the preserving medium is added to bioreactor B, and the Before being added to the retentates of the filtration arrangements, the washing liquid is sterile filtered tangentially over hollow fiber bundles in an overflow. As a result, contamination of the cultures is effectively avoided.
In einer weiteren bevorzugten Ausführungsform der Erfindung wird der pH und der p0_ in den Bioreaktoren über Begasung durch Hohlfasermembranen gesteuert. Derartige Hohlfasern zur blasenfreien Begasung sind beispielsweise aus J. Lehmann et al., BTF-Biotech-Forurr. 2 (1985)3 bekannt.In a further preferred embodiment of the invention, the pH and the p0_ in the bioreactors are controlled by gassing through hollow fiber membranes. Hollow fibers of this type for bubble-free fumigation are described, for example, by J. Lehmann et al., BTF-Biotech-Forurr. 2 (1985) 3.
In einer besonders bevorzugten Ausführungεform der Er¬ findung werden die zur pH- und pO^-Regulierung verwen¬ deten Gase in einem Gasmiεchgefäß rechnergeεteuert ge¬ mischt und gemeinsam durch nur ein Hohlfaεerεystem zu¬ dosiert.In a particularly preferred embodiment of the invention, the gases used for pH and pO ^ regulation are mixed in a computer-controlled manner in a gas mixing vessel and metered in together by only one hollow fiber system.
Die Züchtung von Zellen und Virus verläuft im erfindungs¬ gemäßen Verfahren gekoppelt, wobei gezüchtete Zellen sofort weiter als Wachεtumεgrundlage für Viren verwendet werden. Die gesamte Züchtung von Zellen und Viren läuft: erfindungsgemäß kontinuierlich ab, wodurch die Nach¬ teile einer ansatzweiεen Viruεzüchtung vermieden werden, da eε nicht nach Beendigung eineε Kultur-Zykluε nötig iεt, daε System neu zu beladen und erneut anzufahren. Sobald das erfindungεgemäße Viruεzüchtungεverfahren angelaufen iεt, kann eε ohne Unterbrechungen über lange Zeiträume, zumindest über Wochen, ohne Kontamination verwendet werden, wobei aufgrund der bevorzugt einge¬ bauten Leitrechnerεteuerung kontinuierlich kein beson¬ ders auεgebildetes Perεonal nötig iεt und ein homogeneε Produkt erhalten wird, deεεen Wirkεamkeit garantiert iεt. 1The cultivation of cells and virus is coupled in the method according to the invention, wherein cultivated cells are immediately used further as a basis for the growth of viruses. The entire cultivation of cells and viruses takes place continuously according to the invention, which avoids the disadvantages of a batch of virus cultivation, since it is not necessary to reload and restart the system after a culture cycle has ended. As soon as the virus cultivation method according to the invention has started, it can be used without interruptions over long periods of time, at least for weeks, without contamination, wherein due to the preferably built-in control computer control continuously no specially trained personnel is necessary and a homogeneous product is obtained, its effectiveness guaranteed iεt. 1
Ein weiterer Gegenεtand der Erfindung ist eine Vorrich¬ tung zur Durchführung des Verfahrenε zur kontinuier¬ lichen Züchtung von Virus oder viralem Antigen durch Kultur auf tierischen Zellen in Nährmediumεuεpension, die dadurch gekennzeichnet iεt, daß ein Bioreaktor A, der über eine Leitung mit Nährmedium-Vorratεbehältern und über eine Leitung, welche mit einem Photometer auεgerüstet ist, mit einem Bioreaktor B verbunden iεt, der über eine Leitung mit Erhaltungεmediumbehältern verbunden iεt und der weiter über eine Leitung mit einem erεten Retentatbehälter verbunden ist, aus dem die Zell-Viruε-Suεpension über eine Leitung über eine erεte Filtrationseinrichtung geleitet wird, deren Re- tentat über eine Leitung zurück in den erεten Retentat¬ behälter geführt wird und auε dem erεten Retentatbehälter über eine Leitung auε dem Syεte abgezogen wird, und deren Filtrat über eine Leitung in einen zweiten Reten¬ tatbehälter geführt wird, der über eine Leitung mit einer zweiten Filtrationεeinrichtung verbunden ist, deren Retentat über eine Leitung zurück in den zweiten Retentatbehälter geführt wird und auε dem System über eine Leitung abgezogen wird und deren Filtrat über eine Leitung abgezogen wird, wobei die Photometer und mit einem rechner-geεteuerten Kontrollsyεtem verbunden εind.Another object of the invention is a device for carrying out the method for the continuous cultivation of virus or viral antigen by culture on animal cells in nutrient medium suspension, which is characterized in that a bioreactor A, which is connected via a line to nutrient medium storage containers and via a line which is equipped with a photometer, is connected to a bioreactor B, which is connected via a line to preservation medium containers and which is further connected via a line to a first retentate container from which the cell virus suspension is via a Line is passed through a first filtration device, the retentate of which is led back into the first retentate container via a line and is withdrawn from the first retentate container via a line from the system, and the filtrate is fed via a line into a second retentate container is led, which over e a line is connected to a second filtration device, the retentate of which is fed back into the second retentate container via a line and withdrawn from the system via a line and the filtrate of which is withdrawn via a line, the photometer and being connected to a computer-controlled control system are.
In einer ersten bevorzugten Ausführungεform ist hierbei auch die Leitung von Bioreaktor B in die erste Filtra¬ tionsanordnung mit einem Photometer auεgerüεtet, daε mit dem Kontrollεyεtem verbunden ist. Hierdurch wird eine erneute Zellkonzentrationmesεung ermöglicht, woraus auf die gebildete Viruεmenge geεchlosεen werden kann. In einer zweiten bevorzugten Ausführungεform ist diese Leitung zwischen dem Bioreaktor B und der ersten Fil¬ trationsanordnung mit einem Sammelgefäß, das eine Ultraschallquelle enthält, und gegebenenfalls noch mit einer Pumpe ausgerüstet. Durch die Ultraschallanwendung werden - bei der Zucht von Viren, die keinen lytiεchen Effekt auεüben - die Zellen zerεtört und die gezüchteten Viren freigeεetzt. Auε dem Sammelgefäß wird die Suεpen- εion dann vorzugεweiεe über die Pumpe in den erεten Retentatbehälter geleitet.In a first preferred embodiment, the line from bioreactor B into the first filtration arrangement is also equipped with a photometer that is connected to the control system. This enables a new cell concentration measurement, from which one can deduce the amount of virus formed. In a second preferred embodiment, this line between the bioreactor B and the first filtration arrangement is equipped with a collecting vessel, which contains an ultrasound source, and optionally also with a pump. The use of ultrasound destroys the cells and releases the cultivated viruses when viruses are cultivated which have no lytic effect. From the collection vessel, the suspension is then preferably fed via the pump into the first retentate container.
In ebenfalls bevorzugten Ausführungεformen der Erfindung sind die Zuleitungen aus den Nährmedium- und Erhaltungs¬ medium-Vorratsbehältern und/oder die Zuleitungen zu den Filtrationεeinrichtungen mit Pumpen verεehen, die besonders bevorzugt ebenfalls von dem rechner-geεteuerten Kontrollεyεtem gesteuert werden.In likewise preferred embodiments of the invention, the feed lines from the nutrient medium and maintenance medium storage containers and / or the feed lines to the filtration devices are provided with pumps, which are also preferably controlled by the computer-controlled control system.
In einer weiteren bevorzugten Ausführungsform der Erfindung sind die Bioreaktoren A und B der Vorrichtung mit Hohlfasermembranen versehen, über die der pH-Wert und p02-Gehalt der Zellsuεpenεion durch Zugabe von Gaεen reguliert werden kann. Hierzu kann die Vorrichtung auch noch ein Miεchgefäß für Gaεe enthalten, in dem Gaεe rechnergeεteuert vermischt werden, und dann über das Hohlfaserεystem dosiert werden. Es ist im Rahmen der Erfindung ebenfallε möglich, nicht zwei getrennte Hohlfaεermembranen für die beiden Bioreaktoren einzu¬ setzen, sondern ein verbundenes Hohlfasermembransyεtem in beiden Reaktoren zu verwenden.In a further preferred embodiment of the invention, the bioreactors A and B of the device are provided with hollow fiber membranes, by means of which the pH and p0 2 content of the cell suspension can be regulated by adding gases. For this purpose, the device can also contain a mixing vessel for gas, in which gases are mixed in a computer-controlled manner, and then metered in via the hollow fiber system. It is also possible within the scope of the invention not to use two separate hollow fiber membranes for the two bioreactors, but to use a connected hollow fiber membrane system in both reactors.
In noch einer weiteren bevorzugten Auεführungsform der Erfindung εind die Bioreaktoren A und B mit Magnetrührern ausgestattet, die ein entsprechendeε Vermiεchen und Rühren in dieεen Bioreaktoren bewirken. 3In yet another preferred embodiment of the invention, bioreactors A and B are equipped with magnetic stirrers, which bring about appropriate mixing and stirring in these bioreactors. 3
Um eine Kontamination in der Vorrichtung zu vorlie¬ genden Elementen zu vermeiden, εind die Leitungen zwischen dem Bioreaktor A und B, zwischen dem Bioreak¬ tor B und dem erεten Retentatbehälter, zwiεchen dem ersten Retentatbehälter und dem zweiten Retentatbehäl¬ ter, die Leitungen zur Abführung der Retentate, εowie zur Abführung von niedermolekularen Bestandteilen und Waεεer und εchließlich die Waschflüssigkeit-Zulauf¬ leitungen in einer bevorzugten Auεführungεform der Er¬ findung mit Rückwuchεfallen auεgeεtattet. Weiter sind bevorzugt die Bioreaktoren A und B über Leitungen mit Überdruckventilen auεgeεtattet, die in ein Abluftsicher- heitεgefäß münden. Hierdurch können Druckunterεchiede und Überdrucke, die durch Überleitung von Suεpenεion, bzw. durch Einleitung von Gaεen oder Medien entstehen, auεgeglichen werden. Daε Abluftεicherheitεgefäß dient zur Reinigung der Abluft, so daß keine gefährdenden Viruspartikel entweichen können. Auch die Retentatbe¬ hälter verfüσen in einer bevorzugten Auεführungsform der Erfindung über Druckausgleichεleitungen, die eben¬ falls in daε Abluftεicherheitsgefäß münden. All dieεe Druckauεgleichεleitungen εind bevorzugt mit Hohlfaεer- filtern ausgerüεtet, die bereits eventuell vorhandene Viruspartikel zurückhalten (bei Waεchflüεsigkeit noch keine Rückflußfallen) . In einer weiteren Auεführungsform der Erfindung εind die Retentatbehälter über je eine Leitung, die mit einer Pumpe versehen iεt, mit einem Waεchflüsεigkeitεbehälter verbunden, über den Wasch¬ flüssigkeit in die Retentatbehälter eingeführt werden kann. Die hierfür verwendete Leitung iεt in einer bevorzugten Ausführungsform ebenfallε mit Rückwuchε- fallen verεehen. Die Hohlfaεermembransyεteme zur Begasung im Bioreaktor A und B können ebenfallε über Leitungen, und bevorzugt auch mit Hohlfaεerfilter zur Abtrennung von eventuell vorhandenem Virus, mit dem Abluftsicherheitεgefäß verbunden sein.In order to avoid contamination in the elements to be present, the lines between bioreactor A and B, between bioreactor B and the first retentate container, between the first retentate container and the second retentate container, are the lines for removal the retentates, as well as for the removal of low molecular weight constituents and water and finally the washing liquid feed lines in a preferred embodiment of the invention are equipped with regrowth traps. Furthermore, the bioreactors A and B are preferably equipped with lines with pressure relief valves which open into an exhaust air safety vessel. Differences in pressure and overprints which result from transfer of suspension or from introduction of gases or media can hereby be compensated for. The exhaust air safety vessel is used to clean the exhaust air so that no dangerous virus particles can escape. In a preferred embodiment of the invention, the retentate containers also have pressure equalization lines which also open into the exhaust air safety vessel. All of these pressure equalization lines are preferably equipped with hollow fiber filters which retain any virus particles that may already be present (no reflux traps in the case of liquid). In a further embodiment of the invention, the retentate containers are each connected to a washing liquid container via a line, which is provided with a pump, through which washing liquid can be introduced into the retentate containers. In a preferred embodiment, the line used for this is also provided with regrowth traps. The hollow fiber membrane systems for gassing in bioreactor A and B can also be connected to the exhaust air safety vessel via lines, and preferably also with hollow fiber filter to separate any virus that may be present.
Durch das erfindungεgemäße Verfahren und die hierzu verwendbare er indungεgemäße Vorrichtung wird es er¬ möglicht, auf einfache Weiεe kontinuierlich Viruε oder virale Antigene, die zur Vakzinierung geeignet εind, kontinuierlich zu züchten, wobei dieεeε Verfahren auch von nur wenig ausgebildetem Personal durchgeführt werden kann und über Wochen hinweg zu einem homogenen Produkt führt. Daε erfindungεgemäße Verfahren und die Vorrichtung erεparen aufgrund der kompakten Geräte den Bau großer Laboratorien mit dazugehöriger Infraεtruktur. Daε Syεtem eignet εich auch zum Einbau in vorfabrizierte Laborcontainer, die fertig ausgerüεtet am beiεpiels- weiεe tropischen Standort aufgestellt, nach Anschluß an die Versorgung sofort mit der Produktion beginnen können. Auf diese Weise können auch ad hoc in Problem¬ gebieten bei Seuchenausbruch gegebenenfallε Vakzinen auε lokalεpezifiεchen Erregerεtämmen erzeugt werden. Dieεer Vorteil iεt inεofern von Bedeutung, alε Erreger zahlreicher tropiεcher Seuchen nicht in die Induεtrie- länder verbracht werden dürfen und damit eine zentrale Vakzine-Produktion für den Weltbedarf in einem Induεtrie- land unmöglich iεt (Rinderpeεt, afrikaniεche Schweine- peεt, Blue Tongue Viruε u.a.) . Daε erfindungεgemäß erhaltene Produkt iεt εyεtembedingt von hoher Qualität und Antigenität, frei von εchädlichen Nebenprodukten (niedrigmolekulare Subεtanzen oder Zellreεte) und kann mit nur niedrigen Koεten hergeεtellt werden. HThe method according to the invention and the device suitable for this purpose make it possible to continuously continuously and continuously grow viruε or viral antigens which are suitable for vaccination in a simple manner, which method can also be carried out by only little trained personnel and via Leads to a homogeneous product for weeks. Because of the compact devices, the method and the device according to the invention save the construction of large laboratories with the associated infrastructure. The system is also suitable for installation in prefabricated laboratory containers which, when fully equipped and set up at the tropical location, for example, can begin production immediately after being connected to the supply. In this way, vaccines can also be generated ad hoc in problem areas in the event of an outbreak of epidemics from locally specific pathogen strains. This advantage is important insofar as pathogens of numerous tropical epidemics must not be brought to the industrialized countries and thus central vaccine production for global needs in an industrialized country is impossible (beef pork, African pork pest, blue tongue virus etc.) ). The product obtained according to the invention is of high quality and antigenicity, free of harmful by-products (low-molecular substances or cell residues) and can be manufactured with only low costs. H
Die Figur zeigt eine erfindungεgemäß bevorzugte Vorrich¬ tung mit einem Bioreaktor A 2, in den geεteuert auε Nährmediumvorratsbehältern 4 über eine Leitung 6 , die mit einer Pumpe 8 ausgerüεtet iεt, Nährmedium in den Bioreaktor A 2 eingeführt wird.The figure shows a preferred device according to the invention with a bioreactor A 2, in which nutrient medium is introduced into the bioreactor A 2 into the nutrient medium storage containers 4 via a line 6, which is equipped with a pump 8.
Der Bioreaktor A 2 iεt hierbei weiter mit Hohlf sermem- branen 58 auεgerüεtet, über die Gaεe zur Regulierung deε pH oder p0~ eingeleitet werden können. Zuεätzlich iεt der Bioreaktor A 2 mit einem Magnetruhrer 62 und über eine Leitung 66, die ein Überdruckventil aufweist, mit dem Abluftεicherheitsgefäß 70 verbunden, wobei die Leitung einen Hohlfaserfilter 82 zum Zurückhalten even¬ tuell vorhandener Viren aufweiεt. Der Bioreaktor A 2 iεt über eine Leitung 10, die mit einem Photometer 12 und einer Rückwuchεfalle auεgerüεtet ist, mit dem Bioreaktor B 14 verbunden, in den über eine Leitung 18, die mit einer Pumpe 20 und einer Rückwuchεfalle auεge- rüstet ist, aus den Behältern für Erhaltungsmedium 16 Medium zum Bioreaktor B 14 zugeführt wird. Weiter ist der Bioreaktor B 14 mit einer Hohlfasermembran 60 ausgerüεtet, die zur Begaεung und mit zur Regulierung des pH und pO_ dient. Zusätzlich weiεt der Bioreaktor B 14 einen Magnetruhrer 64 zum Vermiεchen der Inhaltsstoffe auf. Eine Leitung 68 mit einem Überdruckventil dient zum Druckausgleich und führt in das Abluftsicherheits¬ gefäß 70 über einen Hohlfaserfilter 84, in dem eventuell vorhandene Viren zurückgehalten werden. Der Bioreaktor B 14 iεt mit dem erεten Retentatbehälter 26 der ersten Filtrationεanordnung über eine Leitung 22, die mit einem Photometer 24 und einer Rückwuchsfalle ausgerüstet iεt, verbunden, der wiederum über eine Leitung 78 mit einem daran vorhandenen Hohlfaεerfilter 86 mit dem Abluftεicherheitεgefäß 70 zum Druckausgleich verbunden iεt. Retentat auε dem erεten Retentatbehälter 26 wird über die Leitung 36, die mit einer Rückwuchεfalle ausgerüεtet iεt, abgezogen. Auε dem erεten Retentatbe¬ hälter 26 wird über eine Leitung 28, die mit einem Photometer 30 auεgerüεtet iεt, Suεpenεion in den erεten Filter 32 geleitet, und daraus das Retentat über die Leitung 34 in den ersten Retentatbehälter 26 zurückge¬ führt und das Filtrat über die Leitung 38, die mit einer Rückwuchsfalle ausgerüεtet iεt, in den zweiten Retentatbehälter 40 eingeleitet, der wiederum mit einer Druckauεgleichεleitung 80 verεehen ist, die über einen Hohlfaserfilter 86 in das Abluftsicherheitεgefäß 70 mündet. Weiter iεt der zweite Retentatbehälter 40 mit einer Leitung 50 versehen, die eine Rückwuchsfalle enthält und über die Viruskonzentrat abgezogen wird. Aus dem zweiten Retentatbehälter 40 wird über eine Leitung 42, die mit einer Pumpe 44 versehen ist, Sus- penεion in die Filtrationεeinrichtung 46 geführt, deren Retentat über die Leitung 48 in den zweiten Retentatbe¬ hälter 40 zurückgeführt wird, und deren Filtrat über die Leitung 54, die mit einer Rückflußfalle ausgestattet iεt, abgezogen wird. Aus den Waεchflüεsigkeitεbehältern 76 kann ebenfallε rechnergeε euert über Leitungen 72, die mit Rückflußfallen auεgestattet εind und mit einer Pumpe 74 verεehen εind, Waschflüεεigkeit in die Retentat¬ behälter 26,40 zugeleitet werden.The bioreactor A 2 is further equipped with hollow fiber membranes 58, via which gases for regulating the pH or pH can be introduced. In addition, the bioreactor A 2 is connected to the exhaust air safety vessel 70 with a magnetic stirrer 62 and via a line 66 which has a pressure relief valve, the line having a hollow fiber filter 82 for retaining any viruses that may be present. The bioreactor A 2 is connected to the bioreactor B 14 via a line 10, which is equipped with a photometer 12 and a regrowth trap, into which the bioreactor B 14 is connected via a line 18, which is equipped with a pump 20 and a regrowth trap Containers for maintenance medium 16 medium is fed to bioreactor B 14. Furthermore, the bioreactor B 14 is equipped with a hollow fiber membrane 60 which is used for gassing and for regulating the pH and pO_. In addition, the bioreactor B 14 has a magnetic stirrer 64 for mixing the ingredients. A line 68 with a pressure relief valve serves to equalize the pressure and leads into the exhaust air safety vessel 70 via a hollow fiber filter 84, in which viruses which may be present are retained. The bioreactor B 14 is connected to the first retentate container 26 of the first filtration arrangement via a line 22 which is equipped with a photometer 24 and a regrowth trap, which in turn is connected via a line 78 with a hollow fiber filter 86 present thereon Exhaust air safety vessel 70 is connected for pressure equalization. Retentate from the first retentate container 26 is withdrawn via line 36, which is equipped with a regrowth trap. From the first retentate container 26, a suspension 28 is fed into the first filter 32 via a line 28, which is equipped with a photometer 30, and from this the retentate is returned via line 34 to the first retentate container 26 and the filtrate is passed through the Line 38, which is equipped with a regrowth trap, is introduced into the second retentate container 40, which in turn is provided with a pressure equalization line 80 which opens into the exhaust air safety vessel 70 via a hollow fiber filter 86. Furthermore, the second retentate container 40 is provided with a line 50 which contains a regrowth trap and is drawn off via the virus concentrate. From the second retentate container 40, a suspension is fed via a line 42, which is provided with a pump 44, into the filtration device 46, the retentate of which is returned via line 48 to the second retentate container 40, and the filtrate of which is via the line 54, which is equipped with a reflux trap, is withdrawn. Computer-controlled washing lines can also be fed into the retentate containers 26, 40 from the liquid liquid containers 76 via lines 72, which are equipped with reflux traps and provided with a pump 74.
Die Steuerung der Zu- und Ableitungen zu und auε den verεchiedenen Teilen der Vorrichtung wird mit Hilfe eineε Prozeßrechnerε 56 durchgeführ .The control of the supply and discharge lines to and from the various parts of the device is carried out with the aid of a process computer 56.
Das folgende Beiεpiel εoll die Erfindung weiter er¬ läutern. B e i s p i e l 1The following example is intended to explain the invention further. Example 1
Herstellung von BHK 21 Klon 13 Zellen in kontinuier¬ licher Suspensionskultur.Production of BHK 21 clone 13 cells in a continuous suspension culture.
Die Anzucht der Zellen BHK 21 Klon 13 (ECACC Nr. 84100501 (zu beziehen durch Flow Laboratories Meckenheim) ) kann εowohl in Monolayer- alε auch in Rollerflaschen erfol¬ gen. Zur Inokulation müεεen im Fermenter mindestensThe cells BHK 21 clone 13 (ECACC No. 84100501 (to be obtained from Flow Laboratories Meckenheim)) can be grown both in monolayers and in roller bottles. At least inoculation must be done in the fermenter
4 8 x 10 Zellen pro ml vorhanden sein, um e n schnelles4 8 x 10 cells per ml should be present to ensure a fast
Anwachsen der Kultur zu gewährleiεten. Die Verdopplungs¬ zeit der BHK 21 Klon 13 Zellen betrug 24 Stunden. Mit der kontinuierlichen Zellgewinnung konnte nach 3 TagenTo guarantee growth of the culture. The doubling time of the BHK 21 clone 13 cells was 24 hours. With continuous cell recovery, after 3 days
5 begonnen werden, εobald die Zellzahl mindeεtenε 4x10 pro ml betrug. Die Mediumzugabe (9,538 g/1 MEM (minimal ess. media) mit Earl ' ε Spinnerεalzen, 2,2 g/1 NaHCO-., 10,0 ml/1 MEM-Vitaminlöεung, 10,0 ml/1 nicht eεsentielle Aminosäuren, 10,0 %/l foetaleε Kälberεeru , über Hohl¬ faserbündel, die direkt an den Bioreaktor angeεchlossen sind, steril filtriert) betrug zum Fermentationsbeginn 240 ml pro Tag, daε entspricht einer Verdünnung von 1:12,5 und konnte proportional zum Wachεtum bis 1500 ml pro Tag geεteigert werden (1:2) . Die Regulierung erfolgte über ein eingebauteε Photometer. Die Zellen wuchsen ohne Mikrocarrier. Das Reaktionεvolumen des Bioreaktors A betrug 4 1, daε Flüεεigkeitεvolumen 3 1. Die Reaktions¬ temperatur war 37°C, die Rührgeεchwindigkeit 150 U/min, der pH-Wert wurde auf 7,0 und der pO« auf 45% 0_ einer luftkalibrierten Löεung eingestellt. Der Gasgrundstrom betrug hierbei 4,5 l/irtin. Als Vordruck wurden 40 bar N-, 20 mbar 0„ und 30 mbar CO- sowie Luft ohne Regelung eingesetzt. Als Geräte wurden im Beispiel die Folgenden eingesetzt: 1-5 be started as soon as the cell count was at least 4x10 per ml. The medium addition (9.538 g / 1 MEM (minimal ess. Media) with Earl's spinner salts, 2.2 g / 1 NaHCO-., 10.0 ml / 1 MEM vitamin solution, 10.0 ml / 1 non-essential amino acids, 10.0% / l fetal calf serum, sterile filtered through hollow fiber bundles, which are directly connected to the bioreactor, was 240 ml per day at the start of fermentation, which corresponds to a dilution of 1: 12.5 and could be proportional to growth up to 1500 ml per day (1: 2). The regulation was carried out via a built-in photometer. The cells grew without a microcarrier. The reaction volume of bioreactor A was 4 liters, the liquid volume 3 liters. The reaction temperature was 37 ° C., the stirring speed was 150 rpm, the pH was 7.0 and the pO was 45% of an air-calibrated solution set. The basic gas flow was 4.5 l / irtin. 40 bar N, 20 mbar 0 "and 30 mbar CO and air without control were used as the upstream pressure. The following were used as devices in the example: 1-
Herεteller:Manufacturer:
Mediumzugabe: Pumpe IV BCC (By-pasε)Medium addition: Pump IV BCC (By-pasε)
Pumpe III BCCPump III BCC
Temperaturregelung: Modul TEM BCCTemperature control: TEM BCC module
Temperaturfühler: T 100 Lemo FGG. IBTemperature sensor: T 100 Lemo FGG. IB
Externeε Heizgerät: Kälte-Umwälz- HaakeExternal heater: cold circulation Haake
Bad-Thermoεtat KT 2Bath thermostat KT 2
Typ 001-3973Type 001-3973
Interneε Heizgerät: Temperierkorb BCC / IBT eigene EntwicklungInternal heater: temperature control basket BCC / IBT developed in-house
Rührung: Modul SPE BCC Interner Antrieb: im Gefäßdeckel feεt verankert Magnetrührεtab mit aufgeεetzten RührpropellernStirring: SPE BCC module Internal drive: Magnetic stirrer anchored firmly in the vessel lid with attached stirring propellers
Externer Antrieb: im Verεorgungεgehäuse feεt instal¬ lierter Magnetruhrer pH-Regulierung: Modul PH BCC Elektrode: pH-Elektrode Ingold autoklavierbar pH-Bereich 0 bis 12 Typ 465-36-90-K9 p02-Regulierung: Modul P0_ BCC Elektrode: p02-Elektrode Ingold autoklavierbar Typ 7455 / 322756702External drive: magnetic stirrer installed in the supply housing pH regulation: module PH BCC electrode: pH electrode Ingold autoclavable pH range 0 to 12 type 465-36-90-K9 p0 2 regulation: module P0_ BCC electrode: p0 2 - Ingold electrode autoclavable, type 7455/322756702
Gaεverεorgung: Verεion I: -DurchflußregelventileGas supply: Version I: flow control valves
Meßrohr FD-I/8-08-G-5/m Fischer & PortMeasuring tube FD-I / 8-08-G-5 / m Fischer & Port
-Magnetventile BCC-Magnetic valves BCC
Oliven 5 mm 0Olives 5 mm 0
-Gaεmiεchgefäß BCC-Gaεmiεchgefäß BCC
Verεion 2 -Maεεendurchflußregler MKSVersion 2 mass flow controller MKS
-Gaεmiεchgefäß BCC i£-Gaεmiεchgefäß BCC i £
Druckminderer: Typ Nr. D 142/03 Dräger Preßluft: kontinuierlicher LuftεtromPressure reducer: Type No. D 142/0 3 Dräger compressed air: continuous air flow
Hohlfaεer zur Belüftung: Accurel (R) PP Enka- embranaHollow fiber for ventilation: Accurel (R) PP Enkaembrana
Typ S 6/2 hydrophob AkzoType S 6/2 hydrophobic Akzo
Porengröße: max 0,54 μm Wicklung: εenkrecht zum FlüssigkeitsεpiegelPore size: max. 0.54 μm winding: perpendicular to the liquid level
Bedarf: 3 m/1 1 FlüεsigkeitRequirement: 3 m / 1 1 liquid
Niveauregulierung: Modul NIV BCC Elektroden: autoklavierbar BCCLevel control: module NIV BCC electrodes: autoclavable BCC
TeflonüberzugTeflon coating
Dichtebestimmung der Zell-Suspen- εion: Durchflußphotometer BCCDensity determination of the cell suspension: flow photometer BCC
B e i s p i e l 2Example: 2
Kontinuierliche Produktion von ND (Newcaεtle Disease) Vakzine auf BHK 21 ZellenContinuous production of ND (Newcaεtle Disease) vaccine on BHK 21 cells
Als Zellerhaltungsmedium wurde folgendes Medium ver¬ wendet:The following medium was used as the cell maintenance medium:
MEM (minimal eεε. media) mit Earl'ε Spinnersalzen 9,543 g/1 NaHC03 2,2 g/1MEM (minimal eεε. Media) with Earl'ε spinner salts 9.543 g / 1 NaHC0 3 2.2 g / 1
MEM-Vitaminlösung 10,0 ml/1 fötales Kälberserum 2,0 %/lMEM vitamin solution 10.0 ml / 1 fetal calf serum 2.0% / l
Sterilisation des Mediums erfolgt durch Filtration über direkt an den Bioreaktor angeschloεsene Hohlfaserbündel. iέThe medium is sterilized by filtration through hollow fiber bundles connected directly to the bioreactor. iέ
Virusinoculum für den Bioreaktor B (Virus)Virus inoculum for bioreactor B (virus)
Der Viruε ermenter wurde mit einer auf BHK MonolayerThe viruεermenter was with a BHK monolayer
7 gezüchteten Kultur, die m 100 ml 7,1 x 10 ID Viruε enthält, beimpft. Die Infektion der Monolayerkultur war zwei Tage vor der erεten Friεchzellenzugabe in den Bio¬ reaktor erfolgt. Der Zellraεen der Monolayerkultur durfte biε zu dieεem Zeitpunkt der Beimpfung deε Virus- fermenters noch keine Zellysiε aufweiεen. Die eigent¬ liche Freiεetzung der Viren εollte im Fermenter erfolgεn und zwar zum Zeitpunkt der Zellzugabe auε dem Zellreaktor (Reaktor A) .7 inoculated culture containing m 100 ml 7.1 x 10 ID Viruε inoculated. The infection of the monolayer culture had occurred two days before the first addition of fresh cells to the bioreactor. The cell line of the monolayer culture was not allowed to have any cell lyses at the time of inoculation of the virus fermenter. The actual release of the viruses should take place in the fermenter at the time of cell addition from the cell reactor (reactor A).
Zur Inokulation auε Bioreaktor A wurden 6 x 10 Zellen/ml in einer Durchflußmenge von 50 biε 100 ml pro StundeFor inoculation from bioreactor A, 6 × 10 cells / ml were used in a flow rate of 50 to 100 ml per hour
(je nach Zellwachεturn) von Bioreaktor A zu Reaktor B(depending on cell growth) from bioreactor A to reactor B
8 8 bei 3,0 x 10 biε 6,0 x 10 Zellen pro Stunde in Reak¬ tor B zugegeben. Daε Reaktorvolumen deε Bioreaktor B war ebenfalls 4 1, daε Flüεεigkeitsvolumen 3 1, die Temperatur 37°C, die Rührgeεchwindigkeit 150 Umdrehun¬ gen pro Minute, der pH-Wert wurde auf 7,0, der p0_-Wert auf 40,0 % O- in einer luftkalibrierten Löεung einge- εtellt. Der Gaεgrundεtrom betrug 4,0 1 pro Minute, als Vordruck wurde 40 mbar N2, 20 mbar 02, 30 mbar CO- und Luft ohne Regelung deε Vordruckε eingeεetzt. Die Mediumzugabe betrug 2 1 pro Tag und die Zeilzugäbe durchεchnittlich 1,2 x 10 Zellen pro Tag. Die Viruε-8 8 at 3.0 x 10 to 6.0 x 10 cells per hour in reactor B added. The reactor volume of the bioreactor B was likewise 4 1, the liquid volume 3 1, the temperature 37 ° C., the stirring speed 150 revolutions per minute, the pH value became 7.0, the p0_ value 40.0% - Set in an air calibrated solution. The gas flow rate was 4.0 l per minute, and 40 mbar N 2 , 20 mbar 0 2 , 30 mbar CO and air were used as the admission pressure without regulating the admission pressure. The medium addition was 2 1 per day and the line additions on average 1.2 x 10 cells per day. The Viruε-
7 10 ernte ergab 1,0 x 10 ID/ml = 2,0 x 10 ID/Tag, wobei die Ernte erfolgte, wenn 80% der Zellen zerεtört waren.7 10 harvest yielded 1.0 x 10 ID / ml = 2.0 x 10 ID / day, the harvest taking place when 80% of the cells had been destroyed.
Die Geräteauεεtattung des Bioreaktorε 2 entέprach der deε Bioreaktors I, jedoch mit folgenden Besonderheiten: ±1The equipment of the bioreactor 2 corresponded to that of the bioreactor I, but with the following special features: ± 1
Regulierung der Mediumzugabe in Verbindung mit dem Zellzulauf aus dem Reaktor I.Regulation of the medium addition in connection with the cell inflow from the reactor I.
Die im Reaktor I eingebaute Niveauregulierung öffnete zeitverzögert zwei Magnetventile:The level control built into reactor I opened two solenoid valves with a time delay:
Magnetventil 1: Zellfluß zu Reaktor II wurde ermöglicht Magnetventil 2: Magnetventil 1 war geεchloεεen; zum Freiεpülen der Zuleitung von Zellen wurde der Mediumzulauf für Reaktor II geöffnet; öffnungεzeit 5 biε 10 Sekunden.Solenoid valve 1: cell flow to reactor II was made possible. Solenoid valve 2: solenoid valve 1 was closed; to flush the supply line of cells, the medium inlet for reactor II was opened; opening time 5 to 10 seconds.
Abluftentεorgung:Exhaust air disposal:
FirmenCompanies
Fritte: 0,4 μm Porengröße BCCFrit: 0.4 μm pore size BCC
NaOH-Bad: 2%iges LaugenbadNaOH bath: 2% lye bath
Filter: Schwebstoffklasse S Enka Akzo Porengröße 0,05 μm hydrophobFilters: Particle size class S Enka Akzo pore size 0.05 μm hydrophobic
Bestimmung des cyto- pathiεchen Effekts zur Ermittlung des Viruε- titers : Durchfluß-Photometer BCCDetermination of the cytopathic effect for determining the virutiter: flow photometer BCC
Die Abscheidung von Zellresten erfolgte über Zellfilter mit 0,45 nm Porengröße (Akzo MD 020 P2N) in der Filtra¬ tionsanordnung 1 und über Virusfilter 50.000 NMGG (Akzo V 100 I) in der Filtrationsanordnung 2. Zahnradpumpen, ISCell residues were separated using cell filters with 0.45 nm pore size (Akzo MD 020 P2N) in filtration arrangement 1 and virus filters 50,000 NMGG (Akzo V 100 I) in filtration arrangement 2. Gear pumps, IS
Niveauregulierung und Magnetventile εtammten von der Firma BCC. Alε übergeordnete Koordination der Bioreak¬ toren und Filter wurde der induεtrielle Leitrechner FLSl mit angepaßter Software der Firma Münzer und Diehl verwendet. Daε Leitrechnerεyεtem kontrollierte durch Dichtebeεtimmung daε Zellwachεtum im Bioreaktor A und die Viruεvermehrung über den cytopathiεchen (lytiεchen Effekt in Bioreaktor B und εteuert den Nährbodenzulauf zu Bioreaktor A und Zellabgabe von Bioreaktor B optimal. Die variabel drehenden Pumpen deε Filtrationεsystems wurden über den Rechner so gesteuert, daß alles anfal¬ lende Material sofort aufgearbeitet wurde. Level control and solenoid valves came from the company BCC. As a higher-level coordination of the bioreactors and filters, the industrial control computer FLS1 with adapted software from the company Münzer and Diehl was used. The host computer system controlled the density of the cell growth in bioreactor A and the virus increase via the cytopathic (lytic effect in bioreactor B and controls the nutrient inflow to bioreactor A and cell delivery from bioreactor B) all material was immediately processed.

Claims

P a t e n t a n ε p r ü c h eP a t e n t a n ε p r u c h e
Verfahren zur Herεtellung von Viruε oder viralem Antigen durch Kultur auf tierischen Zellen in Nährmedium-Suspension, d a d u r c h g e k e n n z e i c h n e t , daß man in einem erεten Bioreaktor A die tierischen Zellen unter kontinuierlicher Nährmediumzugabe in Suεpen- εion kultiviert, nach Erreichen einer bestimmter. Zellkonzentration die Zellεuεpension in einen zweiten Bioreaktor B kontinuierlich überführt und dort als Nährboden für die Viruεzüchtung unter kontinuierlicher Zugabe eines Erhaltungεmediumε verwendet, nach Erzeugung einer beεtimmten Menge an Viruε in einer erεten Filtrationεanordnung Zellen und Zellreεte von der viruεhaltigen Nähr- mediumsuspenεion kontinuierlich abtrennt, daε Retentat verwirft, im Filtrat in einer zweiten Filtrationεanordnung Virus oder virales Antigen von der Nährmediumsuspenεion kontinuierlich trennt und dabei aufkonzentriert und daε Nährmedium verwirft.Process for the production of viru or viral antigen by culture on animal cells in nutrient medium suspension, so that the animal cells are cultivated in a first bioreactor A with continuous addition of nutrient medium in suspension, after a certain one has been reached. Cell concentration, the cell suspension is continuously transferred to a second bioreactor B and used there as a breeding ground for virus cultivation with the continuous addition of a preservation medium, after generation of a certain amount of virus in a first filtration arrangement, cells and cell residues are continuously separated from the virus-containing nutrient suspension because the retentate is separated , in the filtrate, in a second filtration arrangement, continuously separates virus or viral antigen from the nutrient medium suspension and thereby concentrates and discards the nutrient medium.
Verfahren nach Anεpruch 1 , d a d u r c h g e k e n n z e i c h n e t , daß die Zellen nach Erreichen einer Konzentration von mindestensMethod according to claim 1, so that the cells after reaching a concentration of at least
5 4x10 Zellen pro ml kontinuierlich in den Bioreaktor5 4x10 cells per ml continuously in the bioreactor
B überführt werden.B are transferred.
Verfahren nach Anspruch 1 oder 2, d a d u r c h g e k e n n z e i c h n e t , daß die Filtrationsschritte kontinuierlich erfolgen, sobald im Bioreaktor B mindestens 80 % der Zellen lyεiert sind. 2.0Method according to claim 1 or 2, characterized in that the filtration steps take place continuously as soon as at least 80% of the cells in the bioreactor B have been lysed. 2.0
4. Verfahren nach Anεpruch 1 oder 2, d a d u r c h g e k e n n z e i c h n e t , daß man bei der Züchtung von Viren, die keinen cytopathischen Effekt ausüben, nach einer vorbe¬ stimmten Zeit Zellsuspenεion auε dem Bioreaktor B abzieht, die Zellen durch Ultraschallbehandlung zerεtört und die Suεpenεion dann in die erεte Fil¬ trationεanordnung überführt.4. The method according to claim 1 or 2, characterized in that in the cultivation of viruses which do not have a cytopathic effect, cell suspension is removed from the bioreactor B after a predetermined time, the cells are destroyed by ultrasound treatment and the suspension is then separated into the first one Filtration arrangement transferred.
5. Verfahren nach einem der vorhergehenden Anεprüche, d a d u r c h g e k e n n z e i c h n e t , daß die Retentate der ersten Filtrationεanordnung und/oder zweiten Filtrationsanordnung mit einer Waschflüεsigkeit gewaεchen werden, die alε Filtrat in der zweiten Filtrationεanordnung dem Viruεpro- dukt entzogen und verworfen wird.5. The method as claimed in one of the preceding claims, namely that the retentates of the first filtration arrangement and / or second filtration arrangement are washed with a washing liquid, the filtrate in the second filtration arrangement is removed from the virus product and discarded.
6. Verfahren nach einem der Anεprüche 1 biε 5, d a d u r c h g e k e n n z e i c h n e t , daß die Konzentration der Zellen durch photometriεche Meεεung beεtimmt wird und der Zulauf von Bioreaktor6. The method according to any one of claims 1 to 5, that means that the concentration of the cells is determined by photometric measurement and the inflow of the bioreactor
A zu Bioreaktor B, vom Bioreaktor B in die erεte Filtrationεanordnung εowie die Zugabe von Waεchflüε- εigkeit zu den Retentaten der ersten und zweiten Filtrationεanordnung über einen Rechner geεteuert wird.A to bioreactor B, from bioreactor B into the first filtration arrangement, and the addition of liquid to the retentates of the first and second filtration arrangements is controlled by a computer.
7. Verfahren nach einem der Anεprüche 1 biε 6, d a d u r c h g e k e n n z e i c h n e t , daß in den Filtrationεanordnungen Hohlfaserbündel alε Filter im Überström verwendet werden.7. The method according to one of the claims 1 to 6, that is, that the hollow fiber bundles are used as filters in the overflow in the filtration arrangements.
8. Verfahren nach einem der vorhergehenden Anεprüche, d a d u r c h g e k e n n z e i c h n e t , daß daε Nährmedium vor der Zugabe zu Bioreaktor A, daε Erhaltungεmedium vor der Zugabe zu Bioreaktor B εowie die Waεchflüssigkeit vor Zugabe zu den Retentaten der Filtrationsanordnung tangential über Hohlfaserbündel im Überstrom εteril filtriert wird.8. The method according to any one of the preceding claims, characterized in that the nutrient medium before addition to bioreactor A, the preservation medium before addition to bioreactor B and the washing liquid before addition to the retentates of the filtration arrangement are filtered tangentially over hollow fiber bundles in an overflow εterile.
9. Verfahren nach einem der vorhergehenden Ansprüche, d a d u r c h g e k e n n z e i c h n e t , daß der pH und p02 in den Bioreaktoren über Begaεung über Hohlfaεermembranen gesteuert werden.9. The method according to any one of the preceding claims, characterized in that the pH and p0 2 in the bioreactors are controlled by gassing via hollow fiber membranes.
10. Verfahren nach Anεpruch 9, d a d u r c h g e k e n n z e i c h n e t , daß die zur pH- und p02-Regulierung verwendeten Gaεe in einem Gaεmiεchgefäß rechnergeεteuert gemiεcht und gemein- εam durch nur ein Hohlfaserεyεtem eingeleitet werden.10. The method according to claim 9, characterized in that the gases used for pH and p0 2 regulation are computer-controlled mixed in a gas tank and are introduced together by only one hollow fiber system.
11. Vorrichtung zur Durchführung deε Verfahrens nach Anspruch 1 biε 9, d a d u r c h g e k e n n z e i c h n e t , daß ein Bio¬ reaktor A (2) , der über eine Leitung (6) mit Nährmedium-Vorratεbehältern (4) und über eine Leitung (10) , welche mit einem Photometer (12) auεgerüεtet iεt, mit einem Bioreaktor B (14) verbunden iεt, der wiederum über eine Leitung (18) mit Erhaltungεmediumbehältern (16) und weiter über eine Leitung (22) mit einem erεten Retentatbehälter (26) verbunden iεt, auε dem die Zell-Virus-Suεpen- εion über eine Leitung (28) über eine erεte Filtra¬ tionseinrichtung (32) geleitet wird, deren Retentat über eine Leitung (34) zurück in den ersten Reten¬ tatbehälter (26) geführt wird und aus dem ersten Retentatbehälter (26) über eine Leitung (36) auε dem Syεtem abgezogen wird, und deren Filtrat über eine Leitung (38) in einen zweiten Retentatbehälter (40) geführt wird, der über eine Leitung (42) mit einer zweiten Filtrationseinrichtung (46) verbunden ist, deren Retentat über eine Leitung (48) zurück in den zweiten Retentatbehälter (40) geführt wird und aus dem Syεtem über eine Leitung (50) abge¬ zogen wird und deren Filtrat über eine Leitung (54) abgezogen wird, wobei daε Photometer (12) mit einem Rechner-geεteuerten Kontrollεyεtem (56) verbunden εind.11. Apparatus for performing the method according to claim 1 to 9, characterized in that a bio-reactor A (2), which via a line (6) with nutrient storage containers (4) and via a line (10), which with a Photometer (12) is equipped with a bioreactor B (14), which in turn is connected via a line (18) to preservation medium containers (16) and further via a line (22) to a first retentate container (26) the cell virus suspension is passed via a line (28) via a first filtration device (32), the retentate of which is led back via a line (34) into the first retentate container (26) and out of the first Retentate container (26) is withdrawn from the system via a line (36), and the filtrate is fed via a line (38) into a second retentate container (40) which is connected via a line (42) to a second filtration device (46) whose retentate is fed back into the second retentate container (40) via a line (48) and drawn off from the system via a line (50) and whose filtrate is drawn off via a line (54), the photometer ( 12) are connected to a computer-controlled control system (56).
12. Vorrichtung nach Anspruch 11, d a d u r c h g e k e n n z e i c h n e t , daß die Leitung (22) ebenfallε mit einem Photometer (24) ausgerüεtet iεt, daε mit dem Kontrollsyεtem (56) verbunden iεt.12. The apparatus of claim 11, so that the line (22) is also equipped with a photometer (24) that is connected to the control system (56).
13. Vorrichtung nach Anεpruch 11, d a d u r c h g e k e n n z e i c h n e t , daß die Lei¬ tung (22) mit einem Sammelgefä , daε eine Ultra¬ schallquelle enthält, und dahinter einer Pumpe ausgerüεtet iεt.13. Apparatus according to claim 11, so that the line (22) is equipped with a collecting vessel that contains an ultrasound source and a pump behind it.
14. Vorrichtung nach einem der Anεprüche 11 bis 13, d a d u r c h g e k e n n z e i c h n e t , daß die Leitungen (6, 18, 28, 42) mit Pumpen (8, 20, 30, 44) ausgerüstet sind.14. Device according to one of the claims 11 to 13, so that the lines (6, 18, 28, 42) are equipped with pumps (8, 20, 30, 44).
15. Vorrichtung nach einem der Ansprüche 11 bis 14, d a d u r c h g e k e n n z e i c h n e t , daß die Bioreaktoren A und B (2, 14) mit Hohlfaεer- membranen (58, 60) verεehen εind, über die der pH-Wert und pO„-Gehalt der Zellεuεpenεionen durch Zugabe von Gaεen reguliert werden. 2.315. Device according to one of claims 11 to 14, characterized in that the bioreactors A and B (2, 14) with hollow fiber membranes (58, 60) ver εind, through which the pH and pO "content of the Zellεuεpenεionen by Addition of gases can be regulated. 2.3
16. Vorrichtung nach einem der vorhergehenden Ansprüche, d a d u r c h g e k e n n z e i c h n e t , daß die Bioreaktoren A und B (2, 14) mit Magnetrüh¬ rern (62, 64) auεgeεtattet εind.16. Device according to one of the preceding claims, that the bioreactors A and B (2, 14) are equipped with magnetic stirrers (62, 64).
17. Vorrichtung nach einem der Ansprüche 11 biε 16, d a d u r c h g e k e n n z e i c h n e t , daß die Leitungen (6, 10, 18, 22, 36, 38, 50 und 54) mit Rückwuchεfallen ausgestattet sind.17. The device as claimed in one of claims 11 to 16, that the lines (6, 10, 18, 22, 36, 38, 50 and 54) are equipped with backwash traps.
18. Vorrichtung nach einem der Ansprüche 11 bis 17, d a d u r c h g e k e n n z e i c h n e t , daß die Bioreaktoren A und B (2, 14) mit Leitungen (66, 68) mit Überdruckventilen ausgeεtattet sind, die in ein Abluftsicherheitεgefäß (70) münden.18. Device according to one of claims 11 to 17, so that the bioreactors A and B (2, 14) are equipped with lines (66, 68) with pressure relief valves which open into an exhaust air safety vessel (70).
19. Vorrichtung nach einem der Anεprüche 11 biε 18, d a d u r c h g e k e n n z e i c h n e t , daß eine Leitung (72) mit einer Pumpe (74) vorhan¬ den iεt, über die in den erεten Retentatbehälter19. Device according to one of the claims 11 to 18, which also means that a line (72) with a pump (74) is present, via which into the first retentate container
(26) und den zweiten Retentatbehälter (40) eine Waεchflüssigkeit aus Waschflüsεigkeitεbehältern (76) eingeführt werden kann.(26) and the second retentate container (40), a washing liquid from washing liquid containers (76) can be introduced.
20. Vorrichtung nach einem der Anεprüche 11 biε 19, d a d u r c h g e k e n n z e i c h n e t , daß Druckauεgleichεleitungen (78, 80) an den Retentatbehältern (26, 40) vorhanden sind, die in daε Abluftsicherheitsgefäß (70) münden.20. Device according to one of the claims 11 to 19, so that the pressure equalization lines (78, 80) are present on the retentate containers (26, 40) which open into the exhaust air safety vessel (70).
21. Vorrichtung nach einem der Ansprüche 11 bis 20, d a d u r c h g e k e n n z e i c h n e t , daß die Zuleitungen (66, 68, 78, 80) zum Abluft¬ sicherheitεgefäß mit Hohlfaεerfiltern (82, 84, 86) auεgerüstet sind. 21. Device according to one of claims 11 to 20, so that the feed lines (66, 68, 78, 80) to the exhaust air safety vessel are equipped with hollow fiber filters (82, 84, 86).
EP19890903142 1988-03-11 1989-03-09 Process and device for producing viruses and viral antigens Withdrawn EP0357738A1 (en)

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