CA2071954C - Matrix with adherently bound cells and process for producing viruses/virus antigens - Google Patents
Matrix with adherently bound cells and process for producing viruses/virus antigensInfo
- Publication number
- CA2071954C CA2071954C CA 2071954 CA2071954A CA2071954C CA 2071954 C CA2071954 C CA 2071954C CA 2071954 CA2071954 CA 2071954 CA 2071954 A CA2071954 A CA 2071954A CA 2071954 C CA2071954 C CA 2071954C
- Authority
- CA
- Canada
- Prior art keywords
- virus
- cells
- antigen
- matrix
- bound
- 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.)
- Expired - Lifetime
Links
- 241000700605 Viruses Species 0.000 title claims abstract description 89
- 239000000427 antigen Substances 0.000 title claims abstract description 54
- 102000036639 antigens Human genes 0.000 title claims abstract description 54
- 108091007433 antigens Proteins 0.000 title claims abstract description 54
- 239000011159 matrix material Substances 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims description 25
- 210000004027 cell Anatomy 0.000 claims abstract description 76
- 210000003501 vero cell Anatomy 0.000 claims abstract description 11
- 238000004113 cell culture Methods 0.000 claims abstract description 9
- 230000001419 dependent effect Effects 0.000 claims abstract description 6
- 230000002779 inactivation Effects 0.000 claims abstract description 4
- 238000000746 purification Methods 0.000 claims abstract description 4
- 210000004102 animal cell Anatomy 0.000 claims abstract description 3
- 210000005260 human cell Anatomy 0.000 claims abstract description 3
- 230000010412 perfusion Effects 0.000 claims description 18
- 238000004519 manufacturing process Methods 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 12
- 238000000855 fermentation Methods 0.000 claims description 11
- 230000004151 fermentation Effects 0.000 claims description 11
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 239000012679 serum free medium Substances 0.000 claims description 5
- 241000712891 Arenavirus Species 0.000 claims description 3
- 229920002307 Dextran Polymers 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 241000710831 Flavivirus Species 0.000 claims description 2
- 239000001828 Gelatine Substances 0.000 claims description 2
- 229920000159 gelatin Polymers 0.000 claims description 2
- 235000019322 gelatine Nutrition 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000035899 viability Effects 0.000 claims description 2
- 208000004006 Tick-borne encephalitis Diseases 0.000 claims 4
- 229920002994 synthetic fiber Polymers 0.000 claims 1
- 239000001963 growth medium Substances 0.000 abstract description 6
- 239000000758 substrate Substances 0.000 abstract 3
- 241000710771 Tick-borne encephalitis virus Species 0.000 abstract 2
- 230000010261 cell growth Effects 0.000 abstract 1
- 206010014599 encephalitis Diseases 0.000 abstract 1
- 230000002101 lytic effect Effects 0.000 abstract 1
- 201000011475 meningoencephalitis Diseases 0.000 abstract 1
- 210000002966 serum Anatomy 0.000 abstract 1
- 208000015181 infectious disease Diseases 0.000 description 12
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 210000003837 chick embryo Anatomy 0.000 description 4
- 210000004556 brain Anatomy 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 241000867607 Chlorocebus sabaeus Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000001235 sensitizing effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- 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
- C12N2531/00—Microcarriers
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
- C12N2770/24151—Methods of production or purification of viral material
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Compounds Of Unknown Constitution (AREA)
- Peptides Or Proteins (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
A matrix or substrate bears adherently bound human or animal cells infected with a virus. It has been shown that surface dependent cells useful for virus multiplication remain adherently bound to a matrix even when infected with a virus, produce vi-rus antigens over a relatively long period of time and release them in the culture medium. In order to produce early Summer men-ingo-encephalitis (FSME) virus antigens by cultivating the FSME virus in cell cultures, a surface dependent permanent cell line, preferably the vero-cell line ATTC CCL 81, is inoculated with the FSME virus and the cells are bound to substrates and kept in a non lytic serum free system in conditions that ensure cellular growth, so that antigens are produced. The antigen-containing medi-um is then separated from the substrate bound cells and processed in a known manner by concentration, inactivation and purifi-cation until a galenically acceptable is obtained.
Description
e s ~
~ - 2071954 A matrix with cells adherently bound thereto, as well as a method of producing virus/virus antigen The invention relates to a matrix with human or animal cells adherently bound thereto, as well as to a method of producing virus/virus antigen, in particular tick-borne enceph~litis (TBE) virus antigen.
Infections with the virus of TBE have been observed in Europe since World War II. In Austria, in Southern Germany and in ~7-echoslovakia, several hundred patients are stationarily treated each year because of a TBE infection.
The TBE vlrus is assigned to the group of flaviviruses of the earlier serological group B of the arboviruses, which constitutes a genus of the ,o~aviridae virus group.
Inactivated v~cc~nes against one of the most important and most frequent ence~h~litis pathogens in man, the JAPA~eSe encerhAl itis B virus, have been available for some time. These inactivated v~cc~nes are recovered from the brains of infected mice, purified, and are a~h,l~.tledged to be safe and effective (Hoke et al., N. Engl. J. Med., 319, 608 (1988)).
Slnce 1976 a vacclne against TBE has been avallable and admitted by the health authorities. For pro~llc~ng this vA~ ne, the virus is grown in the - '_ 2071954 brains of infected baby mice, propagated in chick embryo cells, inactivated with formalin and subsequently subjected to an efflcient purification procedure (Heinz et al., J. Med. Vlrol., 6, 103 (1980)).
In the literature a number of possibllities for propagating arboviruses with a view to the possible production of a vaccine have been described. The method mostly used today is the inocculation of chick embryo fibroblasts with a TBE seed virus recovered from a mouse brain, and the cultivation of the inocculated cells. This method reguires a complicated purification of the antigen so as to remove complex, heterological biological material and so as to avoid a sensitizing effect in the persons to be v~ccln~ted when repeatedly administering v~cç~ne doses obtA~ne~ therefrom.
For providing chick embryo cells, one has to depart from SPF (= specific pathogen free) eggs. These SPF eggs must be subjected to a great number of time-consuming examinations to maintain their SPF status prior to each use.
Furthermore, chick embryo cell cultures exhibit only low generatlon numbers in cont~ culturlng, thereby llmltlng the size of batches, the primary culture was difficult to keep sterile and the ~uality of the primary cells with regard to virus propagation and antigen production was not constant.
These disadvantages exist not only with the methods of produelng TBE vlrus antlgen, but exlst qulte generally ln the produetlon of antlgens.
The lnventlon has as lts ob~eet to lmprove the produetlon of vlrus/vlrus antlgen, ln partleular of TBB
vlrus/vlrus antlgen sueh that the above-mentloned dlsad-vantages are ellmlnated, and to provlde a method of growlng vlrus/vlrus antlgen ln cell eultures whleh partleularly enables productlon on a large scale, whereln slmultaneously the eulture can be malntalned sterlle ln a simple manner.
Furthermore, the dellvery of undeslred eellular protelns lnto the eulture supernatant 18 to be mlnlmlzed.
For obtalnlng the above lndleated ob~eets, a matrlx 18 provlded, l.e. a earrler materlal, havlng human or anlmal eells adherently bound thereto, the eells belng lnfeeted wlth vlrus. The lnventlon 18 based on the flndlng that surfaee-dependent eells sultable for vlrus propagatlon remaln adherently bound to a matrlx even ln the vlrus-lnfeeted state, eontlnuously produee vlrus antlgen over relatlvely long perlods of tlme and dellver lt lnto the eulture medlum.
Aeeordlngly, ln one aspeet the present lnventlon provldes a serum-free eell eulture eontalnlng a matrlx wlth human or anlmal eells adherently bound thereto for the produetlon of flavlvlrus/vlrus antlgen or arena vlrus/vlrus antlgen, whlch cells are lnfected wlth TBB vlrus or arena vlrus.
In another aspeet the lnventlon provldes a method of produelng tlek-borne eneephalltls (TBE) vlrus antlgen uslng a matrlx as deflned above, eharaeterlzed ln that surfaee-~., ~ L 24242-492 dependent permanent cells lnoculated wlth the TBE vlrus are kept adherently bound to the matrlx ln a serum-free medium whlle thelr vlablllty 18 malntalned, 80 that there occurs antlgen formatlon ln the cells and antlgen dellvery lnto the medlum, and antlgen-contalnlng medlum 18 separated from the carrler-bound cells.
It 18 posslble to store the matrlx accordlng to the lnventlon loaded wlth lnfected cells for several days at a temperature of between 0C and 8C, l.e. under condltlons under whlch the cell metabollsm and thus the vlrus productlon are stopped. A matrlx thus stored may later on be used for produclng vlrus - 3a -207195~
antigen without any problems, by introducing it lnto a culture medium and ad~usting the respectlve growth conditions. The matrix according to the invention thus constitutes a starting culture capable of being produced in stock at constant quality and activity, whose sterile condition is easy to check and which may be used for virus antigen production at any time.
The binding of the antlgen-producing cells to the carrier furthermore allows for an extremely slmple handling of the virus infected cells that are ready for productlon. Thus it is, e.g., possible to carry out the virus/virus antigen production continuously in a perfusion reactor. Separation of the cells from the antigen-containing medium is substantially facilitated by their being bound to the matrix, whereby the matrix according to the invention simplifies the commercial-scale production of virus/virus antigen.
A preferred embodiment of the matrix according to the invention consists in that Vero cells ATCC CCL 81 are provided as the adherently bound cells, which preferably are provided for the production of TBE virus antigen and thus are infected wlth TBE vlrus.
The cells adherently bound to the matrix may, however, also be infected with flavlvirus or with arena virus.
Glass, cross-llnked dextran, gelatine or synthetic materlal has proved to be well suited as the materlal for the matrix, it being best if the matrix is formed as a microcarrier whose particle diameter preferably is in the range between lOO~m and 3000~m. These microcarriers may have a smooth surface or a porous structure.
A further suitable embodiment of the matrix accordlng to the invention is characterised in that between lx105 and 4x105 cells are adherently bound to its surface per cm thereof.
The invention also relates to a method of producing TBE virus antigen by using the matrix according to the invention, which is charaterised in that the surface-dependent permanent cells, preferably the Vero cells ATCC CCL 81, are inoculated with the TBE
virus, and the cells are kept adherently bound to a matrix in a serum-free medium while maintA1 n1 ng their viability so as to maintain an antigen formation and an antigen delivery into the medium, whereupon the antigen-contAi n~ ng medium is separated from the carrier-bound cells and is pr~ss~ to a galen1cAlly acceptable preparation in a known manner by ~-o~ce~tration, inactivation and purlficatlon.
The Vero cell llne A~CC CCL 81 i8 obt~1ne~ from the kidney t1~?ue of the green monkey (Cercopithec aethiops) and may be kept metAhol1c~11y actlve ln serum-free medium. For such a permanent cell line, a mother seed cell bank and a working seed cell bank are started and all the tests for contaminating substances are carried out. This permanent cell line thus can be prec1sely characterised not only with regard to its freedom from contaminating microorganisms, but also with regard to is growth behaviour, starting culture, propagation behaviour, and, once optimized, may be considered as constant.
With the method according to the invention, preferably Vero cells bound to microcarriers are used.
Thereby a high cell density can be obt~ne~ which, with the primary cell cultures hitherto used, could be obtA~ne~ neither in Roux flasks nor in suspension, and which enables a considerable increase in virus and virus antigen yields per fermentation volume.
An advantageous embodiment of the method according to the invention consists in that virus propagation and antigen formation are carried out in a continuously operated perfusion reactor for a period of at least 5 days, at a temperature of between 34 and 37C, wherein perfusion may be effected at a perfusion rate of from 0.3 to lO v/v/day. Furthermore, in the perfusion reactor there may be provided a cell density of from 2 x 109 to 2 8 101 cells per liter of fermentation volume, the latter in a fl~ 7e~ bed fermenter.
The virus propagation of the lnvention ln a perfusion culture enables a sub~ ial reduction of the - perfusion-rate dep~nAent - dwell time of the - ~ 2071954 virus and of the virus antigen in the medium as compared to that of the cultivatlon in batches. The shorter dwell time causes a much slighter thermal inactivation and thus results in a higher productivity of the method according to the invention. Thus, an antigen conce~tration of from 1 to 10 ~g/ml may be reached and maintA~ned in the perfusion medium.
With the method accordlng to the invention, optimum conditions for cultivation may be ad~usted in a simple manner. Furthermore, substantially less manipulations are required for its execution than with all the known methods, thus making handling of the infectious material safer and enabling continuous and quick working up of the virus and of the virus antigens from the culture medium.
In the following, the production of the virus inoculum, the culture of the cells for the virus and virus antigen production, and the virus and virus antigen production as such will be described in more detail.
1. Virus ~no~ um Cells (e.g. Vero ATCC CCL 81) are cultivated in Roller flasks at 37-C up to confluence snd infected with 1 ml of a seed virus su~rens~on. Starting from the 2nd day after infection, half the medlum i8 replaced with serum-free medlum each day. The medlum supernatants from the 4th to the 8th day contain ~ 2071954 2-5 x 107 p.f.u. per ml and are stored at -20C untll they are used as virus ~oc~lum.
2. Culturing the cells for the virus/virus antigen production Starting from the ATCC CCL 81 working seed cells stored in liquid nitrogen, these cells are propagated in tissue culture flasks until an amount of cells is obt~ne~ which allows ~nocl)lation of a fermenter.
Further culturing of the cells is effected in fermentation vessels at 37C, and as much surface as possible should be provided for adherence of the adheringly growing working seed cells. Such large surfaces are obt~ne~ by using Roller flasks of glass or of pol~s~y~ene of by using microcarriers (MC). MC of cross-linked dextran having a size of between 170~m and 250~m are best suited.
The MC loaded with seed cells are cultured at 37C
until a cell density of from lx105 - 4x105 cells per cm2 has been reAch~. Generally, this cell density is rç~c~e~ after six days. During culturing, the microcarrier is completely overgrown with cells, and finally individual microcarriers may grow together to groups via the cell sheet adhering to their sur~aces.
~ - 2071954 A matrix with cells adherently bound thereto, as well as a method of producing virus/virus antigen The invention relates to a matrix with human or animal cells adherently bound thereto, as well as to a method of producing virus/virus antigen, in particular tick-borne enceph~litis (TBE) virus antigen.
Infections with the virus of TBE have been observed in Europe since World War II. In Austria, in Southern Germany and in ~7-echoslovakia, several hundred patients are stationarily treated each year because of a TBE infection.
The TBE vlrus is assigned to the group of flaviviruses of the earlier serological group B of the arboviruses, which constitutes a genus of the ,o~aviridae virus group.
Inactivated v~cc~nes against one of the most important and most frequent ence~h~litis pathogens in man, the JAPA~eSe encerhAl itis B virus, have been available for some time. These inactivated v~cc~nes are recovered from the brains of infected mice, purified, and are a~h,l~.tledged to be safe and effective (Hoke et al., N. Engl. J. Med., 319, 608 (1988)).
Slnce 1976 a vacclne against TBE has been avallable and admitted by the health authorities. For pro~llc~ng this vA~ ne, the virus is grown in the - '_ 2071954 brains of infected baby mice, propagated in chick embryo cells, inactivated with formalin and subsequently subjected to an efflcient purification procedure (Heinz et al., J. Med. Vlrol., 6, 103 (1980)).
In the literature a number of possibllities for propagating arboviruses with a view to the possible production of a vaccine have been described. The method mostly used today is the inocculation of chick embryo fibroblasts with a TBE seed virus recovered from a mouse brain, and the cultivation of the inocculated cells. This method reguires a complicated purification of the antigen so as to remove complex, heterological biological material and so as to avoid a sensitizing effect in the persons to be v~ccln~ted when repeatedly administering v~cç~ne doses obtA~ne~ therefrom.
For providing chick embryo cells, one has to depart from SPF (= specific pathogen free) eggs. These SPF eggs must be subjected to a great number of time-consuming examinations to maintain their SPF status prior to each use.
Furthermore, chick embryo cell cultures exhibit only low generatlon numbers in cont~ culturlng, thereby llmltlng the size of batches, the primary culture was difficult to keep sterile and the ~uality of the primary cells with regard to virus propagation and antigen production was not constant.
These disadvantages exist not only with the methods of produelng TBE vlrus antlgen, but exlst qulte generally ln the produetlon of antlgens.
The lnventlon has as lts ob~eet to lmprove the produetlon of vlrus/vlrus antlgen, ln partleular of TBB
vlrus/vlrus antlgen sueh that the above-mentloned dlsad-vantages are ellmlnated, and to provlde a method of growlng vlrus/vlrus antlgen ln cell eultures whleh partleularly enables productlon on a large scale, whereln slmultaneously the eulture can be malntalned sterlle ln a simple manner.
Furthermore, the dellvery of undeslred eellular protelns lnto the eulture supernatant 18 to be mlnlmlzed.
For obtalnlng the above lndleated ob~eets, a matrlx 18 provlded, l.e. a earrler materlal, havlng human or anlmal eells adherently bound thereto, the eells belng lnfeeted wlth vlrus. The lnventlon 18 based on the flndlng that surfaee-dependent eells sultable for vlrus propagatlon remaln adherently bound to a matrlx even ln the vlrus-lnfeeted state, eontlnuously produee vlrus antlgen over relatlvely long perlods of tlme and dellver lt lnto the eulture medlum.
Aeeordlngly, ln one aspeet the present lnventlon provldes a serum-free eell eulture eontalnlng a matrlx wlth human or anlmal eells adherently bound thereto for the produetlon of flavlvlrus/vlrus antlgen or arena vlrus/vlrus antlgen, whlch cells are lnfected wlth TBB vlrus or arena vlrus.
In another aspeet the lnventlon provldes a method of produelng tlek-borne eneephalltls (TBE) vlrus antlgen uslng a matrlx as deflned above, eharaeterlzed ln that surfaee-~., ~ L 24242-492 dependent permanent cells lnoculated wlth the TBE vlrus are kept adherently bound to the matrlx ln a serum-free medium whlle thelr vlablllty 18 malntalned, 80 that there occurs antlgen formatlon ln the cells and antlgen dellvery lnto the medlum, and antlgen-contalnlng medlum 18 separated from the carrler-bound cells.
It 18 posslble to store the matrlx accordlng to the lnventlon loaded wlth lnfected cells for several days at a temperature of between 0C and 8C, l.e. under condltlons under whlch the cell metabollsm and thus the vlrus productlon are stopped. A matrlx thus stored may later on be used for produclng vlrus - 3a -207195~
antigen without any problems, by introducing it lnto a culture medium and ad~usting the respectlve growth conditions. The matrix according to the invention thus constitutes a starting culture capable of being produced in stock at constant quality and activity, whose sterile condition is easy to check and which may be used for virus antigen production at any time.
The binding of the antlgen-producing cells to the carrier furthermore allows for an extremely slmple handling of the virus infected cells that are ready for productlon. Thus it is, e.g., possible to carry out the virus/virus antigen production continuously in a perfusion reactor. Separation of the cells from the antigen-containing medium is substantially facilitated by their being bound to the matrix, whereby the matrix according to the invention simplifies the commercial-scale production of virus/virus antigen.
A preferred embodiment of the matrix according to the invention consists in that Vero cells ATCC CCL 81 are provided as the adherently bound cells, which preferably are provided for the production of TBE virus antigen and thus are infected wlth TBE vlrus.
The cells adherently bound to the matrix may, however, also be infected with flavlvirus or with arena virus.
Glass, cross-llnked dextran, gelatine or synthetic materlal has proved to be well suited as the materlal for the matrix, it being best if the matrix is formed as a microcarrier whose particle diameter preferably is in the range between lOO~m and 3000~m. These microcarriers may have a smooth surface or a porous structure.
A further suitable embodiment of the matrix accordlng to the invention is characterised in that between lx105 and 4x105 cells are adherently bound to its surface per cm thereof.
The invention also relates to a method of producing TBE virus antigen by using the matrix according to the invention, which is charaterised in that the surface-dependent permanent cells, preferably the Vero cells ATCC CCL 81, are inoculated with the TBE
virus, and the cells are kept adherently bound to a matrix in a serum-free medium while maintA1 n1 ng their viability so as to maintain an antigen formation and an antigen delivery into the medium, whereupon the antigen-contAi n~ ng medium is separated from the carrier-bound cells and is pr~ss~ to a galen1cAlly acceptable preparation in a known manner by ~-o~ce~tration, inactivation and purlficatlon.
The Vero cell llne A~CC CCL 81 i8 obt~1ne~ from the kidney t1~?ue of the green monkey (Cercopithec aethiops) and may be kept metAhol1c~11y actlve ln serum-free medium. For such a permanent cell line, a mother seed cell bank and a working seed cell bank are started and all the tests for contaminating substances are carried out. This permanent cell line thus can be prec1sely characterised not only with regard to its freedom from contaminating microorganisms, but also with regard to is growth behaviour, starting culture, propagation behaviour, and, once optimized, may be considered as constant.
With the method according to the invention, preferably Vero cells bound to microcarriers are used.
Thereby a high cell density can be obt~ne~ which, with the primary cell cultures hitherto used, could be obtA~ne~ neither in Roux flasks nor in suspension, and which enables a considerable increase in virus and virus antigen yields per fermentation volume.
An advantageous embodiment of the method according to the invention consists in that virus propagation and antigen formation are carried out in a continuously operated perfusion reactor for a period of at least 5 days, at a temperature of between 34 and 37C, wherein perfusion may be effected at a perfusion rate of from 0.3 to lO v/v/day. Furthermore, in the perfusion reactor there may be provided a cell density of from 2 x 109 to 2 8 101 cells per liter of fermentation volume, the latter in a fl~ 7e~ bed fermenter.
The virus propagation of the lnvention ln a perfusion culture enables a sub~ ial reduction of the - perfusion-rate dep~nAent - dwell time of the - ~ 2071954 virus and of the virus antigen in the medium as compared to that of the cultivatlon in batches. The shorter dwell time causes a much slighter thermal inactivation and thus results in a higher productivity of the method according to the invention. Thus, an antigen conce~tration of from 1 to 10 ~g/ml may be reached and maintA~ned in the perfusion medium.
With the method accordlng to the invention, optimum conditions for cultivation may be ad~usted in a simple manner. Furthermore, substantially less manipulations are required for its execution than with all the known methods, thus making handling of the infectious material safer and enabling continuous and quick working up of the virus and of the virus antigens from the culture medium.
In the following, the production of the virus inoculum, the culture of the cells for the virus and virus antigen production, and the virus and virus antigen production as such will be described in more detail.
1. Virus ~no~ um Cells (e.g. Vero ATCC CCL 81) are cultivated in Roller flasks at 37-C up to confluence snd infected with 1 ml of a seed virus su~rens~on. Starting from the 2nd day after infection, half the medlum i8 replaced with serum-free medlum each day. The medlum supernatants from the 4th to the 8th day contain ~ 2071954 2-5 x 107 p.f.u. per ml and are stored at -20C untll they are used as virus ~oc~lum.
2. Culturing the cells for the virus/virus antigen production Starting from the ATCC CCL 81 working seed cells stored in liquid nitrogen, these cells are propagated in tissue culture flasks until an amount of cells is obt~ne~ which allows ~nocl)lation of a fermenter.
Further culturing of the cells is effected in fermentation vessels at 37C, and as much surface as possible should be provided for adherence of the adheringly growing working seed cells. Such large surfaces are obt~ne~ by using Roller flasks of glass or of pol~s~y~ene of by using microcarriers (MC). MC of cross-linked dextran having a size of between 170~m and 250~m are best suited.
The MC loaded with seed cells are cultured at 37C
until a cell density of from lx105 - 4x105 cells per cm2 has been reAch~. Generally, this cell density is rç~c~e~ after six days. During culturing, the microcarrier is completely overgrown with cells, and finally individual microcarriers may grow together to groups via the cell sheet adhering to their sur~aces.
3. Virus/virus antigen production When the cell density indicated has been re~-h~, the cells bound to MCs are infected with the virus 1 ~oC~l um (1-0.01 pfu/cell, preferably 0.1 pfu/cell) to 207195~
produce the matrix accordlng to the invention. The matrix according to the invention may be stored for several days at a temperature of between 0C and 8C or it may immediately be used for the production of virus antigen.
For the production of antigen, the MCs loaded with lnfected cells are introduced into a perfusion reactor.
From this time of virus infection onwards, only serum-free medium will be used in the culture, which is pumped continuously through the perfusion reactor, while the cells cultured on the microcarriers are retained in the reactor by a retention arrangement.
Starting from the 2nd day post infection, virus antigen in a high concentration is present and may be continuously recovered therefrom for at least lO days.
The method according to the invention will be explained in more detail by way of the following Examples. Determination of the virus antigen was effected in all the Examples with an antigen-ELISA.
Example 1 Vero cells ATCC CCL 81 were cultured in a 6-l-fermenter on microcarriers (Cytodex 3 of Pharmacia) at 370C up to a cell n~ ~r of 2X106 per ml of culture medium (DMEM - D~llhPc~s Eagle Medium), and a) was infected with TBE virus (0.1 pfu/cell), and virus propagation was effected in batches.
_ g _ '-- 2071~54 Table 1 Days post infection virus/virus antigen ~g/ml 2 0.20 3 0.70 4 1.60 2.70 6 4.00 7 3.80 8 2.90 The productivity amounted to 4 mg of virus/virus antigen per 1 of fermentation volume.
b) was infected with TBE virus (0.1 pfu/cell) and culture medium was continuously perfused at O.S
volumes/fermenter volume/day.
Table 2 Days post infection virus/virus antigen ~g/ml 2 0.30 3 1.60 4 4.50 4.50 6 2.50 7 3.20 E
8 2.90 9 2.50 2.30 ~ 20719S4 The productivity amounted to 13.7 mg of virus/virus antigen per 1 of fermentation volume.
c) was infected with TBE virus (0.1 pfu/cell) and culture medium (DMEM) was continuously perfused at 1 volume/fermenter volume/day.
Table 3 Days post infection virus/virus antigen ~g/ml 2 0.45 3 1.40 4 2.00 2.00 6 1.70 7 1.60 8 1.10 9 1.10 The productivity amounted to 12.4 mg of virus/virus antigen per 1 of fermentation volume.
Example 2 Vero cells (ATCC CCL 81) were cultured in as 40-1-fermenter on microcarriers (Cytodex 3 of Pharmacla) at 37C up to a cell number of 2X108 cells/ml and, after infection with TBE virus (0.1 pfu/cell), contl n~o.-~l y perfused with medlum (DMEM) (0.33 vol/fermenter volume/day).
~ 20719~4 Table 4 Days post infectlon virus/virus antigen ~g/ml 2 1.60 3 3.50 4 5.00 4.30 6 4.00 7 2.90 8 2.70 9 2.10 2.00 The productivity amounted to 10.7 mg of virus/virus antigen per 1 of fermentation volume.
Example 3 Vero cells (ATCC CCL 81) were cultured in a 40-1-fermenter on microcarriers (Cytodex 3 of Pharmacla) at 37C up to a cell number of 3X106 cells /ml and continuously perfused with medium (DMEM) (1 vol/fermenter volume/day) after infection with TBE
virus (0.1 pfu/cell).
20719~4 Table 5 Days post infection Virus/virus antigen ~g/ml 2 1.10 3 3.80 4 3.90 3.00 6 2.30 7 2.20 8 2.00 9 3.15**) 2.30 **) The perfusion rate was reduced to 0.5 v/fermenter volume/day.
The productivity amounted to 21.7 mg of virus/virus antigen per l of fermentation volume.
Example 4 Vero cells (ATCC CCL 81) were cultured in a 150-l-fermenter on microcarriers (Cytodex 3 of Pharmacia) at 37C up to 2X106 cells/ml and continuously perfused with medium (DMEM) (O.33 vol/fermenter volume/day) after $nfection with TBE virus (0.1 pfu/cell).
` ~ 20719~4 Table 6 Days post infection Virus/Virus antigen ~g/ml 2 0.20 3 1.90 4 2.40 4.80 6 5.40 7 4.10 8 4.40 9 3.20 4.50 The productivity amounted to 14.7 mg of virus/virus antigen per 1 of fermentation volume.
produce the matrix accordlng to the invention. The matrix according to the invention may be stored for several days at a temperature of between 0C and 8C or it may immediately be used for the production of virus antigen.
For the production of antigen, the MCs loaded with lnfected cells are introduced into a perfusion reactor.
From this time of virus infection onwards, only serum-free medium will be used in the culture, which is pumped continuously through the perfusion reactor, while the cells cultured on the microcarriers are retained in the reactor by a retention arrangement.
Starting from the 2nd day post infection, virus antigen in a high concentration is present and may be continuously recovered therefrom for at least lO days.
The method according to the invention will be explained in more detail by way of the following Examples. Determination of the virus antigen was effected in all the Examples with an antigen-ELISA.
Example 1 Vero cells ATCC CCL 81 were cultured in a 6-l-fermenter on microcarriers (Cytodex 3 of Pharmacia) at 370C up to a cell n~ ~r of 2X106 per ml of culture medium (DMEM - D~llhPc~s Eagle Medium), and a) was infected with TBE virus (0.1 pfu/cell), and virus propagation was effected in batches.
_ g _ '-- 2071~54 Table 1 Days post infection virus/virus antigen ~g/ml 2 0.20 3 0.70 4 1.60 2.70 6 4.00 7 3.80 8 2.90 The productivity amounted to 4 mg of virus/virus antigen per 1 of fermentation volume.
b) was infected with TBE virus (0.1 pfu/cell) and culture medium was continuously perfused at O.S
volumes/fermenter volume/day.
Table 2 Days post infection virus/virus antigen ~g/ml 2 0.30 3 1.60 4 4.50 4.50 6 2.50 7 3.20 E
8 2.90 9 2.50 2.30 ~ 20719S4 The productivity amounted to 13.7 mg of virus/virus antigen per 1 of fermentation volume.
c) was infected with TBE virus (0.1 pfu/cell) and culture medium (DMEM) was continuously perfused at 1 volume/fermenter volume/day.
Table 3 Days post infection virus/virus antigen ~g/ml 2 0.45 3 1.40 4 2.00 2.00 6 1.70 7 1.60 8 1.10 9 1.10 The productivity amounted to 12.4 mg of virus/virus antigen per 1 of fermentation volume.
Example 2 Vero cells (ATCC CCL 81) were cultured in as 40-1-fermenter on microcarriers (Cytodex 3 of Pharmacla) at 37C up to a cell number of 2X108 cells/ml and, after infection with TBE virus (0.1 pfu/cell), contl n~o.-~l y perfused with medlum (DMEM) (0.33 vol/fermenter volume/day).
~ 20719~4 Table 4 Days post infectlon virus/virus antigen ~g/ml 2 1.60 3 3.50 4 5.00 4.30 6 4.00 7 2.90 8 2.70 9 2.10 2.00 The productivity amounted to 10.7 mg of virus/virus antigen per 1 of fermentation volume.
Example 3 Vero cells (ATCC CCL 81) were cultured in a 40-1-fermenter on microcarriers (Cytodex 3 of Pharmacla) at 37C up to a cell number of 3X106 cells /ml and continuously perfused with medium (DMEM) (1 vol/fermenter volume/day) after infection with TBE
virus (0.1 pfu/cell).
20719~4 Table 5 Days post infection Virus/virus antigen ~g/ml 2 1.10 3 3.80 4 3.90 3.00 6 2.30 7 2.20 8 2.00 9 3.15**) 2.30 **) The perfusion rate was reduced to 0.5 v/fermenter volume/day.
The productivity amounted to 21.7 mg of virus/virus antigen per l of fermentation volume.
Example 4 Vero cells (ATCC CCL 81) were cultured in a 150-l-fermenter on microcarriers (Cytodex 3 of Pharmacia) at 37C up to 2X106 cells/ml and continuously perfused with medium (DMEM) (O.33 vol/fermenter volume/day) after $nfection with TBE virus (0.1 pfu/cell).
` ~ 20719~4 Table 6 Days post infection Virus/Virus antigen ~g/ml 2 0.20 3 1.90 4 2.40 4.80 6 5.40 7 4.10 8 4.40 9 3.20 4.50 The productivity amounted to 14.7 mg of virus/virus antigen per 1 of fermentation volume.
Claims (16)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A serum-free cell culture containing a matrix with human or animal cells adherently bound thereto for the production of flavivirus/virus antigen or arena virus/virus antigen, which cells are infected with TBE virus or arena virus.
2. A cell culture according to claim 1, characterized in that the matrix is comprised of glass, cross-linked dextran, gelatine or synthetic material.
3. A cell culture according to claim 2, characterized in that the matrix is a microcarrier.
4. A cell culture according to claim 1, 2 or 3 charac-terized in that Vero cells ATCC CCL 81 are the adherently bound cells.
5. A cell culture according to claim 1, 2 or 3, characterized in that between 1x105 and 4x105 cells are adherently bound to the surface of the matrix per cm2 thereof.
6. A cell culture according to claim 4, characterized in that between 1x105 and 4x105 cells are adherently bound to the surface of the matrix per cm2 thereof.
7. A method of producing tick-borne encephalitis (TBE) virus antigen using a matrix characterized in that surface-dependent permanent cells inoculated with the TBE virus are kept adherently bound to the matrix in a serum-free medium while their viability is maintained, so that there occurs antigen formation in the cells and antigen delivery into the medium, and antigen-containing medium is separated from the carrier-bound cells.
8. A method according to claim 7, characterized in that the cells bound to the matrix are Vero cells ATCC CCL 81.
9. A method according to claim 7, characterized in that virus propagation and antigen formation are carried out in a continuously operated perfusion reactor for a period of at least 5 days at a temperature of between 34 and 37°C.
10. A method according to claim 8, characterized in that virus propagation and antigen formation are carried out in a continuously operated perfusion reactor for a period of at least 5 days at a temperature of between 34 and 37°C.
11. A method according to claim 9, characterized in that the perfusion 18 carried out at a perfusion rate of from 0.3 to 10 v/v/day.
12. A method according to claim 10, characterized in that the perfusion is carried out at a perfusion rate of from 0.3 to 10 v/v/day.
13. A method according to claim 9, characterized in that in the perfusion reactor a cell density of from 2x109 to 2x1010 cells per liter of fermentation volume is provided, the latter in a fluidized bed fermenter.
14. A method according to claim 10, characterized in that in the perfusion reactor a cell density of from 2x109 to 2x1010 cells per liter of fermentation volume is provided, the latter in a fluidized bed fermenter.
15. A method according to any one of claims 7 to 14, characterized in that a virus/virus antigen concentration of from 1 to 10 µg/ml is maintained in the medium.
16. A method according to any one of claims 7 to 14, characterized in that the separated antigen-containing medium is processed to a galenically acceptable preparation by concentration, inactivation and purification.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT2928/89A AT393356B (en) | 1989-12-22 | 1989-12-22 | METHOD FOR PRODUCING TBE VIRUS ANTIGES |
| ATA2928/89 | 1989-12-22 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2071954A1 CA2071954A1 (en) | 1991-06-23 |
| CA2071954C true CA2071954C (en) | 1996-06-18 |
Family
ID=3542524
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2071954 Expired - Lifetime CA2071954C (en) | 1989-12-22 | 1990-12-21 | Matrix with adherently bound cells and process for producing viruses/virus antigens |
Country Status (16)
| Country | Link |
|---|---|
| EP (1) | EP0506714B1 (en) |
| JP (1) | JP2633391B2 (en) |
| AT (2) | AT393356B (en) |
| CA (1) | CA2071954C (en) |
| CZ (1) | CZ281804B6 (en) |
| DE (1) | DE59007659D1 (en) |
| DK (1) | DK0506714T3 (en) |
| ES (1) | ES2067916T3 (en) |
| FI (1) | FI98377C (en) |
| HR (1) | HRP921354A2 (en) |
| HU (1) | HU213886B (en) |
| NO (1) | NO310305B1 (en) |
| RU (1) | RU2082757C1 (en) |
| SK (1) | SK659090A3 (en) |
| WO (1) | WO1991009935A1 (en) |
| YU (1) | YU242390A (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE542891T1 (en) * | 1994-11-10 | 2012-02-15 | Baxter Healthcare Sa | METHOD FOR PRODUCING BIOLOGICAL PRODUCTS IN PROTEIN-FREE CULTURE |
| FR2737412B1 (en) * | 1995-08-01 | 1997-10-24 | Pasteur Merieux Serums Vacc | PROCESS FOR PRODUCING A VACCINE AGAINST JAPANESE ENCEPHALITIS VIRUS AND VACCINE OBTAINED THEREBY |
| CA2228128C (en) * | 1995-08-01 | 2011-03-29 | Pasteur Merieux Serums & Vaccins | Method for industrially producing a japanese encephalitis vaccine, and resulting vaccine |
| KR20010032699A (en) * | 1998-10-05 | 2001-04-25 | 무따이 마사히꼬 | Enhanced immunogen for inactivated vaccine for infection with japanese encephalitis viruses and process for producing the same |
| AT409379B (en) | 1999-06-02 | 2002-07-25 | Baxter Ag | MEDIUM FOR PROTEIN- AND SERUM-FREE CELL CULTURE |
| US6951752B2 (en) | 2001-12-10 | 2005-10-04 | Bexter Healthcare S.A. | Method for large scale production of virus antigen |
| US6855535B2 (en) * | 2001-12-10 | 2005-02-15 | Baxter Healthcare S.A. | Method of large scale production of Hepatitis A virus |
| JP2007068401A (en) * | 2003-08-07 | 2007-03-22 | Chemo Sero Therapeut Res Inst | West nile virus vaccine |
| US9359629B2 (en) * | 2007-12-27 | 2016-06-07 | Baxalta Incorporated | Cell culture processes |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AT358167B (en) * | 1978-12-22 | 1980-08-25 | Immuno Ag | METHOD FOR PRODUCING EARLY SUMMER MENINGOENZEPHALITIS VIRUS (FSME VIRUS) VACCINES |
| SE445116B (en) * | 1979-09-12 | 1986-06-02 | Pharmacia Fine Chemicals Ab | MAKE CULTURE CELLS ON MICROBATORS WITH FIBRONECTINE LAYERS |
| NO161446C (en) * | 1981-03-13 | 1989-08-16 | Damon Biotech Inc | PROCEDURE FOR CULTING CELLS RELATED TO ANCHORING. |
| SE8103138L (en) * | 1981-05-19 | 1982-11-20 | Pharmacia Fine Chemicals Ab | MICROBATCHERS FOR CULTURE OF ANCHORING-DEPENDENT CELLS |
| CA1206900A (en) * | 1981-12-21 | 1986-07-02 | Raymond L. Downs | Hollow glass shell microcarrier for growth of cell cultures, and method of shell manufacture |
| AT385203B (en) * | 1985-04-26 | 1988-03-10 | Immuno Ag | METHOD FOR PRODUCING AN EARLY SUMMER MENINGOCEPHALITIS VIRUS (TBE VIRUS) VACCINE |
| DD274336A3 (en) * | 1985-10-03 | 1989-12-20 | Npo Biolar | Process for obtaining a microcarrier substance for cell cultivation |
-
1989
- 1989-12-22 AT AT2928/89A patent/AT393356B/en not_active IP Right Cessation
-
1990
- 1990-12-21 ES ES91900666T patent/ES2067916T3/en not_active Expired - Lifetime
- 1990-12-21 CZ CS906590A patent/CZ281804B6/en not_active IP Right Cessation
- 1990-12-21 AT AT91900666T patent/ATE113652T1/en not_active IP Right Cessation
- 1990-12-21 YU YU242390A patent/YU242390A/en unknown
- 1990-12-21 SK SK6590-90A patent/SK659090A3/en not_active IP Right Cessation
- 1990-12-21 HU HU9202009A patent/HU213886B/en unknown
- 1990-12-21 RU SU905052769A patent/RU2082757C1/en not_active IP Right Cessation
- 1990-12-21 CA CA 2071954 patent/CA2071954C/en not_active Expired - Lifetime
- 1990-12-21 DE DE59007659T patent/DE59007659D1/en not_active Expired - Fee Related
- 1990-12-21 EP EP19910900666 patent/EP0506714B1/en not_active Expired - Lifetime
- 1990-12-21 WO PCT/AT1990/000128 patent/WO1991009935A1/en active IP Right Grant
- 1990-12-21 JP JP50113291A patent/JP2633391B2/en not_active Expired - Fee Related
- 1990-12-21 DK DK91900666T patent/DK0506714T3/en not_active Application Discontinuation
-
1992
- 1992-06-18 FI FI922851A patent/FI98377C/en active
- 1992-06-19 NO NO19922422A patent/NO310305B1/en not_active IP Right Cessation
- 1992-11-25 HR HRP921354 patent/HRP921354A2/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| CZ281804B6 (en) | 1997-02-12 |
| FI922851A0 (en) | 1992-06-18 |
| FI98377B (en) | 1997-02-28 |
| WO1991009935A1 (en) | 1991-07-11 |
| ATE113652T1 (en) | 1994-11-15 |
| FI922851A (en) | 1992-06-18 |
| EP0506714A1 (en) | 1992-10-07 |
| SK279236B6 (en) | 1998-08-05 |
| CA2071954A1 (en) | 1991-06-23 |
| CZ659090A3 (en) | 1996-11-13 |
| SK659090A3 (en) | 1998-08-05 |
| FI98377C (en) | 1997-06-10 |
| JP2633391B2 (en) | 1997-07-23 |
| HRP921354A2 (en) | 1996-02-29 |
| YU242390A (en) | 1993-05-28 |
| NO922422L (en) | 1992-08-17 |
| EP0506714B1 (en) | 1994-11-02 |
| AT393356B (en) | 1991-10-10 |
| NO310305B1 (en) | 2001-06-18 |
| HU213886B (en) | 1997-11-28 |
| ES2067916T3 (en) | 1995-04-01 |
| JPH05502581A (en) | 1993-05-13 |
| NO922422D0 (en) | 1992-06-19 |
| RU2082757C1 (en) | 1997-06-27 |
| HUT65410A (en) | 1994-06-28 |
| HU9202009D0 (en) | 1992-10-28 |
| ATA292889A (en) | 1991-03-15 |
| DE59007659D1 (en) | 1994-12-08 |
| DK0506714T3 (en) | 1995-04-18 |
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