SK12882000A3 - Medium and method for viral propagation and growth - Google Patents

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

Technical field

The invention relates to an environment for propagation and multiplication of viruses and to a method for propagation and multiplication of viruses on cells in culture, in particular for the production of vaccines.

BACKGROUND OF THE INVENTION

The production of viral vaccines, whether attenuated, inactivated, subunits or recombinants, involves large-scale virus production.

This production can be carried out depending on the nature of the virus on various carriers that allow the virus to replicate; for example, for most commercially available influenza vaccines, virus proliferation occurs on seeded hen eggs, while for some Japanese encephalitis vaccines, this operation is performed on mouse brains.

The use of such carriers encounters many problems: availability, reproducibility, but mainly safety due to possible contamination by viruses, myoplasm or any other undesirable elements coming from the carrier. Thus, efforts are being made to leave such carriers, and wherever possible, cell cultures are used which are infected with a small amount of the virus to be produced and which are then maintained under conditions that promote virus replication within the infected cells as well as propagation of the infection. from one cell to another. The viruses produced are then collected either from the cell bath culture medium when they are intracellular viruses that leave the cell (which has the advantage of allowing several virus collections with the same cell culture) or by processing the cells themselves when they are intracellular viruses.

Good quality cell cultures are required for these methods; therefore, many of the prior development work is concerned with improving cell cultures and, in particular, the culture media used. To reduce the risk of cell contamination by myoplasm, viruses, bovine spongy encephalopathy agents or

PV-1288-00,79 / Täfl 2.11.2000 by any other unusual transmissible agents, the use of serum-free and even protein-free culture media has been proposed (WO 96/15231). The use of an animal serum-free cell culture medium but containing plant extracts is described (French Patent Specification FR 2 732347).

When using such cell culture environments, the safety of the products obtained using the cells so produced increases.

However, when it comes to the production of viruses forming part of the vaccine compositions, the procedures following this phase of cell culture should also exhibit the maximum degree of safety themselves.

Usually (for example, U.S. Pat. No. 4,525,349), when cells are used in a culture medium for virus production, once the culture phases have been carried out, the medium used to cultivate the cells is removed and replaced with a new environment in which the cells some washing time for washing: this new environment is then removed: this cell washing process may possibly be repeated several times.

A new environment is then introduced into the cell culture which first allows the cells to survive and be infected with viruses, and secondly, elements that are sufficient as carriers for the replication of these viruses must be available. However, as is known in the literature ("Protein-free culture of Vero cells: A substrate for replication of human pathogenic viruses" Cell Biology International, Vol. 17, No. 9, pp. 885-895, 1993), although cells cultivate in a protein-free environment, culture medium used according to /

The prior art at this stage of virus multiplication and propagation typically comprises fetal calf serum or, to reduce pollution problems, at least human albumin. The use of human albumin at this stage of the process for producing viral vaccines is considered without risk to the quality of the vaccine. However, in order to completely eliminate all hazards and theoretically, it would be desirable to have an environment suitable for propagation and multiplication of viruses on a carrier consisting of a cell culture medium which is free of human or animal protein and recombinant origin but which nevertheless allows production results. which are compatible with industrial requirements not to increase the cost of producing virus-containing vaccines grown on cell cultures.

PV-1288-00,79 / Ta / 12.11.2000

In particular, it is an object of the invention to provide such an environment. It is also an object of the present invention to provide such an environment and method for propagation and multiplication of viruses which optionally allows virus inactivation.

SUMMARY OF THE INVENTION

According to the invention, the medium for propagation and multiplication of viruses on cells in the culture medium is based on the fact that it is free of human and animal protein and of recombinant origin and contains plant extracts.

According to one embodiment of the invention, the virus propagation and multiplication medium comprises proteins or glycoproteins extracted from potatoes and cucumbers having a mitogenic potential and a molecular weight of 1000 to 200,000 daltons.

According to another embodiment of the invention, the medium comprises plant hydrolysates.

According to yet another embodiment of the invention, the proteins or glycoproteins are extracted from potatoes.

The invention also relates to proteins and glycoproteins obtained by the method

- the plants are washed and ground,

- the product is diluted in the aquatic environment,

the mixture obtained is thermally coagulated, and

- the product is purified by chromatography.

According to the invention, plant hydrolysates are obtained by chemical and / or

I by enzymatic hydrolysis of raw materials such as cotton, soybeans, wheat or rice. Particularly good results are obtained with products of the HyPep ™ series (commercial products of Quest International), in particular products containing a high proportion of peptides with a molecular weight of less than 1000 daltons.

According to the invention, the virus propagation and multiplication environment is particularly suitable for multiplying viruses on Vera cells.

The invention also relates to an environment for propagation and multiplication of viruses, particularly suitable for multiplication of Japanese encephalitis, infant and rabies viruses.

PV-1288-00,79 / TS / 12.11.2000

According to the invention, a method for producing viral vaccines in which viruses are produced in cell culture is based on the fact that the virus propagation and multiplication phase is carried out in the presence of an environment which is free of human and animal protein and recombinant origin and which contains plant extracts.

The invention is illustrated in more detail by the following detailed description.

The medium or medium and the method of the invention are suitable for the production of all viruses that are capable of replicating in a cell culture medium. In particular, they may be viruses belonging to the following families: orthomyxovirus, para myxovirus, reovirus, picomavirus, flavivirus, arenavirus, herpesvirus, poxvirus and adenoviruses. They may also be recombinant viruses. The invention is particularly suitable for the production of viruses which may form part of vaccine compositions and in particular for human vaccines where safety requirements are maximized. These may include, but are not limited to, childhood, rabies, Japanese encephalitis, yellow fever, rubella, mumps, Deng fever or measles, various forms of jaundice, AIDS or smallpox, a group of herpes-causing viruses, respiratory syncytial virus, rotavirus or influenza virus.

The invention is particularly suitable for the production of viruses that are the cause of childhood rabies, rabies, Japanese encephalitis, rubella, smallpox, jaundice type A, influenza, Deng's fever, measles or mumps.

The cells that can be used to produce the viruses of the invention are all cells that can be multiplied in the culture medium and that are accessible to the virus to be produced. These may be Vero cells, CV-1, LLC-MK2, MDCK, MDBK, WI-38, MRC5 (human fibroblasts), or BHK21 cells. Particularly good results are obtained with cultures of Vero or MRC5 cells.

Cell culture can be performed in a variety of ways: using a cell cultivator, roller flasks, Roux flasks, Multitrays ™ and CellCubes ™ For industrial purposes, it is preferred to grow cells in cell cultivators using microcarriers such as Cytodex ™ beads.

The cultivation of the cells can be carried out under various conditions, especially with regard to the environment used; according to the invention, the method is possible even if the cell culture medium used comprises serum or at least human or animal proteins; in this case, however, a thorough washing of the cells before

PV-1288-00,79 / ΤϊΛ 2.11.2000 by infecting them so as not to lose the advantage of the method according to the invention, which is the elimination of the danger of contamination. However, it is preferred to use cultures obtained from a culture medium which is free of proteins of human or animal origin.

Depending on the cells used and the viruses to be produced, the most advantageous time of infection of the virus with the virus vaccine may vary. Generally, this operation is performed in the middle or at the end of the exponential growth phase; indeed, it has been shown that under these conditions the results obtained are particularly satisfactory.

According to the invention, the culture medium is used to propagate and multiply the virus without human and animal or recombinant proteins, but contains proteins or glycoproteins extracted from potatoes or cucumbers having a mitogenic potential and a molecular weight of 1000 to 200,000 daltons and / or hydrolyzates of plant extracts. This medium is a medium that may contain all or part of the elements commonly used for virus propagation, such as HAM F 12, but which is supplemented by plant extracts obtained, for example, by

- reduces plant marrow,

- the pulp obtained is treated with a solvent,

- the solvent is evaporated,

- the extract obtained is purified by chromatography.

The myogenic potential of proteins or glycoproteins suitable for the purposes of the invention can be evaluated, in particular, using assays for analysis of cell growth and mitogenic activity, such as the M.T.T.

The plant extracts suitable according to the invention are in particular those described in French patent specification FR 2 732347. It is also possible to use the commercial products GCR1003, TCR1005 or BCR1008 (Biomedia) alone or in combination or the product Prolifix (same company) containing all nutrient products of the conventional HAM F 12 culture medium supplemented with appropriate plant extracts.

Alternatively, a conventional culture medium and HAM F 12 supplemented with plant hydrolysates such as HyPep ™ products may be used in an amount of 5 g / L.

PV-1288-00,79 / TS / l 2.11.2000

It is also possible to use a culture medium containing both proteins or glycoproteins obtained by heat coagulation of the plant pulp and the above-mentioned plant hydrolysates.

In addition, it is possible to add to this environment any other element that can improve or facilitate one of the following operations leading to vaccine production, as long as a slight modification of this environment is possible without changing its characteristics.

The invention is illustrated by the following examples.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Production of Japanese encephalitis vaccines

Vero cells are used to produce viruses that cause Japanese encephalitis. The cells used are derived from a strain distributed by ATCC (American Type Culture Collection) under number ATCC-CCL 81VERO F 1415, which is in passage 124 and has been routinely transferred to passage 137. The desired product is strain P3 virus at 88 passage supplied by NVSI.

A 15 liter cell cultivator tank is filled with Iscove medium supplemented with calf serum containing Cytodex 1 ™ microcarriers at 3.5 g / L. The medium is inoculated with a Vero cell vaccine of 200,000 cells per ml.

The culture is amplified and subcultured twice a week every 4 and 5 days. The concentration of cells obtained is 2 x 10 ⁶ to 3 x 10 ⁶ per ml. The cell suspension obtained is then rinsed with virus propagation media to remove the remaining calf serum as much as possible.

In order to test the medium according to the invention in comparison with the medium according to the prior art, a parallel process is carried out in several identical cell cultivators with two different media:

- medium according to the invention consisting of medium HAM F 12 (Life Technologies Gibco) supplemented with medium Prolifix II (Biomedia) in a ten-fold concentration

PV-1288-00, 79 / TSZI 2.11.2000 compared to their 1996 catalog in proportions of 1 volume of 10-fold concentrated Prolifix II per 10 volumes of HAM F 12.

a prior art medium consisting of Iscove medium (Life Technologies Gibco) supplemented with human albumin to achieve a final 0.3% albumin concentration in the medium.

After washing, a viral vaccine in an amount corresponding to an MOI (Multiplicity Of Infection) of 1/6000 is introduced into each cell cultivator.

The contents of the cell cultivators are then allowed to stir for 3 days at 37 ° C, harvesting 1 to recover the virus propagation medium which has become a viral suspension which is returned to each cell cultivator with the same amount of fresh medium of the same nature; it is harvested every 24 hours until 5 are collected. Each harvest is filtered through a filter with the lowest value of 0.22 gm and stored at + 5 ° C until mixed with 4 additional harvestings from the same cell cultivator.

After 5 collections, the collections were mixed and this mixture was concentrated by filtration 100-fold. The mixture consisting of five collections from the same cell cultivator forms a batch.

To determine the amount of virus produced, a test for the infectious activity of each batch produced is performed. The assay is carried out in two different ways: either infectious activity measured on the cell layer or infectious activity measured in mice by intracerebral inoculation of mice, observation of animals for 14 days and calculation of LD50. .

Results are expressed as log 10 infectious particles per liter of cell cultivator. The mean value obtained by titration for the different batches produced using the medium of the invention is 8.84, while the mean value obtained from the batches produced using the prior art medium is 7.85. The mean values of the titration results for mice for the lots of the invention are 9.22, while the mean of the lots produced using the prior art medium is 9.30. Thus, it has been unexpectedly found that the results obtained with the medium of the invention are equivalent to those obtained with the prior art medium, and one would expect the albumin deficiency, which is considered to be a significant protein in the transport of nutrient elements within the cell. will result in a significant decrease in the amount of virus produced.

PV-1288-00,79 / Ti / 12.11.2000

The batches obtained are then inactivated with formaldehyde solution in an amount corresponding to the final concentration in the 1/4000 mixture and left at room temperature for 14 days with stirring. Verification of inactivation by inspection of protocol recommended by WHO (Technical Bulletin 771 of 1988) showed that the batches produced comply with WHO recommendations.

These inactivated lots are purified by ion exchange chromatography and gel filtration to remove any contained viral proteins and residual impurities that are undesirable in the vaccines. The resulting mixture is formulated to produce vaccine doses. The good quality of the vaccines produced is verified by an immunogenicity test in mice according to the WHO recommendations for Japanese encephalitis vaccines, which is based on the determination of the amount of neutralizing antibodies produced in immunized mice. Since the activity of the vaccines produced by the method of the invention is not lower than that of the prior art, the possibility of using the method of the invention to produce a viral vaccine under conditions of increased safety, excluding albumin, is confirmed.

Example 2

Comparison of different media according to the invention

150 cm ³ flasks filled with 50 ml Iscove medium supplemented with 4% calf serum are used for comparative testing of different media. This medium is inoculated with Vera cells at 250,000 cells / cm ³ . The flasks are allowed to stand for 4 to 5 days at 37 ° C, sprayed with virus propagation and multiplication media.

The media used are:

- medium A which is a prior art medium consisting of Iscove medium (Life Technologies Gibco) supplemented with human albumin to a final albumin concentration of 0.3%,

- medium B, consisting of HAM F 12 medium supplemented with the following mitogenic molecules:

* BCR 1008 at 10 ^-3 g / L; * TCR 1005 at 10 ^-4 g / L; * GCR 1003 at 10 ^-4 g / L;

PV-1288-00.79 / TS / I2.11.2000

- environment C, consisting of the above medium (HAM F 12, supplemented by BCR

1008, TCR 1005 and GCR 1003), but also containing 1 g / L HyPep

4602, which consists of gluten hydrolyzate (a product of Questel

International)

- medium C 'which is identical to medium C but with a HyPep 4602 content of 5 g / i,

- medium D, consisting of HAM F 12 medium, supplemented with 1 g / l HyPep 4602 but free of BCR 1008, TCR 1005 or GCR 1003 mitogenic molecules,

DS medium identical to medium D, but having a HyPep 4602 of 5 g / l.

After rinsing, Japanese encephalitis virus vaccine, identical to the vaccine of the preceding example, is introduced into each flask in an amount corresponding to MO11 / 6000.

The contents of the flasks are then allowed to stand at 37 ° C for 3 days before the first harvest. Further collections are carried out as in the previous example.

Tests for infectious titer determination are performed on BHK21 cells with the following results expressed as TCID 50 / ml (log 10);

environment 4.6 environment B 5.1 environment C 5.0 environment C ' 5.6 environment D 5.4 D 'environment 6.3

These results confirm that the medium of the invention makes it possible to obtain yields that are as good, if not better, than the prior art medium containing albumin.

Example 3

Manufacture of rabies vaccine

PV-1288-00,79 / TSZI2.11.2000

In a two-liter cultivator, a culture of Vero cells is prepared in a conventional environment and after washing, the cells are infected with rabies virus with MO11 / 1000 and maintained in an environment identical to that described in Example 1 (HAM F 12 supplemented with Profilix ™). II); the same experiment is performed in exactly the same manner in another cultivator of the same volume, with the only difference that the virus propagation and multiplication medium is according to the prior art and contains human albumin. Harvesting takes place sequentially on the 4th, 5th, 6th and 7th day after infection. The totals of the collections obtained using the medium according to the invention make it possible to obtain comparable results for both environments; infectious titers of the obtained viruses are similarly identical.

These results thus demonstrate the possibility of growing rabies vaccine on cells.

Example 4

Production of polio vaccine

Tests for the production of type 1 polyomyelitis virus on Vero cell culture are performed. Virus infectious titers obtained using the virus propagation and multiplication environment of the invention having the composition of Example 1 are equivalent to the average of the infectious titers obtained from the collections performed with the medium used to produce commercially available vaccines.

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Industrial usability

Production of a vaccine against various viruses using a human albumin free environment.

Claims (7)

NEW PATENT CLAIMS Use of a culture medium which is free of human or animal protein and of recombinant origin and which contains plant extracts for propagation and multiplication of viruses in cell culture. The use according to claim 1 of an environment comprising mitogenic proteins or glycoproteins extracted from potatoes or cucumbers. The use according to claim 1 or 2 of plant extracts obtained so as to: - the plants are washed and ground, - the product is diluted in the aquatic environment, the mixture obtained is thermally coagulated, and - the product is purified by chromatography. Use according to claims 1 to 3 of a culture medium containing hydrolyzates of plant extracts. Use according to claims 1 to 4 as a carrier for propagation and multiplication of Vera cells. ) Use according to claims 1 to 5 for the production of Japanese encephalitis, rabies, polio, hepatitis A, influenza, Deng fever, mumps, smallpox and rubella. Use according to claims 1 to 6 for the production of Japanese encephalitis viruses for human vaccines.