NO310305B1 - Matrix with adherently bound cells, as well as the method of producing late-summer meningoencephalitis (FSME) virus antigen - Google Patents
Matrix with adherently bound cells, as well as the method of producing late-summer meningoencephalitis (FSME) virus antigen Download PDFInfo
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- NO310305B1 NO310305B1 NO19922422A NO922422A NO310305B1 NO 310305 B1 NO310305 B1 NO 310305B1 NO 19922422 A NO19922422 A NO 19922422A NO 922422 A NO922422 A NO 922422A NO 310305 B1 NO310305 B1 NO 310305B1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2531/00—Microcarriers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
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- 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
Abstract
Description
Foreliggende oppfinnelsen vedrører en serumfri cellekultur som er særpreget ved at den inneholder en matrise med adherent bundede humane eller animalske celler, for produksjon av Flavi-virus/virusantigen eller Arena-virus/virusantigen, hvilke celler er infisert med FSME-virus eller Arena-virus. Oppfinnelsen omfatter også en fremgangsmåte ved fremstilling av forsommer-meningoencefalitis-(FSME)-virus-antigen under anvendelse av den ovennevnte matrise slik som angitt i krav 6. The present invention relates to a serum-free cell culture which is characterized by the fact that it contains a matrix of adherently bound human or animal cells, for the production of Flavi virus/virus antigen or Arena virus/virus antigen, which cells are infected with FSME virus or Arena virus . The invention also includes a method for the production of early summer meningoencephalitis (FSME) virus antigen using the above-mentioned matrix as stated in claim 6.
Infeksjoner med virus fra forsommer-meningoencefalitis (FSME) har vært iakttatt i Europa siden 2. verdenskrig. I Østerrike, Sydtyskland og i Tsjekoslovakia behandles stasjonært hvert år flere hundre pasienter for en FSME-infeksjon. Infections with early summer meningoencephalitis (FSME) virus have been observed in Europe since World War II. In Austria, southern Germany and Czechoslovakia, several hundred patients are treated on an inpatient basis every year for an FSME infection.
FSME-viruset tilhører familien Flavivirus, den tidligere serologiske gruppe B av Arbovirus, som er en slekt fra virusfamilien Togaviridae. The FSME virus belongs to the family Flavivirus, the former serological group B of Arbovirus, which is a genus of the virus family Togaviridae.
Mot en av de viktigste og hyppigste encefalitis-sykdoms-kilder hos mennesket, det japanske encefalitis-B-virus, har det i lengre tid vært tilgjengelig vaksiner. Disse vaksiner utvinnes fra hjernen av infiserte mus, renses, og de er anerkjent som sikre og virksomme (Hoke et al., N.Engl.J.-Med., 319, 608 (1988). Vaccines have been available for a long time against one of the most important and frequent sources of encephalitis disease in humans, the Japanese encephalitis B virus. These vaccines are extracted from the brains of infected mice, purified, and are recognized as safe and effective (Hoke et al., N.Engl.J.-Med., 319, 608 (1988).
Siden 1976 er det tilgjengelig og godkjent fra helsemyn-dighetene en vaksine mot FSME. For fremstilling av denne vaksine dyrkes virus i hjernen av infiserte baby-mus, formeres i høneembryonalceller, inaktiveres med formalin og underkastes derefter en effektiv renseprosess (Heinz et al., J.Med.Virol., 6, 103 (1980)). Since 1976, a vaccine against FSME has been available and approved by the health authorities. For the production of this vaccine, virus is grown in the brains of infected baby mice, propagated in chicken embryonic cells, inactivated with formalin and then subjected to an efficient purification process (Heinz et al., J.Med.Virol., 6, 103 (1980)).
I litteraturen er det beskrevet en rekke muligheter for formering av arbovirus med hensyn til en mulig vaksinefrem-stilling. Den idag mest anvendte metode er podning av høneembryonalfibroblaster med en fra musenjerne frembragt FSME-modervirus, og kultivering av de podede celler. Denne metode krever en kostbar rensning av antigenet for å fjerne komplekst, heterologt biologisk materiale, og for å unngå ved gjentatt administrasjon av derav frembragte vaksine-doser en sensitiverende effekt hos de vaksinerte. In the literature, a number of possibilities for propagation of arboviruses are described with regard to a possible vaccine production. The method most used today is inoculation of chicken embryonic fibroblasts with an FSME mother virus produced from mouse brains, and cultivation of the inoculated cells. This method requires an expensive purification of the antigen to remove complex, heterologous biological material, and to avoid a sensitizing effect in the vaccinated by repeated administration of the resulting vaccine doses.
Ved fremstilling av høneembryonalceller må man gå ut fra SPF(= specific pathogen free)-egg. Disse SPF-egg må for opprettholdelse av deres SPF-status underkastes, før hvert bruk, et stort antall langvarige undersøkelser. When producing hen embryonic cells, SPF (= specific pathogen free) eggs must be used as a starting point. In order to maintain their SPF status, these SPF eggs must be subjected, before each use, to a large number of long-term examinations.
Dessuten viser høneembryonalcellekulturer bare små genera-sjonstall ved videredyrkning, hvorved det settes grenser for chargestørrelsen, en vanskelig sterilholdelse av primærkulturdyrkningen og ingen konstant kvalitet av primærcellene med hensyn til virusformering og antigenproduksjon. In addition, hen embryonic cell cultures only show small generation numbers during further cultivation, whereby limits are set for the batch size, a difficult sterile maintenance of the primary culture cultivation and no constant quality of the primary cells with regard to virus propagation and antigen production.
Disse ulemper foreligger ikke bare ved fremgangsmåter ved produksjon av FSME-virusantigen, men helt generelt ved antigenfremstillingen. These disadvantages exist not only in methods for the production of FSME virus antigen, but in general in antigen production.
Oppgaven for foreliggende oppfinnelse består i å fremskaffe en serumfri cellekultur for å forbedre produksjonen av FSME-virus/virusantigen, slik at de ovenfor nevnte ulemper fjernes og en fremgangsmåte ved dyrkning av virus/virus-antigen i cellekulturer tilveiebringes, som spesielt muliggjør produksjon i stor teknisk målestokk, hvorved kulturen på enkel måte samtidig kan holdes steril. Dessuten skal avgivelsen av uønkskede celleproteiner til kultur-resten minimeres. The task of the present invention is to provide a serum-free cell culture to improve the production of FSME virus/virus antigen, so that the above-mentioned disadvantages are removed and a method for growing virus/virus antigen in cell cultures is provided, which in particular enables production on a large scale technical scale, whereby the culture can be kept sterile at the same time in a simple way. Furthermore, the release of unwanted cell proteins to the culture residue must be minimized.
For å løse denne oppgave tilveiebringes en matrise, dvs. et bærermateriale med adherent bundede humane eller animalske celler, hvorved cellene er infisert med virus. Oppfinnelsen beror på den erkjennelse at overflateavhengige celler som er egnet til virusformering, selv i virusinfisert tilstand forblir adherent bundet til en matrise, produserer kontinuerlig over relativt lang tid virusantigen, og avgir dette til kulturmediet. To solve this task, a matrix is provided, i.e. a carrier material with adherently bound human or animal cells, whereby the cells are infected with viruses. The invention is based on the recognition that surface-dependent cells which are suitable for virus propagation, even in a virus-infected state, remain adherently bound to a matrix, continuously produce virus antigen over a relatively long time, and release this into the culture medium.
Det er mulig å oppbevare den med infiserte celler anrikede matrise ifølge oppfinnelsen i noen dager ved en temperatur på mellom 0°C og 8°C, altså under betingelser hvor celle-metabolismen og dermed virusproduksjonen er forhindret. En således oppbevart matrise kan i det følgende uten problemer anvendes til virusantigenproduksjon ved innføring i et kulturmedium og innstilling av de respektive kultiverings-betingelser. Den serumfri cellekultur med matrisen ifølge oppfinnelsen utgjør således en utgangskultur som kan produseres for lagring med konstant kvalitet og aktivitet, hvis tilstand lett kan testes med hensyn til sterilitet, og som til en hver tid kan trekkes frem til virusantigenpro-duks jon . It is possible to store the matrix enriched with infected cells according to the invention for a few days at a temperature of between 0°C and 8°C, i.e. under conditions where cell metabolism and thus virus production is prevented. A matrix stored in this way can in the following be used without problems for virus antigen production by introducing it into a culture medium and setting the respective cultivation conditions. The serum-free cell culture with the matrix according to the invention thus constitutes a starting culture which can be produced for storage with constant quality and activity, the condition of which can be easily tested with regard to sterility, and which can be drawn up at any time for virus antigen production.
Bindingen av de antigenproduserende celler til bæreren muliggjør dessuten en meget enkel håndtering av de virusin-fiserte og produksjonsklare celler. Således er det f.eks. mulig å gjennomføre virus/virusantigenproduksjonen kontinuerlig i en perfusjonsreaktor. Separasjonen av cellene fra antigenholdig medium blir vesentlig lettere på grunn av bindingen til matrisen, idet matrisen forenkler produksjonen av virus/virusantigen i stor teknisk målestokk. The binding of the antigen-producing cells to the carrier also enables very simple handling of the virus-infected and production-ready cells. Thus, it is e.g. possible to carry out virus/virus antigen production continuously in a perfusion reactor. The separation of the cells from the antigen-containing medium becomes significantly easier due to the binding to the matrix, as the matrix facilitates the production of virus/virus antigen on a large technical scale.
En foretrukken utførelsesform av den serumfri cellekultur med matrisen ifølge oppfinnelsen består i at det som adherent bundede celler anvendes Vero-celler ATCC CCL 81 som anvendes fortrinnsvis ved produksjon av forsommer-meningo-encafalitis-(FSME)-virus-antigen og således er infisert med FSME-virus. A preferred embodiment of the serum-free cell culture with the matrix according to the invention consists in Vero cells ATCC CCL 81 being used as adherent cells, which are preferably used in the production of early summer meningo-encephalitis (FSME) virus antigen and are thus infected with FSME virus.
De på matrisen adherent bundede celler kan imidlertid også være infisert med Flavivirus eller med Arenavirus. However, the cells adhered to the matrix may also be infected with Flavivirus or with Arenavirus.
Som materiale for matrisen har glass, fornettet dekstran, gelatin eller plast vist seg som godt egnet, hvorved matrisen best er utformet som mikrobærer hvis partikkel-diameter fortrinnsvis ligger mellom 100/im og 3000/xm. Disse mikrobærere kan ha en glatt overflate eller en porøs strukturering. As material for the matrix, glass, cross-linked dextran, gelatin or plastic have proven to be well suited, whereby the matrix is best designed as a microcarrier whose particle diameter is preferably between 100 µm and 3000 µm. These microcarriers can have a smooth surface or a porous structure.
En ytterligere hensiktsmessig utførelsesform av matrisen er at det på dennes overflate er adherent bundet mellom lxl0<5 >og 4xl0<4> celler pr. cm<2.>A further suitable embodiment of the matrix is that on its surface between lxl0<5> and 4xl0<4> cells per cm<2.>
Oppfinnelsen vedrører også en fremgangsmåte ved produksjon av f orsommer-meningoencef alitis- (FSME) -virus-antigen under anvendelse av matrisen beskrevet ovenfor og som er karakterisert ved at overflateavhengige permanente celler, fortrinnsvis Vero-cellene ATTC CCL 81, podes med FSME-viruset, og at cellene i et serumfritt medium under opprettholdelse av sin levedyktighet holdes adherent bundet til en matrise for å opprettholde en antigendannelse og en antigenavgivelse til mediet, hvorefter det antigenholdige medium skilles fra cellene som er bundet til bæreren, og opparbeides på kjent måte til et galenisk aksepterbart preparat ved oppkonsentrering, inaktivering og rensning. The invention also relates to a method for the production of early summer meningoencephalitis (FSME) virus antigen using the matrix described above and which is characterized in that surface-dependent permanent cells, preferably the Vero cells ATTC CCL 81, are inoculated with the FSME virus , and that the cells in a serum-free medium, while maintaining their viability, are kept adherently bound to a matrix to maintain antigen formation and antigen delivery to the medium, after which the antigen-containing medium is separated from the cells bound to the carrier, and processed in a known manner into a galenically acceptable preparation by concentration, inactivation and purification.
Vero-cellelinjen ATCC CCL 81 utvinnes fra nyrevev hos den grønne marekatt (Cercopithecus aethiops), og kan holdes metabolsk aktiv i serumfritt medium. For en slik permanent cellelinje anlegges det en moderstamcellebank og en arbeid-sstamcellebank, og alle undersøkelser gjennomføres på kontaminerende substanser. Denne permanente cellelinje er således nøyaktig karakteriserbar ikke bare med henblikk på frihet av kontaminerende mikroorganismer, men også på vekstforhold, kultivering, formeringsforhold og - når engang optimert - skal betraktes som konstant. The Vero cell line ATCC CCL 81 is obtained from kidney tissue of the green marten (Cercopithecus aethiops), and can be kept metabolically active in serum-free medium. For such a permanent cell line, a maternal stem cell bank and a working stem cell bank are set up, and all investigations are carried out on contaminating substances. This permanent cell line is thus precisely characterizable not only with regard to freedom from contaminating microorganisms, but also on growth conditions, cultivation, propagation conditions and - once optimized - must be considered constant.
Ved fremgangsmåten ifølge oppfinnelsen anvendes det fortrinnsvis Vero-celler som er bundet på mikrobærere. Således kan det oppnås en høy celletetthet som ved de hittil brukte primære cellekulturer hverken kunne oppnås i Roux-flasker eller i suspensjon, og som muliggjør en betraktelig utbyt-teøkning av virus og virusantigen pr. fermentasjonsvolum. In the method according to the invention, Vero cells which are attached to microcarriers are preferably used. Thus, a high cell density can be achieved which with the primary cell cultures used up until now could not be achieved either in Roux flasks or in suspension, and which enables a considerable increase in the yield of virus and virus antigen per fermentation volume.
En fordelaktig utførelsesform av fremgangsmåten ifølge oppfinnelsen består i at virusformeringen og antigendannelsen gjennomføres i en kontinuerlig dreven perfusjonsreaktor i løpet av minst 5 dager, ved en temperatur mellom 34 og 37°C, idet perfusjonen kan gjennomføres med en perfusjonsrate på 0,3 til 10 v/v/dag. I perfusjonsreaktoren kan man dessuten sørge for en celletetthet på 2 x IO<9> til 2 x 10<10 >celler pr. liter fermentasjonsvolum, sistnevnte ved en fluidisert bed-fermentator. An advantageous embodiment of the method according to the invention consists in the virus propagation and antigen formation being carried out in a continuously operated perfusion reactor during at least 5 days, at a temperature between 34 and 37°C, the perfusion being carried out with a perfusion rate of 0.3 to 10 v /v/day. In the perfusion reactor, a cell density of 2 x 10<9> to 2 x 10<10 >cells per liter fermentation volume, the latter in a fluidized bed fermenter.
Virusformeringen ifølge oppfinnelsen i en perfusjonskultur muliggjør en, i sammenligning med en satsvis kultivering, vesentlig forkortelse av den av perfusjonsraten forutbe-stemte oppholdstid av viruset og virusantigenet i mediet. På grunn av den kortere oppholdstid oppnår man en vesentlig mindre termisk inaktivering og således en høyere produk-tivitet av fremgangsmåten ifølge oppfinnelsen. Således kan det i perfusjonsmediet oppnås og opprettholdes en antigen-konsentrasjon på 1 til 10 /xg/ml. The virus propagation according to the invention in a perfusion culture enables, in comparison with a batch cultivation, a significant shortening of the residence time of the virus and virus antigen in the medium, predetermined by the perfusion rate. Due to the shorter residence time, a significantly smaller thermal inactivation is achieved and thus a higher productivity of the method according to the invention. Thus, an antigen concentration of 1 to 10 µg/ml can be achieved and maintained in the perfusion medium.
Ved fremgangsmåten ifølge oppfinnelsen kan man på enkel måte innstille de for kultiveringen optimale betingelser. Dessuten er det for gjennomføringen vesentlig mindre manipulasjoner nødvendige enn ved alle de kjente fremgangsmåter, noe som betyr en større sikkerhet i omgang med det infektiøse materiale og muliggjør en kontinuerlig rask opparbeidelse av virus og virusantigen fra kulturmediet. With the method according to the invention, the optimum conditions for the cultivation can be easily set. Moreover, significantly less manipulation is required for the implementation than with all the known methods, which means greater safety in dealing with the infectious material and enables a continuous, rapid processing of virus and virus antigen from the culture medium.
Fremstillingen av virusinokulumet, dyrkningen av cellene for virus- hhv. virusantigenproduksjon, og den egentlige virus- hhv. virusantigenproduksjon skal beskrives nærmere i det følgende. The preparation of the virus inoculum, the cultivation of the cells for the virus or virus antigen production, and the actual virus or virus antigen production will be described in more detail below.
1. Virusinokulum 1. Virus inoculum
Celler (f.eks. Vero ATCC CCL 81) dyrkes i rulleflasker ved 37°C inntil konfluens og infiseres med 1 ml av en moder-virussuspensjon. Fra 2. dag efter infeksjon, gjennomføres daglig en halv medieveksel med serumfritt medium. Medie-restene fra 4. til 8. dag inneholder 2-5 x IO<7> p.f.u. pr. ml og lagres ved -20°C til de skal brukes som virusinokulum. Cells (eg Vero ATCC CCL 81) are grown in roller bottles at 37°C until confluent and infected with 1 ml of a parent virus suspension. From the 2nd day after infection, half a medium exchange with serum-free medium is carried out daily. The media residues from the 4th to the 8th day contain 2-5 x IO<7> p.f.u. per ml and stored at -20°C until they are to be used as virus inoculum.
2. Dyrkning av celler for virus/virusantigenproduksjon 2. Cultivation of cells for virus/virus antigen production
Ut fra ATCC CCL 81 arbeidsmodercellene som er lagret i flytende nitrogen, gjennomføres en formering av disse celler i vevskulturflasker inntil man oppnår en cellemengde som tillater å inokulere en fermentator. Den videre kultivering av cellene skjer i fermentasjonskar ved 37°C, hvorved de adherent voksende arbeidsmodercellene skal ha mest mulig overflate til disposisjon for adhesjon. Slike store overflater er disponible ved bruk av rulleflasker av glass eller polystyrol eller ved bruk av mikrobærere (MC). Best egnet er MC av fornettet dekstran med en størrelse mellom 170 fim og 250^m. From the ATCC CCL 81 working mother cells that are stored in liquid nitrogen, a multiplication of these cells is carried out in tissue culture flasks until a cell quantity is obtained that allows inoculating a fermenter. The further cultivation of the cells takes place in fermentation vessels at 37°C, whereby the adherently growing worker mother cells must have as much surface as possible available for adhesion. Such large surfaces are available by using roller bottles made of glass or polystyrene or by using microcarriers (MC). Most suitable is MC of cross-linked dextran with a size between 170 µm and 250 µm.
De med moderceller anrikede MC kultiveres ved 37°C til det oppnås en celletetthet på 1.10<5> - 4.IO<5> celler pr. cm<2>. Denne celletetthet oppnås generelt efter seks dager. Under kultiveringen kommer det til en fullstendig overvoksing av mikrobærerne med celler, idet til slutt noen mikrobærere kan voske sammen til grupper over cellene som sitter fast på deres overflate. The mother cell-enriched MCs are cultured at 37°C until a cell density of 1.10<5> - 4.10<5> cells per cm<2>. This cell density is generally achieved after six days. During the cultivation, the microcarriers become completely overgrown with cells, as eventually some microcarriers can coalesce into groups above the cells stuck to their surface.
3. Virus/virusantigenproduksjon 3. Virus/virus antigen production
Efter at den angitte celletetthet er oppnådd, infiseres de celler med virusinokulumet som er bundet til MC (1-0,01 pfu/celle, fortrinnsvis 0,1 pfu/celle) for å danne den aktuelle matrise. Matrisen ifølge oppfinnelsen kan lagres ved en temperatur mellom 0°C og 8°C eller kan anvendes straks i virusantigenproduksjonen. After the indicated cell density is achieved, the cells are infected with the virus inoculum bound to the MC (1-0.01 pfu/cell, preferably 0.1 pfu/cell) to form the appropriate matrix. The matrix according to the invention can be stored at a temperature between 0°C and 8°C or can be used immediately in virus antigen production.
For antigenproduksjon innføres de med infiserte celler anrikede MC i en perfusjonsreaktor. Fra dette tidspunkt av virusinfeksjonen anvendes i kulturen bare serumfritt medium som pumpes kontinuerlig gjennom perfusjonsreaktoren, mens cellene som kultiveres på mikrobærerne holdes tilbake i reaktoren ved hjelp av en tilbakeholdelsesanordning. I det utløpende kulturmediet foreligger fra 2. dag efter infeksjon virusantigen i høy konsentrasjon og kan utvinnes derfra kontinuerlig i minst 10 dager. For antigen production, MCs enriched with infected cells are introduced into a perfusion reactor. From this point of the virus infection, only serum-free medium is used in the culture, which is continuously pumped through the perfusion reactor, while the cells cultivated on the microcarriers are held back in the reactor by means of a retention device. In the expiring culture medium, virus antigen is present in high concentration from the 2nd day after infection and can be extracted from it continuously for at least 10 days.
Med de efterfølgende eksempler skal fremgangsmåten ifølge oppfinnelsen forklares ytterligere. Bestemmelsen av virusantigenet skjer i alle eksempler med et antigen-ELISA. With the following examples, the method according to the invention will be further explained. The determination of the virus antigen takes place in all examples with an antigen ELISA.
Eksempel 1 Example 1
Vero-celler ATCC CCL 81 ble kultivert i en 6-1-fermentator på mikrobærer (Cytodex 3 fra firma Pharmacia) ved 37°C til et celleantall på 2xl0<6> pr. ml kulturmedium (DMEM = Dulg-becco's Eagle Medium) og Vero cells ATCC CCL 81 were cultivated in a 6-1 fermenter on microcarriers (Cytodex 3 from the company Pharmacia) at 37°C to a cell number of 2x10<6> per ml culture medium (DMEM = Dulg-becco's Eagle Medium) and
a) infisert med FSME-virus (0,1 pfu/celle), og vi-rusf ormeringen ble gjennomført satsvis a) infected with FSME virus (0.1 pfu/cell), and the virus transformation was carried out batchwise
Produktiviteten pr. 1 fermentasjonsvolum var 4 mg virus/virusantigen. The productivity per 1 fermentation volume was 4 mg virus/virus antigen.
b) infisert med FSME-virus (0,1 pfu/celle) og kulturmedium (DMEM) ble perfundert kontinuerlig med 0,5 b) infected with FSME virus (0.1 pfu/cell) and culture medium (DMEM) was perfused continuously with 0.5
volum/fermentatorvolum/dag volume/fermenter volume/day
Produktiviteten pr. 1 fermentasjonsvolum var 13,7 mg virus/virusantigen. The productivity per 1 fermentation volume was 13.7 mg virus/virus antigen.
c) infisert med FSME-virus (0,1 pfu/celle), og kultur medium (DMEM) ble perfundert kontinuerlig med 1 c) infected with FSME virus (0.1 pfu/cell), and culture medium (DMEM) was perfused continuously with 1
volum/fermentatorvolum/dag volume/fermenter volume/day
Produktiviteten pr. 1 fermentasjonsvolum var 12,4 mg virus/virusantigen. The productivity per 1 fermentation volume was 12.4 mg virus/virus antigen.
Eksempel 2 Example 2
Vero-celler (ATCC CCL 81) ble i en 40-1-fermentator kultivert på mirkobærer (Cytodex 3 fra firma Phamracia) ved 37°C til et celleantall på 2 x IO<6> celler/ml, og ble efter infeksjon med FSME-virus (0,1 pfu/celle) perfundert kontinuerlig med medium (DMEM) (0,33 vol/fermentatorvolum/dag). Vero cells (ATCC CCL 81) were cultivated in a 40-1 fermenter on microcarriers (Cytodex 3 from the company Phamracia) at 37°C to a cell number of 2 x 10<6> cells/ml, and after infection with FSME -virus (0.1 pfu/cell) perfused continuously with medium (DMEM) (0.33 vol/fermenter volume/day).
Produktiviteten pr. 1 fermentasjonsvolum var 10,7 mg virus/virusantigen. The productivity per 1 fermentation volume was 10.7 mg virus/virus antigen.
Eksempel 3 Example 3
Vero-celler (ATCC CCL 82) ble i en 40-1-fermentator kultivert på mirkobærer (Cytodex 3 fra firma Pharmacia) ved 37°C til et celleantall på 3 x 10<6> celler/ml, og ble efter infeksjon med FSME-virus (0,1 pfu/celle) perfundert kontinuerlig med medium (DMEM) (1 vol/fermentatorvolum/dag). Produktiviteten pr. 1 fermentasjonsvolum var 21,7 mg virus/virusantigen. Vero cells (ATCC CCL 82) were cultivated in a 40-1 fermenter on microcarriers (Cytodex 3 from the company Pharmacia) at 37°C to a cell number of 3 x 10<6> cells/ml, and after infection with FSME -virus (0.1 pfu/cell) perfused continuously with medium (DMEM) (1 vol/fermenter volume/day). The productivity per 1 fermentation volume was 21.7 mg virus/virus antigen.
Eksempel 4 Example 4
Vero-celler (ATCC CCL 81) ble i 150-1-fermentator på mikrobærer (Cytodex 3 fra firma Pharmacia) kultivert ved 37°C til 2 x 10<6>/ml, og ble efter infeksjon med FSME-virus (0,1 pfu/celle) perfundert kontinuerlig med medium (DMEM) (0,33 vol/fermentatorvolum/dag). Vero cells (ATCC CCL 81) were cultured in a 150-1 fermenter on microcarriers (Cytodex 3 from the company Pharmacia) at 37°C to 2 x 10<6>/ml, and after infection with FSME virus (0, 1 pfu/cell) perfused continuously with medium (DMEM) (0.33 vol/fermenter volume/day).
Produktiviteten pr. 1 fermentasjonsvolum var 14,7 mg virus/virusantigen. The productivity per 1 fermentation volume was 14.7 mg virus/virus antigen.
Claims (10)
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 |
| PCT/AT1990/000128 WO1991009935A1 (en) | 1989-12-22 | 1990-12-21 | Matrix with adherently bound cells and process for producing viruses/virus antigens |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| NO922422D0 NO922422D0 (en) | 1992-06-19 |
| NO922422L NO922422L (en) | 1992-08-17 |
| NO310305B1 true NO310305B1 (en) | 2001-06-18 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NO19922422A NO310305B1 (en) | 1989-12-22 | 1992-06-19 | Matrix with adherently bound cells, as well as the method of producing late-summer meningoencephalitis (FSME) virus antigen |
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| 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) |
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| JP3158157B2 (en) * | 1994-11-10 | 2001-04-23 | バクスター・アクチエンゲゼルシャフト | Biopharmaceutical production in protein-free culture |
| AU709584B2 (en) * | 1995-08-01 | 1999-09-02 | Pasteur Merieux Serums Et Vaccins | Industrial production process for a vaccine against Japanese encephalitis, and vaccine obtained |
| 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 |
| AU743546B2 (en) * | 1998-10-05 | 2002-01-31 | Research Foundation For Microbial Diseases Of Osaka University, The | 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 |
| US6855535B2 (en) * | 2001-12-10 | 2005-02-15 | Baxter Healthcare S.A. | Method of large scale production of Hepatitis A virus |
| US6951752B2 (en) | 2001-12-10 | 2005-10-04 | Bexter Healthcare S.A. | Method for large scale production of virus antigen |
| JP2007068401A (en) * | 2003-08-07 | 2007-03-22 | Chemo Sero Therapeut Res Inst | West nile virus vaccine |
| PT2235197T (en) * | 2007-12-27 | 2017-10-11 | Baxalta Inc | 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
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1990
- 1990-12-21 CA CA 2071954 patent/CA2071954C/en not_active Expired - Lifetime
- 1990-12-21 SK SK6590-90A patent/SK659090A3/en not_active IP Right Cessation
- 1990-12-21 JP JP50113291A patent/JP2633391B2/en not_active Expired - Fee Related
- 1990-12-21 WO PCT/AT1990/000128 patent/WO1991009935A1/en active IP Right Grant
- 1990-12-21 DK DK91900666T patent/DK0506714T3/en not_active Application Discontinuation
- 1990-12-21 HU HU9202009A patent/HU213886B/en unknown
- 1990-12-21 YU YU242390A patent/YU242390A/en unknown
- 1990-12-21 DE DE59007659T patent/DE59007659D1/en not_active Expired - Fee Related
- 1990-12-21 RU SU905052769A patent/RU2082757C1/en not_active IP Right Cessation
- 1990-12-21 CZ CS906590A patent/CZ281804B6/en not_active IP Right Cessation
- 1990-12-21 ES ES91900666T patent/ES2067916T3/en not_active Expired - Lifetime
- 1990-12-21 EP EP19910900666 patent/EP0506714B1/en not_active Expired - Lifetime
- 1990-12-21 AT AT91900666T patent/ATE113652T1/en not_active IP Right Cessation
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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 |
|---|---|
| HRP921354A2 (en) | 1996-02-29 |
| CZ281804B6 (en) | 1997-02-12 |
| HU213886B (en) | 1997-11-28 |
| NO922422D0 (en) | 1992-06-19 |
| FI922851A (en) | 1992-06-18 |
| SK279236B6 (en) | 1998-08-05 |
| EP0506714A1 (en) | 1992-10-07 |
| WO1991009935A1 (en) | 1991-07-11 |
| HUT65410A (en) | 1994-06-28 |
| JP2633391B2 (en) | 1997-07-23 |
| FI98377C (en) | 1997-06-10 |
| CZ659090A3 (en) | 1996-11-13 |
| DE59007659D1 (en) | 1994-12-08 |
| YU242390A (en) | 1993-05-28 |
| JPH05502581A (en) | 1993-05-13 |
| FI922851A0 (en) | 1992-06-18 |
| DK0506714T3 (en) | 1995-04-18 |
| HU9202009D0 (en) | 1992-10-28 |
| ATE113652T1 (en) | 1994-11-15 |
| CA2071954A1 (en) | 1991-06-23 |
| SK659090A3 (en) | 1998-08-05 |
| FI98377B (en) | 1997-02-28 |
| NO922422L (en) | 1992-08-17 |
| EP0506714B1 (en) | 1994-11-02 |
| RU2082757C1 (en) | 1997-06-27 |
| CA2071954C (en) | 1996-06-18 |
| ES2067916T3 (en) | 1995-04-01 |
| ATA292889A (en) | 1991-03-15 |
| AT393356B (en) | 1991-10-10 |
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