WO1999047648A2 - Medium and method for viral propagation and growth - Google Patents

Medium and method for viral propagation and growth Download PDF

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Publication number
WO1999047648A2
WO1999047648A2 PCT/FR1999/000578 FR9900578W WO9947648A2 WO 1999047648 A2 WO1999047648 A2 WO 1999047648A2 FR 9900578 W FR9900578 W FR 9900578W WO 9947648 A2 WO9947648 A2 WO 9947648A2
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WIPO (PCT)
Prior art keywords
medium
cells
viruses
multiplication
viral
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PCT/FR1999/000578
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French (fr)
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WO1999047648A3 (en
Inventor
Pierre Heimindinger
Original Assignee
Aventis Pasteur
Merial S.A.S.
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.)
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Publication date
Application filed by Aventis Pasteur, Merial S.A.S. filed Critical Aventis Pasteur
Priority to BR9908744A priority Critical patent/BR9908744A/en
Priority to SK1288-2000A priority patent/SK12882000A3/en
Priority to AU27352/99A priority patent/AU752577B2/en
Priority to NZ506872A priority patent/NZ506872A/en
Priority to JP2000536831A priority patent/JP2002506636A/en
Priority to KR1020007010026A priority patent/KR20010072556A/en
Priority to EP99907713A priority patent/EP1062324A2/en
Priority to IL13825099A priority patent/IL138250A0/en
Priority to EA200000934A priority patent/EA200000934A1/en
Priority to HU0101081A priority patent/HUP0101081A3/en
Priority to CA 2322619 priority patent/CA2322619A1/en
Publication of WO1999047648A2 publication Critical patent/WO1999047648A2/en
Publication of WO1999047648A3 publication Critical patent/WO1999047648A3/en

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    • 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
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/13—Poliovirus
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/205—Rhabdoviridae, e.g. rabies virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0043—Medium free of human- or animal-derived components
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/76—Undefined extracts from plants
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus
    • C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus
    • C12N2760/20151—Methods of production or purification of viral material
    • 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
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • 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
    • C12N2770/32011—Picornaviridae
    • C12N2770/32611—Poliovirus
    • C12N2770/32634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2770/32011—Picornaviridae
    • C12N2770/32611—Poliovirus
    • C12N2770/32651—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

Definitions

  • the present invention relates to a medium and to a method of viral propagation and multiplication on cells in culture, in particular for the production of vaccines.
  • viral vaccines whether they are simply attenuated, inactivated, subunit or recombinant, involves the production, on a large scale, of viruses.
  • This production can be carried out, depending on the nature of the virus, on different supports allowing virus replication; thus, for example, for most influenza vaccines currently marketed, the proliferation of viruses is carried out on eggs of embryonated hens, while for certain vaccines against Japanese encephalitis, this step is carried out on mouse brains.
  • the use of such supports poses many problems: availability, reproducibility, but above all safety due to possible contamination by viruses, mycoplasmas or any other undesirable element originating from the support.
  • the culture medium used in the prior art during this step of viral multiplication and propagation contains usually fetal calf serum or to limit contamination problems, at least human albumin.
  • the use of human albumin at this stage of a viral vaccine manufacturing process is considered to be safe for the quality of the vaccine.
  • the object of the invention is in particular to propose such a medium.
  • Another object of the invention is to propose a medium and a method for viral propagation and multiplication then making it possible, when desired, to carry out a step of inactivation of the viruses.
  • the present invention relates to a medium for viral propagation and multiplication on cells in culture, characterized in that it is devoid of human or animal proteins, even of recombinant origin, and in that '' it includes elements of plant origin.
  • the viral propagation and multiplication medium comprises proteins or glycoproteins extracted from potatoes or cucumbers, having mitogenic potential and whose molecular weight is between 1,000 and 200,000 daltons.
  • the medium according to the present invention comprises plant hydrolysates.
  • the proteins or glycoproteins are extracted from potatoes.
  • the proteins or glycoproteins are obtained in the following manner:
  • plant hydrolysates are obtained by chemical and / or enzymatic hydrolysis of raw materials such as cotton, soy, wheat or rice. Particularly good results have been obtained with the products of the HyPep TM range supplied by the company QUEST INTERNATIONAL, in particular with products comprising a high proportion of peptides whose molecular mass is less than 1000 daltons.
  • the viral propagation and multiplication medium according to the invention is particularly suitable for viral multiplication on VERO cells.
  • the viral propagation and multiplication medium according to the invention is particularly suitable for the multiplication of Japanese encephalitis viruses, of poliomyelitis viruses, of rabies viruses.
  • the present invention also relates to a process for the manufacture of viral vaccines comprising a phase of viral production on cells in culture, characterized in that one proceeds to the phase of propagation and multiplication of the viruses in the presence of a medium devoid of human or animal protein, even of recombinant origin, and comprising plant extracts.
  • the medium and the method according to the invention are suitable for the production of all viruses capable of replicating on cell cultures. It can in particular be viruses belonging to the following families: orthomyxovirus, paramyxovirus, reovirus, picornavirus, flavivirus, arenavirus, herpesvirus, poxvirus, and adenoviruses. They can also be recombinant viruses.
  • the invention is particularly suitable for the production of viruses capable of entering into the composition of vaccines, and in particular human vaccines for which the safety requirements are maximum.
  • the present invention is particularly advantageous for the production of the viruses responsible for poliomyelitis, rabies, Japanese encephalitis, rubella, chickenpox, hepatitis A, influenza, Dengue fever, measles, mumps.
  • the cells capable of being used for the production of virus according to the invention are all cells capable of multiplying in culture and permissive to the virus which it is desired to produce. They can in particular be VERO cells, CV-1 cells, LLC-MK2 cells, MDCK cells, MDBK cells, WI-38 cells, MRC5 cells (human fibroblasts), or even BHK21 cells. Particularly good results have been obtained with VERO cell cultures or MRC5 cell cultures.
  • Cell culture can be carried out according to the various techniques usually used: in cytocultors, in roller bottles, on Roux boxes, on Multitray, on Cell-Cube. It is preferred, for industrial reasons, to carry out cell culture in cytocultors, using microcarriers constituted for example by Cytodex TM beads.
  • This cell culture can be carried out under different conditions, in particular as regards the medium used; it is indeed possible to implement the present invention while the culture medium used for the cell growth phase contained serum or at least human or animal proteins; in this case, of course, it will be necessary to carry out a perfect washing of the cells before their infection, so as not to lose the advantage provided by the implementation of the invention as regards the absence of risk of contamination.
  • the most opportune time to infect the virus with a virus inoculum may vary. In general, this is done more in the middle or at the end of the exponential growth phase; it has been observed, in fact, that under these conditions, the results obtained were particularly satisfactory.
  • the culture medium used for the viral propagation and multiplication step is a medium devoid of human, animal or recombinant protein, but comprising proteins or glycoproteins extracted from potatoes or cucumber, having a potential mitogenic and whose molecular weight is between 1000 and 200,000 daltons and / or hydrolysates of plant extracts.
  • This medium is a medium which may include all or part of the elements conventionally used for viral multiplication, such as the HAM F 12 medium, but supplemented with plant extracts, obtained for example in the following manner: reduction of the vegetable pulp, treatment of the pulp obtained with a solvent, evaporation of the solvent,
  • the mitogenic potential of proteins or glycoproteins suitable for the purposes of the invention can be assessed in particular by means of tests for analysis of cell growth and mitogenic activity, such as tests for staining with M.T.T.
  • the plant extracts suitable for the purposes of the invention are in particular those described in patent application FR 2732347. It is also possible to use the products sold by the company Biomedia under the name GCR1003, TCR1005, or BCR1008, alone or in combination, or alternatively the PROLIFIX product also marketed by this Company and comprising all the nutrients of the conventional HAM F 12 medium supplemented with appropriate plant extracts.
  • a conventional HAM F 12 medium supplemented with plant hydrolysates such as HyPep TM products at a rate of 5 g / 1. It is also possible to use a medium comprising both the proteins or glycoproteins obtained by thermocoagulation of the plant pulp and plant hydrolysates such as those mentioned above.
  • VERO cells are used to produce the virus responsible for Japanese encephalitis.
  • the cells used are cells derived from the strain distributed by the ATCC (American Type Culture Collection) under the number ATCC-CCL 81- VERO F 1415 which is at the 124th passage and which is brought to the 137th passage in a conventional manner .
  • the virus which it is desired to produce is the virus of the P3 strain at 88 TM passage supplied by the NVSI.
  • the tank of a 15 liter cytocultor is filled with ISCOVE medium supplemented with 4% calf serum containing Cytodex I TM microcarriers at a concentration of 3.5 g / l.
  • the medium is inoculated with an inoculum of VERO cells at the rate of 200,000 cells / ml.
  • the culture is amplified and subcultured 2 times a week every 4-5 days.
  • the cell concentrations that are obtained are between 2 and 3.10 6 per ml.
  • the cell suspension obtained is then rinsed with the viral propagation medium in order to eliminate the maximum of residual calf serum.
  • a medium according to the invention consisting of HAM F 12 medium (supplied by Life Technologies GIBCO) added by PROLIFIX II medium supplied by the company BIOMEDIA, at a concentration 10 times that indicated in their 1996 catalog, in a proportion of 1 volume of PROLIFIX II concentrated 10 times for 10 volumes of HAM F 12,
  • a medium according to the prior art consisting of the ISCOVE medium (supplied by Life Technologies GIBCO) added with human albumin in order to obtain a final concentration of albumin in the medium of 0.3%.
  • an inoculum of virus is introduced into each of the cytocultors, in an amount corresponding to an MOI (Multiplicity Of Infection) of 1/6000.
  • the content of the cytocultors is then kept stirring at a temperature of 37 ° C. for 3 days, then the CTe is harvested by recovery of the viral propagation medium which has become a viral suspension, which is replaced in each of the cytocultors with an equal amount of fresh medium of the same nature; the propagation medium is then harvested every 24 hours, this up to 5 harvests.
  • Each of the crops is filtered on a filter whose cutoff threshold is 0.22 ⁇ m, and stored at + 5 ° C while waiting to be mixed with the 4 other crops from the same cytocultor. When the 5 harvests have been carried out, they are mixed, then this mixture is concentrated by filtration to a factor of 100.
  • the mixture consisting of the 5 harvests from the same cytocultor constitutes a batch.
  • an infectious activity test is carried out for each of the batches produced.
  • the test is carried out in 2 different ways: either in infectious activity measured on the cell table, or in infectious activity measured in mice by intracerebral inoculation in mice, observation of the animals for 14 days and calculation of LD50.
  • the results are expressed in Log 10 of infectious particles per liter of cytocultor.
  • the average of the results obtained for the various batches produced using a medium according to the invention is 8.84 while the average obtained for the batches produced using a medium according to the prior art is 7.85. If we consider the titration on mice, the average for the batches produced according to the invention is this time 9.22 while the average of the batches produced with a medium according to the prior art is 9.30.
  • the batches obtained were then inactivated by the addition of a formaldehyde solution in an amount making it possible to have a final concentration in the mixture of 1/4000 and maintained at room temperature with continuous stirring for 14 days. Verification of inactivation by control according to the protocol recommended by WHO (Technical Report 771 of 1988) showed that the batches produced complied with WHO recommendations.
  • 150 cm 3 'flasks are used which are filled with 50 ml of ISCOVE medium supplemented with 4% calf serum. This medium is seeded with an inoculum of VERO cells at the rate of 250,000 cells / cm 2 '. The vials are left for 4-5 days at 37 ° C and then rinsed using the viral multiplication and propagation medium.
  • the media used are as follows:
  • a medium A which is a medium according to the prior art constituted by the ISCOVE medium (supplied by Life Technologies GIBCO) added with human albumin in order to obtain a final concentration of albumin in the medium of 0.3% ,
  • a medium B consisting of HAM F 12 medium supplemented with the following mitogenic molecules: * BCR 1008 at a rate of 10 ⁇ 3 g / 1
  • a medium C consisting of the medium mentioned above (HAM F 12 added with BCR 1008, TCR 1005, GCR 1003) but also comprising lg / 1 of the product HYPEP 4602 consisting of gluten hydrolyzate and supplied by the QUESTEL International Company,
  • a medium D consisting of HAM F 12 medium supplemented with HYPEP 4602 at a rate of lg / 1, but devoid of the mitogenic molecules BCR 1008, TCR 1005, or
  • an inoculum of Japanese encephalitis virus identical to that of the previous example is introduced into each of the bottles in an amount corresponding to an MOI of 1/6000.
  • the content of the vials is then maintained for 3 days at 37 ° C before it proceeds to the harvest ere.
  • the following harvests are carried out in the same manner as described in the previous example.
  • the infectious titre determination tests are carried out on BHK21 cells and give the following results, expressed in DICC 50 / ml (Log 10):
  • Middle B 5.1
  • Middle C 5.0
  • a culture of VERO cells is carried out in a cytocultor of 21 in conventional medium, then after washing, the cells are infected with rabies virus with an MOI of 1/1000, and the cells are maintained in a medium identical to that described in Example 1 (ie HAM FI 2 medium supplemented with PROLIFIX TM II); the same experiment is carried out in exactly the same way, in another cytocultor of the same capacity, with the only difference being the use of a viral propagation and multiplication medium of the prior art comprising human albumin.
  • Harvests are carried out successively 4, 5, 6 and 7 days after infection.
  • the counts carried out on the crops obtained thanks to the medium according to the invention and on the crops obtained thanks to the medium according to the prior art made it possible to obtain results comparable with the 2 media; similarly, the infectious titers of the viruses obtained were equivalent.
  • Polio type 1 virus production assays were performed on cultured VERO cells.
  • the infectious titers of the viruses obtained using a viral propagation and multiplication medium according to the invention having the composition described in Example 1 were equivalent to the average of the infectious titers obtained with the harvests carried out with the medium used for the manufacture of the currently marketed vaccines.

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Abstract

The invention concerns a medium for viral propagation and growth on cultivated cells, characterised in that it is free of human or animal proteins and it comprises proteins or glycoproteins extracted from potatoes or cucumber, having a mitogen potential and whereof the molecular weight ranges between 1000 and 200000 daltons and/or plant extract hydrolysates.

Description

MILIEU ET PROCEDE DE PROPAGATION ET DE MULTIPLICATION VIRALESVIRAL PROPAGATION AND MULTIPLICATION MEDIUM AND METHOD
La présente invention est relative à un milieu et à un procédé de propagation et de multiplication virales sur des cellules en culture, notamment pour la production de vaccins.The present invention relates to a medium and to a method of viral propagation and multiplication on cells in culture, in particular for the production of vaccines.
La production de vaccins viraux, qu'ils soient simplement atténués, inactivés, sous- unitaires ou recombinants, implique la production, à grande échelle, de virus. Cette production peut être effectuée, selon la nature du virus, sur différents supports permettant la réplication du virus ; ainsi, par exemple, pour la plupart des vaccins contre la grippe actuellement commercialisés, la prolifération des virus est effectuée sur des œufs de poules embryonnés, alors que pour certains vaccins contre l'encéphalite japonaise, cette étape est effectuée sur des cerveaux de souris. Or, l'utilisation de tels supports pose de nombreux problèmes : disponibilité, reproductibilité, mais surtout sécurité du fait des contaminations possibles par des virus, des mycoplasmes ou tout autre élément indésirable provenant du support. De plus en plus, on cherche donc à s'affranchir de tels supports et on utilise, à chaque fois que cela est possible, des cultures cellulaires que l'on infecte par une faible quantité du virus à produire et que l'on maintient ensuite dans des conditions favorables à la réplication des virus à l'intérieur des cellules infectées, ainsi qu'à la propagation de l'infection d'une cellule à l'autre. Les virus produits sont ensuite récoltés, soit à partir du milieu de culture dans lequel baignent les cellules lorsqu'il s'agit de virus intracellulaires quittant la cellule après leur bourgeonnement (ce qui a notamment pour avantage de permettre de réaliser plusieurs récoltes de virus avec les mêmes cellules en culture), soit par traitement des cellules elles-mêmes lorsqu'il s'agit de virus intracellulaires.The production of viral vaccines, whether they are simply attenuated, inactivated, subunit or recombinant, involves the production, on a large scale, of viruses. This production can be carried out, depending on the nature of the virus, on different supports allowing virus replication; thus, for example, for most influenza vaccines currently marketed, the proliferation of viruses is carried out on eggs of embryonated hens, while for certain vaccines against Japanese encephalitis, this step is carried out on mouse brains. However, the use of such supports poses many problems: availability, reproducibility, but above all safety due to possible contamination by viruses, mycoplasmas or any other undesirable element originating from the support. Increasingly, efforts are therefore being made to get rid of such supports and cell cultures are used, whenever possible, which are infected with a small amount of the virus to be produced and which are then maintained. under conditions favorable to the replication of viruses inside infected cells, as well as to the spread of infection from one cell to another. The viruses produced are then harvested, either from the culture medium in which the cells are immersed in the case of intracellular viruses leaving the cell after their budding (which in particular has the advantage of making it possible to carry out several virus harvests with the same cells in culture), or by treatment of the cells themselves when it comes to intracellular viruses.
Ces techniques impliquent de disposer de cultures cellulaires de bonnes qualités ; de nombreux développements de l'art antérieur ont donc porté sur l'amélioration des cultures cellulaires et notamment sur les milieux de culture utilisés. Afin de limiter les risques de contamination des cellules par des mycoplasmes, des virus , des agents de Pencéphalopathie bovine spongiforme ou tout autre agent transmissible non conventionnel, il a été proposé d'utiliser des milieux de culture dépourvus de sérum, et même de protéine, ainsi que cela est décrit dans la demande de brevet WO 96/15231. De façon alternative, la demande FR 2732347 propose l'utilisation de milieu de culture cellulaire dépourvu de sérum animal, mais contenant des extraits végétaux.These techniques involve having good quality cell cultures; many developments in the prior art have therefore focused on improving cell cultures and in particular on the culture media used. In order to limit the risks of contamination of the cells by mycoplasmas, viruses, agents of bovine spongiform encephalopathy or any other non-conventional transmissible agent, it has been proposed to use culture media devoid of serum, and even of protein, as described in the Patent application WO 96/15231. Alternatively, application FR 2732347 proposes the use of cell culture medium devoid of animal serum, but containing plant extracts.
Ainsi, grâce à l'utilisation de tels milieux pour l'étape de culture des cellules, la sécurité des produits obtenus à l'aide des cellules ainsi produites est accrue. Cependant, lorsqu'il s'agit de produire des virus entrant dans la composition de vaccins, les étapes consécutives à cette phase de culture cellulaire, doivent, elles aussi, présenter le maximum de sécurité. En effet, de manière classique, et ainsi que cela est décrit dans le brevet US 4525349 lorsqu'on utilise des cellules en culture pour la production de virus, une fois la phase de culture des cellules effectuée, le milieu qui a été utilisé pour la culture cellulaire est évacué puis remplacé par un nouveau milieu dans lequel les cellules baignent pendant un moment afin d'être lavées ; ce nouveau milieu est ensuite éliminé ; cette opération de lavage des cellules peut éventuellement être renouvelée un certain nombre de fois.Thus, thanks to the use of such media for the cell culture step, the safety of the products obtained using the cells thus produced is increased. However, when it is a question of producing viruses entering into the composition of vaccines, the stages consecutive to this phase of cell culture, must, themselves, also present the maximum of safety. Indeed, conventionally, and as described in US Pat. No. 4,525,349 when cells in culture are used for the production of virus, once the cell culture phase has been carried out, the medium which has been used for the cell culture is removed and replaced by a new medium in which the cells are immersed for a while in order to be washed; this new medium is then eliminated; this cell washing operation can possibly be repeated a certain number of times.
On introduit, ensuite, dans la culture cellulaire un nouveau milieu qui va permettre aux cellules d'une part de survivre et d'être infectées par les virus et d'autre part, de disposer d'éléments suffisants pour être le support de la réplication de ces virus. Or, ainsi que cela est décrit dans l'article « Protein-free culture of Vero cells : A substrate for réplication of human pathogenic viruses », Cell BiologyA new medium is then introduced into cell culture which will allow cells on the one hand to survive and be infected by viruses and on the other hand, to have sufficient elements to support replication of these viruses. However, as described in the article "Protein-free culture of Vero cells: A substrate for replication of human pathogenic viruses", Cell Biology
International, Vol.l7 N°9,1993 pp885-895, même dans le cas où la culture cellulaire est réalisée en milieu sans protéine, le milieu de culture utilisé dans l'art antérieur lors de cette étape de multiplication et de propagation virales contient généralement du sérum de veau foetal ou pour limiter les problèmes de contamination , du moins de l'albumine humaine. L'utilisation de l'albumine humaine, à ce stade d'un procédé de fabrication de vaccins viraux, est considérée sans risque pour la qualité du vaccin. Cependant, dans le but d'éliminer complètement tout risque, même théorique, il serait souhaitable de pouvoir disposer d'un milieu apte à la propagation et à la multiplication de virus sur un support constitué par des cellules en culture, dépourvu de protéine humaine ou animale, même d'origine recombinante, mais permettant cependant des rendements de production compatibles avec les exigences industrielles afin de ne pas accroître les coûts de production des vaccins comprenant des virus produits sur cellules en culture.International, Vol.l7 N ° 9,1993 pp885-895, even in the case where cell culture is carried out in a protein-free medium, the culture medium used in the prior art during this step of viral multiplication and propagation contains usually fetal calf serum or to limit contamination problems, at least human albumin. The use of human albumin at this stage of a viral vaccine manufacturing process is considered to be safe for the quality of the vaccine. However, in order to completely eliminate any risk, even theoretical, it would be desirable to be able to have a medium suitable for the propagation and multiplication of viruses on a support constituted by cells in culture, devoid of human protein or animal, even of recombinant origin, but nevertheless allowing production yields compatible with industrial requirements so as not to not increase the production costs of vaccines comprising viruses produced on cells in culture.
Le but de l'invention est notamment de proposer un tel milieu.The object of the invention is in particular to propose such a medium.
Un autre but de l'invention est de proposer un milieu et un procédé de propagation et de multiplication virales permettant ensuite, lorsque cela est souhaité, de procéder à une étape d'inactivation des virus.Another object of the invention is to propose a medium and a method for viral propagation and multiplication then making it possible, when desired, to carry out a step of inactivation of the viruses.
Pour atteindre ces différents buts, la présente invention a pour objet un milieu de propagation et de multiplication virales sur des cellules en culture, caractérisé en ce qu'il est dépourvu de protéines humaines ou animales , même d'origine recombinante, et en ce qu'il comprend des éléments d'origine végétale.To achieve these various aims, the present invention relates to a medium for viral propagation and multiplication on cells in culture, characterized in that it is devoid of human or animal proteins, even of recombinant origin, and in that '' it includes elements of plant origin.
Selon une caractéristique de l'invention, le milieu de propagation et de multiplication virales comprend des protéines ou glycoprotéines extraites de pommes de terre ou de concombre, ayant un potentiel mitogène et dont le poids moléculaire est compris entre 1 000 et 200 000 daltons.According to a characteristic of the invention, the viral propagation and multiplication medium comprises proteins or glycoproteins extracted from potatoes or cucumbers, having mitogenic potential and whose molecular weight is between 1,000 and 200,000 daltons.
Selon un mode particulier de réalisation, le milieu selon la présente invention comprend des hydrolysats végétaux.According to a particular embodiment, the medium according to the present invention comprises plant hydrolysates.
Selon une caractéristique particulière de l'invention, les protéines ou glycoprotéines sont extraites de pommes de terre.According to a particular characteristic of the invention, the proteins or glycoproteins are extracted from potatoes.
Selon une autre caractéristique de l'invention, les protéines ou glycoprotéines sont obtenues de la manière suivante :According to another characteristic of the invention, the proteins or glycoproteins are obtained in the following manner:
- lavage et broyage des végétaux,- washing and grinding of plants,
- dilution en milieu aqueux,- dilution in an aqueous medium,
- thermocoagulation du mélange obtenu , - purification par chromatographie.- thermocoagulation of the mixture obtained, - purification by chromatography.
Selon une caractéristique de l'invention, les hydrolysats végétaux sont obtenus par hydrolyse chimique et/ou enzymatique de matières premières telles que le coton, le soja, le blé ou le riz. De particulièrement bons résultats ont été obtenus avec les produits de la gamme HyPep ™ fournis par la Société QUEST INTERNATIONAL, en particulier avec les produits comprenant une forte proportion de peptides dont la masse moléculaire est inférieure à 1000 daltons.According to a characteristic of the invention, plant hydrolysates are obtained by chemical and / or enzymatic hydrolysis of raw materials such as cotton, soy, wheat or rice. Particularly good results have been obtained with the products of the HyPep ™ range supplied by the company QUEST INTERNATIONAL, in particular with products comprising a high proportion of peptides whose molecular mass is less than 1000 daltons.
Selon une autre caractéristique, le milieu de propagation et de multiplication virales selon l'invention est particulièrement adapté à la multiplication virale sur cellules VERO.According to another characteristic, the viral propagation and multiplication medium according to the invention is particularly suitable for viral multiplication on VERO cells.
Selon une autre caractéristique, le milieu de propagation et de multiplication virales selon l'invention est particulièrement approprié à la multiplication des virus de l'encéphalite japonaise, des virus de la poliomyélite, des virus de la rage.According to another characteristic, the viral propagation and multiplication medium according to the invention is particularly suitable for the multiplication of Japanese encephalitis viruses, of poliomyelitis viruses, of rabies viruses.
La présente invention a également pour objet un procédé de fabrication de vaccins viraux comportant une phase de production virale sur cellules en culture, caractérisé en ce qu'on procède à la phase de propagation et de multiplication des virus en présence d'un milieu dépourvu de protéine humaine ou animale même d'origine recombinante , et comprenant des extraits végétaux.The present invention also relates to a process for the manufacture of viral vaccines comprising a phase of viral production on cells in culture, characterized in that one proceeds to the phase of propagation and multiplication of the viruses in the presence of a medium devoid of human or animal protein, even of recombinant origin, and comprising plant extracts.
La présente invention sera mieux comprise à la lecture de la description qui va suivre.The present invention will be better understood on reading the description which follows.
Le milieu et le procédé selon l'invention sont adaptés à la production de tous les virus aptes à se répliquer sur des cultures cellulaires. Il peut notamment s'agir des virus appartenant aux familles suivantes : orthomyxovirus, paramyxovirus, reovirus, picornavirus, flavivirus, arenavirus, herpesvirus, poxvirus, et les adénovirus. Il peut également s'agir de virus recombinants. De manière particulière, l'invention est particulièrement adaptée à la production de virus susceptibles de rentrer dans la composition de vaccins, et notamment de vaccins humains pour lesquels les exigences de sécurité sont maximales. Il peut en particulier s'agir des virus de la poliomyélite, de la rage, de l'encéphalite japonaise, de la fièvre jaune, de la rubéole, des oreillons, de la Dengue, de la rougeole, des différentes hépatites, du SIDA, de la varicelle, de l'herpès, des maladies provoquées par le virus syncytial respiratoire, le cytomegalovirus, l'EBV, les rotavirus ou la grippe.The medium and the method according to the invention are suitable for the production of all viruses capable of replicating on cell cultures. It can in particular be viruses belonging to the following families: orthomyxovirus, paramyxovirus, reovirus, picornavirus, flavivirus, arenavirus, herpesvirus, poxvirus, and adenoviruses. They can also be recombinant viruses. In particular, the invention is particularly suitable for the production of viruses capable of entering into the composition of vaccines, and in particular human vaccines for which the safety requirements are maximum. It can in particular be the polio viruses, rabies, Japanese encephalitis, yellow fever, rubella, mumps, Dengue, measles, various hepatitis, AIDS, chickenpox, herpes, diseases caused by respiratory syncytial virus, cytomegalovirus, EBV, rotavirus or influenza.
La présente invention est particulièrement avantageuse pour la production des virus responsables de la poliomyélite, de la rage, de l'encéphalite japonaise, de la rubéole, de la varicelle, de l'hépatite A, de la grippe, de la Dengue, de la rougeole, des oreillons.The present invention is particularly advantageous for the production of the viruses responsible for poliomyelitis, rabies, Japanese encephalitis, rubella, chickenpox, hepatitis A, influenza, Dengue fever, measles, mumps.
Les cellules susceptibles d'être utilisées pour la production de virus selon l'invention sont toutes les cellules aptes à se multiplier en culture et permissives au virus que l'on souhaite produire. Il peut notamment s'agir de cellules VERO, de cellules CV-1, de cellules LLC-MK2, de cellules MDCK, de cellules MDBK, de cellules WI-38, de cellules MRC5 (fibroblastes humains), ou encore de cellules BHK21. On a obtenu de particulièrement bons résultats avec des cultures de cellules VERO ou des cultures de cellules MRC5. La culture cellulaire peut être effectuée selon les différentes techniques habituellement utilisées : en cytoculteurs, en flacons roulants, sur boîtes de Roux, sur Multitray , sur Cell-Cube . On préfère, pour des raisons industrielles, effectuer la culture cellulaire en cytoculteurs, en utilisant des microsupports constitués par exemple par des billes de Cytodex™. Cette culture cellulaire peut être réalisée dans différentes conditions , notamment en ce qui concerne le milieu utilisé ; il est en effet possible, de mettre en œuvre la présente invention alors que le milieu de culture utilisé pour la phase de croissance cellulaire contenait du sérum ou du moins des protéines humaines ou animales ; dans ce cas, bien sûr, il sera nécessaire de procéder à un lavage parfait des cellules avant leur infection, afin de ne pas perdre l'avantage apporté par la mise en œuvre de l'invention en ce qui concerne l'absence de risque de contamination. Cependant, on préfère utiliser des cultures cellulaires obtenues en utilisant un milieu de culture cellulaire dépourvu également de protéines d'origine humaine ou animale. Selon la nature des cellules utilisées et des virus à produire, le moment le plus opportun pour procéder à l'infection des virus par un inoculum du virus peut varier. En général, on procède à cette opération plutôt au milieu ou en fin de la phase exponentielle de croissance ; on a remarqué, en effet, que dans ces conditions, les résultats obtenus étaient particulièrement satisfaisants.The cells capable of being used for the production of virus according to the invention are all cells capable of multiplying in culture and permissive to the virus which it is desired to produce. They can in particular be VERO cells, CV-1 cells, LLC-MK2 cells, MDCK cells, MDBK cells, WI-38 cells, MRC5 cells (human fibroblasts), or even BHK21 cells. Particularly good results have been obtained with VERO cell cultures or MRC5 cell cultures. Cell culture can be carried out according to the various techniques usually used: in cytocultors, in roller bottles, on Roux boxes, on Multitray, on Cell-Cube. It is preferred, for industrial reasons, to carry out cell culture in cytocultors, using microcarriers constituted for example by Cytodex ™ beads. This cell culture can be carried out under different conditions, in particular as regards the medium used; it is indeed possible to implement the present invention while the culture medium used for the cell growth phase contained serum or at least human or animal proteins; in this case, of course, it will be necessary to carry out a perfect washing of the cells before their infection, so as not to lose the advantage provided by the implementation of the invention as regards the absence of risk of contamination. However, it is preferred to use cell cultures obtained using a cell culture medium also devoid of proteins of human or animal origin. Depending on the nature of the cells used and the viruses to be produced, the most opportune time to infect the virus with a virus inoculum may vary. In general, this is done more in the middle or at the end of the exponential growth phase; it has been observed, in fact, that under these conditions, the results obtained were particularly satisfactory.
Selon l'invention, le milieu de culture utilisé pour l'étape de propagation et de multiplication virales est un milieu dépourvu de protéine humaine , animale ou recombinante, mais comprenant des protéines ou glycoprotéines extraites de pommes de terre ou de concombre, ayant un potentiel mitogène et dont le poids moléculaire est compris entre 1000 et 200 000 daltons et/ou des hydrolysats d'extraits végétaux. Ce milieu est un milieu pouvant comprendre tout ou partie des éléments classiquement utilisés pour la multiplication virale, tel que le milieu HAM F 12, mais additionné d'extraits végétaux, obtenus par exemple de la manière suivante : -réduction de la pulpe végétale, -traitement de la pulpe obtenue par un solvant, -évaporation du solvant,According to the invention, the culture medium used for the viral propagation and multiplication step is a medium devoid of human, animal or recombinant protein, but comprising proteins or glycoproteins extracted from potatoes or cucumber, having a potential mitogenic and whose molecular weight is between 1000 and 200,000 daltons and / or hydrolysates of plant extracts. This medium is a medium which may include all or part of the elements conventionally used for viral multiplication, such as the HAM F 12 medium, but supplemented with plant extracts, obtained for example in the following manner: reduction of the vegetable pulp, treatment of the pulp obtained with a solvent, evaporation of the solvent,
-purification de l'extrait obtenu par chromatographie. Le potentiel mitogène des protéines ou glycoprotéines convenant aux fins de l'invention peut être apprécié grâce notamment à des tests d'analyse de croissance cellulaire et d'activité mitogénique, tels que les tests de coloration au M.T.T.-purification of the extract obtained by chromatography. The mitogenic potential of proteins or glycoproteins suitable for the purposes of the invention can be assessed in particular by means of tests for analysis of cell growth and mitogenic activity, such as tests for staining with M.T.T.
Les extraits végétaux convenant aux fins de l'invention sont notamment ceux décrits dans la demande de brevet FR 2732347. On peut également utiliser les produits commercialisés par la Société Biomedia sous la dénomination GCR1003, TCR1005, ou BCR1008 , seuls ou en combinaison, ou encore le produit PROLIFIX commercialisé également par cette Société et comprenant tous les éléments nutritifs du milieu classique HAM F 12 additionné des extraits végétaux appropriés.The plant extracts suitable for the purposes of the invention are in particular those described in patent application FR 2732347. It is also possible to use the products sold by the company Biomedia under the name GCR1003, TCR1005, or BCR1008, alone or in combination, or alternatively the PROLIFIX product also marketed by this Company and comprising all the nutrients of the conventional HAM F 12 medium supplemented with appropriate plant extracts.
Alternativement, il est possible d'utiliser un milieu classique HAM F 12 additionné d'hydrolysats végétaux tels que les produits HyPep™ à raison de 5 g/1. II est possible également d'utiliser un milieu comprenant à la fois les protéines ou glycoprotéines obtenues par thermocoagulation de la pulpe de végétaux et des hydrolysats végétaux tels que ceux mentionnés ci-dessus.Alternatively, it is possible to use a conventional HAM F 12 medium supplemented with plant hydrolysates such as HyPep ™ products at a rate of 5 g / 1. It is also possible to use a medium comprising both the proteins or glycoproteins obtained by thermocoagulation of the plant pulp and plant hydrolysates such as those mentioned above.
De plus, il est possible d'ajouter à ces milieux tout autre élément susceptible d'améliorer ou de faciliter une des étapes conduisant à l'obtention d'un vaccin, de même qu'il est possible de modifier légèrement ce milieu sans en changer les caractéristiques.In addition, it is possible to add to these environments any other element capable of improving or facilitating one of the steps leading to obtaining a vaccine, just as it is possible to slightly modify this environment without changing it. characteristics.
Les exemples qui suivent illustrent, de façon non limitative des modes de réalisation de l'invention.The examples which follow illustrate, without limitation, embodiments of the invention.
EXEMPLE 1 PRODUCTION DE VACCINS CONTRE L'ENCEPHALITE JAPONAISE.EXAMPLE 1 PRODUCTION OF VACCINES AGAINST JAPANESE ENCEPHALITIS.
On utilise pour produire le virus responsable de l'encéphalite japonaise des cellules VERO. Les cellules utilisées sont des cellules issues de la souche distribuée par l'ATCC ( American Type Culture Collection) sous le N° ATCC-CCL 81- VERO F 1415 qui est au 124e e passage et qui est amené au 137eme passage de façon conventionnelle. Le virus que l'on souhaite produire est le virus de la souche P3 au 88 ™ passage fourni par le NVSI.VERO cells are used to produce the virus responsible for Japanese encephalitis. The cells used are cells derived from the strain distributed by the ATCC (American Type Culture Collection) under the number ATCC-CCL 81- VERO F 1415 which is at the 124th passage and which is brought to the 137th passage in a conventional manner . The virus which it is desired to produce is the virus of the P3 strain at 88 ™ passage supplied by the NVSI.
On remplit la cuve d'un cytoculteur de 15 litres avec du milieu ISCOVE additionné de 4% de sérum de veau contenant des microporteurs Cytodex I™ à une concentration de 3,5g/l. On ensemence le milieu par un inoculum de cellules VERO à raison de 200 000 cellules /ml.The tank of a 15 liter cytocultor is filled with ISCOVE medium supplemented with 4% calf serum containing Cytodex I ™ microcarriers at a concentration of 3.5 g / l. The medium is inoculated with an inoculum of VERO cells at the rate of 200,000 cells / ml.
La culture est amplifiée et subcultivée 2 fois par semaine tous les 4-5 jours. Les concentrations cellulaires que l'on obtient sont comprises entre 2 et 3.106 par ml. On rince ensuite la suspension cellulaire obtenue avec le milieu de propagation virale dans le but d'éliminer le maximum de sérum de veau résiduel. Afin de tester un milieu selon l'invention comparativement à un milieu de l'art antérieur, on effectue la même opération en parallèle, dans plusieurs cytoculteurs identiques ,avec 2 milieux différents : - un milieu selon l'invention constitué par le milieu HAM F 12 (fourni par Life Technologies GIBCO) additionné par du milieu PROLIFIX II fourni par la Société BIOMEDIA , à une concentration 10 fois celle indiquée à leur catalogue de 1996, dans une proportion de 1 volume de PROLIFIX II concentré 10 fois pour 10 volumes de HAM F 12,The culture is amplified and subcultured 2 times a week every 4-5 days. The cell concentrations that are obtained are between 2 and 3.10 6 per ml. The cell suspension obtained is then rinsed with the viral propagation medium in order to eliminate the maximum of residual calf serum. In order to test a medium according to the invention compared with a medium of the prior art, the same operation is carried out in parallel, in several identical cytocultors, with 2 different media: a medium according to the invention consisting of HAM F 12 medium (supplied by Life Technologies GIBCO) added by PROLIFIX II medium supplied by the company BIOMEDIA, at a concentration 10 times that indicated in their 1996 catalog, in a proportion of 1 volume of PROLIFIX II concentrated 10 times for 10 volumes of HAM F 12,
- un milieu selon l'art antérieur constitué par le milieu ISCOVE( fourni par Life Technologies GIBCO) additionné par de l'albumine humaine afin d'obtenir une concentration finale en albumine dans le milieu de 0,3%.- A medium according to the prior art consisting of the ISCOVE medium (supplied by Life Technologies GIBCO) added with human albumin in order to obtain a final concentration of albumin in the medium of 0.3%.
Après rinçage, un inoculum de virus est introduit dans chacun des cytoculteurs, en quantité correspondant à une MOI (Multiplicity Of Infection) de 1/6000. Le contenu des cytoculteurs est ensuite maintenu sous agitation à une température de 37°C pendant 3 jours, puis on procède à la lCTe récolte par récupération du milieu de propagation virale qui est devenu une suspension virale, que l'on remplace dans chacun des cytoculteurs par une quantité égale de milieu frais de même nature ; on effectue ensuite toutes les 24 heures une récolte du milieu de propagation, ceci jusqu'à 5 récoltes. Chacune des récoltes est filtrée sur filtre dont le seuil de coupure est de 0,22μm, et stockée à +5°C en attendant d'être mélangée avec les 4 autres récoltes issues du même cytoculteur. Lorsque les 5 récoltes ont été effectuées, elles sont mélangées, puis ce mélange est concentré par filtration jusqu'à un facteur de 100. Le mélange constitué par les 5 récoltes issues d'un même cytoculteur constitue un lot.After rinsing, an inoculum of virus is introduced into each of the cytocultors, in an amount corresponding to an MOI (Multiplicity Of Infection) of 1/6000. The content of the cytocultors is then kept stirring at a temperature of 37 ° C. for 3 days, then the CTe is harvested by recovery of the viral propagation medium which has become a viral suspension, which is replaced in each of the cytocultors with an equal amount of fresh medium of the same nature; the propagation medium is then harvested every 24 hours, this up to 5 harvests. Each of the crops is filtered on a filter whose cutoff threshold is 0.22 μm, and stored at + 5 ° C while waiting to be mixed with the 4 other crops from the same cytocultor. When the 5 harvests have been carried out, they are mixed, then this mixture is concentrated by filtration to a factor of 100. The mixture consisting of the 5 harvests from the same cytocultor constitutes a batch.
Afin de déterminer la quantité de virus produits, on réalise pour chacun des lots produits un test de l'activité infectieuse. Le test est réalisé de 2 façons différentes : soit en activité infectieuse mesurée sur nappe cellulaire, soit en activité infectieuse mesurée sur souris par inoculation intracérébrale aux souris, observation des animaux pendant 14 jours et calcul de DL50 .In order to determine the quantity of virus produced, an infectious activity test is carried out for each of the batches produced. The test is carried out in 2 different ways: either in infectious activity measured on the cell table, or in infectious activity measured in mice by intracerebral inoculation in mice, observation of the animals for 14 days and calculation of LD50.
Les résultats sont exprimés en Log 10 de particules infectieuses par litre de cytoculteur. Lorsqu'on considère le titrage sur cellules, la moyenne des résultats obtenus pour les différents lots produits en utilisant un milieu selon l'invention est de 8,84 alors que la moyenne obtenue pour les lots produits en utilisant un milieu selon l'art antérieur est de 7,85. Si on considère le titrage sur souris, la moyenne pour les lots produits selon l'invention est cette fois de 9,22 alors que la moyenne des lots produits avec un milieu selon l'art antérieur est de 9,30. On constate donc, de façon inattendue , que les résultats obtenus avec le milieu selon l'invention sont équivalents à ceux obtenus avec un milieu de l'art antérieur, alors qu'on s'attendait à ce que le défaut d'albumine qui est une protéine considérée comme jouant un rôle important dans le transport des éléments nutritifs à l'intérieur de la cellule, ait pour conséquence une réduction importante de la quantité de virus produits.The results are expressed in Log 10 of infectious particles per liter of cytocultor. When considering the titration on cells, the average of the results obtained for the various batches produced using a medium according to the invention is 8.84 while the average obtained for the batches produced using a medium according to the prior art is 7.85. If we consider the titration on mice, the average for the batches produced according to the invention is this time 9.22 while the average of the batches produced with a medium according to the prior art is 9.30. It is therefore found, unexpectedly, that the results obtained with the medium according to the invention are equivalent to those obtained with a medium of the prior art, whereas it was expected that the defect in albumin which is a protein considered to play an important role in the transport of nutrients inside the cell, results in a significant reduction in the amount of virus produced.
Les lots obtenus ont ensuite été inactivés par l'ajout d'une solution de formaldéhyde en quantité permettant d'avoir une concentration finale dans le mélange de 1/4000 et maintenue à température ambiante sous agitation continue pendant 14 jours. La vérification de l'inactivation par un contrôle selon le protocole recommandé par l'OMS ( Rapport technique 771 de 1988) a montré que les lots produits étaient conformes aux recommandations de l'OMS.The batches obtained were then inactivated by the addition of a formaldehyde solution in an amount making it possible to have a final concentration in the mixture of 1/4000 and maintained at room temperature with continuous stirring for 14 days. Verification of inactivation by control according to the protocol recommended by WHO (Technical Report 771 of 1988) showed that the batches produced complied with WHO recommendations.
Ces lots inactivés ont ensuite été purifiés par chromatographie échangeuse d'ions et par gel filtration afin d'en éliminer toutes les protéines virales et les impuretés résiduelles non souhaitées dans les vaccins. Le mélange obtenu a ensuite été formulé pour conduire à la production de doses vaccinales. La bonne qualité des vaccins produits a été vérifiée grâce au test d'immunogénicité sur souris recommandé par l'OMS pour les vaccins contre l'encéphalite japonaise, consistant à déterminer le taux d'anticorps meutralisants produits par des souris immunisées. L'activité des vaccins produits selon le procédé de l'invention n'étant pas inférieure à celle des vaccins de l'art antérieur, cela confirme qu'il est possible, grâce au milieu et au procédé selon l'invention, de produire des vaccins viraux dans des conditions de sécurité accrue, en s' affranchissant de l'utilisation d'albumine.These inactivated batches were then purified by ion-exchange chromatography and by gel filtration in order to remove all the viral proteins and residual impurities not desired in the vaccines. The mixture obtained was then formulated to lead to the production of vaccine doses. The good quality of the vaccines produced was verified using the immunogenicity test on mice recommended by the WHO for vaccines against Japanese encephalitis, which consists of determining the level of neutralizing antibodies produced by immunized mice. The activity of the vaccines produced according to the method of the invention not being inferior to that of the vaccines of the prior art, this confirms that it is possible, thanks to the medium and to the method according to the invention, to produce viral vaccines in conditions of increased safety, by avoiding the use of albumin.
EXEMPLE 2 : COMPARAISON DE DIFFERENTS MILIEUX SELON L'INVENTION.EXAMPLE 2 COMPARISON OF DIFFERENT MEDIA ACCORDING TO THE INVENTION
On utilise pour tester différents milieux de façon comparative, des flacons de 150 cm3' que l'on remplit par 50 ml de milieu ISCOVE additionné de 4 % de sérum de veau. Ce milieu est ensemencé par un inoculum de cellules VERO à raison de 250000 cellules / cm2' . Les flacons sont laissés 4-5 jours à 37 ° C puis sont rincés au moyen du milieu de multiplication et de propagation virales. Les milieux utilisés sont les suivants :To compare different media, 150 cm 3 'flasks are used which are filled with 50 ml of ISCOVE medium supplemented with 4% calf serum. This medium is seeded with an inoculum of VERO cells at the rate of 250,000 cells / cm 2 '. The vials are left for 4-5 days at 37 ° C and then rinsed using the viral multiplication and propagation medium. The media used are as follows:
- un milieu A qui est un un milieu selon l'art antérieur constitué par le milieu ISCOVE( fourni par Life Technologies GIBCO) additionné par de l'albumine humaine afin d'obtenir une concentration finale en albumine dans le milieu de 0,3%,a medium A which is a medium according to the prior art constituted by the ISCOVE medium (supplied by Life Technologies GIBCO) added with human albumin in order to obtain a final concentration of albumin in the medium of 0.3% ,
- un milieu B constitué par du milieu HAM F 12 additionné des molécules mitogènes suivantes : * BCR 1008 à raison de 10 ~3 g/1- a medium B consisting of HAM F 12 medium supplemented with the following mitogenic molecules: * BCR 1008 at a rate of 10 ~ 3 g / 1
* TCR 1005 à raison de 10 ~4 g/1* TCR 1005 at a rate of 10 ~ 4 g / 1
* GCR 1003 à raison de 10"4g/l.* GCR 1003 at a rate of 10 "4 g / l.
- un milieu C constitué par le milieu cité ci-dessus ( HAM F 12 additionné de BCR 1008, de TCR 1005, de GCR 1003) mais comprenant en outre lg/1 du produit HYPEP 4602 constitué par de l'hydrolysat de gluten et fourni par la Société QUESTEL International,- a medium C consisting of the medium mentioned above (HAM F 12 added with BCR 1008, TCR 1005, GCR 1003) but also comprising lg / 1 of the product HYPEP 4602 consisting of gluten hydrolyzate and supplied by the QUESTEL International Company,
- un milieu C identique au milieu C mais dans lequel la concentration en HYPEP 4602 est de 5g/l- a medium C identical to medium C but in which the concentration of HYPEP 4602 is 5g / l
- un milieu D constitué par du milieu HAM F 12 additionné de HYPEP 4602 à raison de lg/1, mais dépourvu des molécules mitogènes BCR 1008, TCR 1005, oua medium D consisting of HAM F 12 medium supplemented with HYPEP 4602 at a rate of lg / 1, but devoid of the mitogenic molecules BCR 1008, TCR 1005, or
GCR 1003.GCR 1003.
- un milieu D' identique au milieu D mais dans lequel la concentration en HYPEP 4602 est de 5g/l.- A medium D 'identical to medium D but in which the concentration of HYPEP 4602 is 5g / l.
Après rinçage,un inoculum de virus de l'encéphalite japonaise identique à celui de l'exemple précédent, est introduit dans chacun des flacons en quantité correspondant à une MOI de 1/6000.After rinsing, an inoculum of Japanese encephalitis virus identical to that of the previous example, is introduced into each of the bottles in an amount corresponding to an MOI of 1/6000.
Le contenu des flacons est ensuite maintenu pendant 3 jours à 37°C avant que l'on procède à la lere récolte. Les récoltes suivantes sont effectuées de la même manière que décrite à l'exemple précédent. Les tests de détermination de titre infectieux sont effectués sur cellules BHK21 et donnent les résultats suivants, exprimés en DICCso/ml ( Log 10) :The content of the vials is then maintained for 3 days at 37 ° C before it proceeds to the harvest ere. The following harvests are carried out in the same manner as described in the previous example. The infectious titre determination tests are carried out on BHK21 cells and give the following results, expressed in DICC 50 / ml (Log 10):
Milieu A : 4,6Middle A: 4.6
Milieu B :5,1 Milieu C : 5,0Middle B: 5.1 Middle C: 5.0
Milieu C : 5,6Middle C: 5.6
Milieu D : 5,4Middle D: 5.4
Milieu D' : 6,3.Middle: 6.3.
Ces résultats confirment que les milieux selon l'invention permettent d'obtenir d'aussi bons rendements, si ce n'est meilleurs que les milieux selon l'art antérieur qui contiennent de l'albumine.These results confirm that the media according to the invention make it possible to obtain as good yields, if not better than the media according to the prior art which contain albumin.
EXEMPLE 3 PRODUCTIONDE VACCIN CONTRE LARAGEEXAMPLE 3 VACCINE AGAINST LARAGE PRODUCTION
On réalise une culture de cellules VERO dans un cytoculteur de 21 en milieu classique, puis après lavage, on infecte les cellules par du virus de la rage avec une MOI de 1/1000, et on maintient les cellules dans un milieu identique à celui décrit à l'exemple 1 (soit milieu HAM FI 2 supplémenté en PROLIFIX™ II) ; la même expérience est réalisée de façon parfaitement identique, dans un autre cytoculteur de même capacité, avec comme seule différence l'utilisation d'un milieu de propagation et de multiplication virales de l'art antérieur comprenant de l'albumine humaine. On effectue des récoltes successivement 4, 5 , 6 et 7 jours après l'infection. Les numérations effectuées sur les récoltes obtenues grâce au milieu selon l'invention et sur les récoltes obtenues grâce au milieu selon l'art antérieur ont permis d'obtenir des résultats comparables avec les 2 milieux ; de même, les titres infectieux des virus obtenus étaient équivalents.A culture of VERO cells is carried out in a cytocultor of 21 in conventional medium, then after washing, the cells are infected with rabies virus with an MOI of 1/1000, and the cells are maintained in a medium identical to that described in Example 1 (ie HAM FI 2 medium supplemented with PROLIFIX ™ II); the same experiment is carried out in exactly the same way, in another cytocultor of the same capacity, with the only difference being the use of a viral propagation and multiplication medium of the prior art comprising human albumin. Harvests are carried out successively 4, 5, 6 and 7 days after infection. The counts carried out on the crops obtained thanks to the medium according to the invention and on the crops obtained thanks to the medium according to the prior art made it possible to obtain results comparable with the 2 media; similarly, the infectious titers of the viruses obtained were equivalent.
Ces résultats démontrent donc la possibilité de produire sur cellules du virus contre la rage.These results therefore demonstrate the possibility of producing rabies virus cells.
EXEMPLE 4 PRODUCTION DE VACCIN CONTRE LA POLIOMYELITE.EXAMPLE 4 PRODUCTION OF POLIOMYELITIS VACCINE.
On a effectué des essais de production de virus de la poliomyélite de type 1 sur des cellules VERO en culture. Les titres infectieux des virus obtenus en utilisant un milieu de propagation et de multiplication virales selon l'invention ayant la composition décrite à l'exemple 1 étaient équivalents à la moyenne des titres infectieux obtenus avec les récoltes effectuées avec le milieu utilisé pour la fabrication des vaccins actuellement commercialisés. Polio type 1 virus production assays were performed on cultured VERO cells. The infectious titers of the viruses obtained using a viral propagation and multiplication medium according to the invention having the composition described in Example 1 were equivalent to the average of the infectious titers obtained with the harvests carried out with the medium used for the manufacture of the currently marketed vaccines.

Claims

REVENDICATIONS
1. Milieu de propagation et de multiplication virales sur des cellules en culture, caractérisé en ce qu'il est dépourvu de protéine humaine ou animale, même d'origine recombinante, et en ce qu'il comprend des extraits végétaux.1. Medium for viral propagation and multiplication on cells in culture, characterized in that it is devoid of human or animal protein, even of recombinant origin, and in that it comprises plant extracts.
2. Milieu selon la revendication 1, caractérisé en ce qu'il comprend des protéines ou glycoprotéines mitogènes extraites de pommes de terre ou de concombre.2. Medium according to claim 1, characterized in that it comprises mitogenic proteins or glycoproteins extracted from potatoes or cucumber.
3. Milieu selon une des revendications précédentes, caractérisé en ce que les protéines ou glycoprotéines sont obtenues de la manière suivante :3. Medium according to one of the preceding claims, characterized in that the proteins or glycoproteins are obtained in the following manner:
• réduction de la pulpe de pomme de terre ou de concombre, traitement de la pulpe par un solvant, B traitement par thermocoagulation,• reduction of the potato or cucumber pulp, treatment of the pulp with a solvent, B treatment by thermocoagulation,
• purification par chromatographie.• purification by chromatography.
4. Milieu selon une des revendications précédentes, caractérisé en ce qu'il comprend des hydrolysats d'extraits végétaux.4. Medium according to one of the preceding claims, characterized in that it comprises hydrolysates of plant extracts.
5. Milieu selon une des revendications précédentes, caractérisé en ce que les cellules utilisées pour le support de la propagation et de la multiplication virales sont des cellules VERO.5. Medium according to one of the preceding claims, characterized in that the cells used for the support of viral propagation and multiplication are VERO cells.
ό.Milieu selon une des revendications précédentes, caractérisé en ce que les virus produits sont des virus de l'encéphalite japonaise, de la rage, de la poliomyélite, de l'hépatite A, de la grippe, de la Dengue, de la rougeole, des oreillons, de la varicelle, de la rubéole.ό.A medium according to one of the preceding claims, characterized in that the viruses produced are viruses of Japanese encephalitis, rabies, poliomyelitis, hepatitis A, influenza, Dengue, measles , mumps, chickenpox, rubella.
7. Milieu selon une de revendications précédentes, caractérisé en ce que les virus produits sont des virus de l'encéphalite japonaise destinés à être utilisés dans des vaccins humains. 7. Medium according to one of the preceding claims, characterized in that the viruses produced are Japanese encephalitis viruses intended for use in human vaccines.
8. Procédé de fabrication de vaccins viraux comportant une phase de production virale sur cellules en culture, caractérisé en ce qu'on procède à la phase de propagation et de multiplication des virus en présence d'un milieu dépourvu de protéine humaine ou animale, même d'origine recombinante, mais comprenant des extraits végétaux.8. A method of manufacturing viral vaccines comprising a phase of viral production on cells in culture, characterized in that one proceeds to the phase of propagation and multiplication of viruses in the presence of a medium devoid of human or animal protein, even of recombinant origin, but comprising plant extracts.
9. Procédé selon la revendication précédente, caractérisé en ce que le milieu de propagation et de multiplication virales comprend des protéines ou glycoprotéines extraites de pommes de terre ou de concombre, ayant un potentiel mitogène et dont le poids moléculaire est compris entre 1 000 et 200 000 daltons.9. Method according to the preceding claim, characterized in that the viral propagation and multiplication medium comprises proteins or glycoproteins extracted from potatoes or cucumber, having a mitogenic potential and whose molecular weight is between 1000 and 200 000 daltons.
10. Procédé selon une des revendications 8 ou 9, caractérisé en ce que le milieu de propagation et de multiplication virales comprend des hydrolysats végétaux. 10. Method according to one of claims 8 or 9, characterized in that the viral propagation and multiplication medium comprises plant hydrolysates.
PCT/FR1999/000578 1998-03-13 1999-03-15 Medium and method for viral propagation and growth WO1999047648A2 (en)

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