Classifications

• • 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
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  • 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
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  • 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
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  • 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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Citations (3)

• 1998
  • 1998-03-13 FR FR9803333A patent/FR2775983B1/fr not_active Expired - Fee Related
• 1999
  • 1999-03-15 EA EA200000934A patent/EA200000934A1/ru unknown
  • 1999-03-15 AU AU27352/99A patent/AU752577B2/en not_active Ceased
  • 1999-03-15 EP EP99907713A patent/EP1062324A2/fr not_active Withdrawn
  • 1999-03-15 HU HU0101081A patent/HUP0101081A3/hu unknown
  • 1999-03-15 IL IL13825099A patent/IL138250A0/xx unknown
  • 1999-03-15 CA CA 2322619 patent/CA2322619A1/en not_active Abandoned
  • 1999-03-15 WO PCT/FR1999/000578 patent/WO1999047648A2/fr not_active Application Discontinuation
  • 1999-03-15 TR TR200002641T patent/TR200002641T2/xx unknown
  • 1999-03-15 CN CN99803965A patent/CN1301297A/zh active Pending
  • 1999-03-15 PL PL34355799A patent/PL343557A1/xx not_active Application Discontinuation
  • 1999-03-15 SK SK1288-2000A patent/SK12882000A3/sk unknown
  • 1999-03-15 NZ NZ506872A patent/NZ506872A/en unknown
  • 1999-03-15 KR KR1020007010026A patent/KR20010072556A/ko not_active Application Discontinuation
  • 1999-03-15 BR BR9908744A patent/BR9908744A/pt not_active IP Right Cessation
  • 1999-03-15 JP JP2000536831A patent/JP2002506636A/ja not_active Withdrawn

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