WO2011093692A1 - Method for newcastle disease virus propagation in a bioreactor - Google Patents

Method for newcastle disease virus propagation in a bioreactor Download PDF

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Publication number
WO2011093692A1
WO2011093692A1 PCT/MY2011/000006 MY2011000006W WO2011093692A1 WO 2011093692 A1 WO2011093692 A1 WO 2011093692A1 MY 2011000006 W MY2011000006 W MY 2011000006W WO 2011093692 A1 WO2011093692 A1 WO 2011093692A1
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Prior art keywords
bioreactor
virus
cells
culture
propagation
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PCT/MY2011/000006
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French (fr)
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Maizirwan Mel
Noorhayati Esa
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International Islamic University Malaysia
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • 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
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18151—Methods of production or purification of viral material

Definitions

  • This invention relates to a method for virus propagation in a bioreactor, and more particularly, to a method for propagating Newcastle Disease Virus in a bioreactor using Vero cells.
  • Newcastle Disease Virus is a virus that causes the Newcastle disease in chickens, turkeys and other birds.
  • the Newcastle disease is highly contagious in birds and is of particular importance in domestic poultry due to their high susceptibility to infection.
  • the NDV virus is therefore a threat to the poultry industry, resulting in a global problem with severe economic implications.
  • it is possible to produce a vaccine against the virus.
  • the virus In humans who have been exposed to infected birds, the virus causes mild conjunctivitis and influenza-like symptoms but otherwise poses no hazard to human health.
  • the NDV virus has also been studied as a potential anticancer agent due to its selective toxicity towards human tumour cells. Therefore, a method for large-scale propagation of the NDV virus, whether for production of vaccines or other therapeutic agents such as anticancer agents, is beneficial.
  • the cost of the eggs and the cost of isolating and incubating the infected eggs result in high production costs for the vaccine. Furthermore, some people are allergic to egg protein, making egg-based products unacceptable for them. If the NDV virus is to be used as a potential therapeutic agent in humans, it would be preferable to avoid egg-based systems. It is therefore desirable to find an alternative method to large scale production of the NDV. virus that does not involve the use of eggs.
  • bioreactor based culture system An alternative to the traditional chicken egg-based culture system is the bioreactor based culture system.
  • This system uses cells in bioreactor culture to propagate the virus. Vaccine can be produced in a shorter time, control of process parameters is easier and eggs are not required.
  • the process begins when a cell line is infected with the seed virus. The critical step is the availability of the seed virus. Once the virus is propagated and harvested, the downstream processing parameters for purification, filling, and packaging of the vaccine are similar to current pharmaceutical methodologies with egg-based methodologies.
  • the bioreactor based culture process is suitable for large-scale manufacture of vaccine and the process parameters can be run routinely and more cost effectively.
  • the cell line used must be able to propagate the virus in large quantities, and must be rapid and efficient in expressing the desired virus.
  • the cell line should also be able to grow in a chemically defined synthetic medium. It should also be scalable for industrial processes.
  • a possible cell line is the Vero cell line. This cell line was derived from the kidney of a normal African green monkey and has been used for the industrial production of viral vaccines in preference to primary monkey kidney cells because of availability and the reduced risk of contamination by endogenous viruses.
  • a method for using the Vera ceil line for propagation of Newcastle Disease Virus has not been elucidated to date.
  • the objectives above can be achieved by using the method for virus propagation in a bioreactor (10) of the invention, which comprises the steps of: firstly, culturing a cell in a bioreactor to produce more cells to form a bioreactor culture (1 1 ); then, inoculating the cells with virus (12); and subsequently, incubating the virus in the bioreactor culture (13); wherein the virus is Newcastle Disease Virus (NDV) and the cells are Vero cells.
  • NDV Newcastle Disease Virus
  • Fig. 1 is a flow chart the method for virus propagation in a bioreactor.
  • Fig. 2 is a graph showing the growth profile of Vero cells in T-flask.
  • Fig. 3 is a graph showing the growth profile of Vera cells in spinner vessel.
  • Fig. 4 is a graph showing the growth profile of Vero cells in stirred tank bioreactor.
  • Fig. 5 is a graph showing the haemagglutination (HA) titre over time in T-flask, spinner vessel and stirred tank bioreactor.
  • Fig. 6 is a graph showing the TCID 50 over time in T-flask, spinner vessel and stirred tank bioreactor.
  • the words “include,” “including,” and “includes” mean including, but not limited to.
  • the words “a” or “an” mean “at least one” and the word “plurality” means one or more, unless otherwise mentioned.
  • NDV Newcastlele Disease Virus
  • the words “Vero cells” or “Vero cell line” are used to refer to the Vero cell line derived from the kidney of the normal African green monkey, including any variants or modifications of said cell line.
  • common reference numerals will be used throughout the figures when referring to the same or similar features common to the figures. The present invention will now be described with reference to Figs. 1 -6.
  • the present invention relates to a method for virus propagation in a bioreactor (10) comprising the steps of: firstly, culturing a cell in a bioreactor to produce more cells to form a bioreactor culture (1 ); then, inoculating the cells with virus (12); and subsequently, incubating the virus in the bioreactor culture (13); wherein the virus is Newcastle Disease Virus (NDV) and the cells are Vero cells.
  • a bioreactor 10 comprising the steps of: firstly, culturing a cell in a bioreactor to produce more cells to form a bioreactor culture (1 ); then, inoculating the cells with virus (12); and subsequently, incubating the virus in the bioreactor culture (13); wherein the virus is Newcastle Disease Virus (NDV) and the cells are Vero cells.
  • NDV Newcastle Disease Virus
  • the method of the present invention may be applied using any type of bioreactor.
  • Preferred bioreactors are T-flask, spinner vessel and stirred tank bioreactor. It is further preferred that the bioreactor is provided with means to monitor and control the process parameters such as temperature, agitation speed, pH, and dissolved oxygen content (p0 2 ).
  • the step of culturing the cell in the bioreactor to produce more cells to form the bioreactor culture (11 ) comprises the steps of: firstly, adding a cell culture medium into the bioreactor; then, removing cells from a stock cell culture; then, transferring the cells from the stock cell culture into the bioreactor; then, mixing the cells with the cell culture medium in the bioreactor; and subsequently, incubating the cells in the bioreactor until confluence is achieved.
  • the step of culturing the cell in the bioreactor to produce more cells to form the bioreactor culture (11 ) may further include the step of adding, microcarriers to the bioreactor.
  • the cell culture medium used in the present invention preferably comprises- of a nutrient medium and a serum.
  • the nutrient medium may. be Dulbecco's Modified Eagle's Medium (DMEM). More preferably, the nutrient medium is RPMI-1640 medium.
  • the preferred serum is fetal, bovine serum, more particularly at 10% concentration. It will be obvious to persons skilled in the art that other additives common to the art may be added to the. cell culture medium such as pH buffer agents (e.g. sodium bicarbonate) and antimicrobial agents (e.g. antibiotic agents like penicillin with streptomycin).
  • pH buffer agents e.g. sodium bicarbonate
  • antimicrobial agents e.g. antibiotic agents like penicillin with streptomycin
  • the microcarriers of the preferred embodiment are dextran-based microcarriers.
  • the next step of inoculating the cells with virus (12) preferably comprises the steps of: firstly, removing the cell culture medium from the bioreactor; then, washing the cells of the cell culture; then, adding the virus to the cell culture; then, incubating the virus with the cell culture for up to one hour; and subsequently, adding a -fresh supply of the cell culture medium to the bioreactor.
  • the cells of the cell culture are washed using phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the virus used for inoculating the cells ( 2) has preferably passed through up to six serial passages prior to inoculation of the cells with the virus (12).
  • the virus is incubated in the bioreactor culture (13), preferably for 2-3 days post inoculation.
  • the incubation of the virus in the bioreactor culture (13) may be carried out without control of incubation parameters, it is preferred that the incubation is maintained under controlled parameters comprising of temperature, agitation speed, pH, and dissolved oxygen content (p0 2 ).
  • the temperature is 37°C.
  • the bioreactor is a spinner vessel
  • the preferred agitation speed is preferably 30 rpm.
  • the bioreactor is a stirred tank bioreactor
  • the preferred agitation speed is 70 rpm.
  • the preferred pH for the incubation of the virus in the bioreactor culture (13) is pH 7.2.
  • the dissolved oxygen content (p0 2 ) is 30%. It would be obvious to persons skilled in the art that the method for virus propagation in a bioreactor (10) of the present invention should be carried out using appropriate aseptic techniques to avoid risk of contamination.
  • the cultures described below were prepared using RPMI-1640 nutrient medium supplemented with 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • Vero cells were sub-cultured by removing the cell culture medium from the stock cell culture in a 25 cm 2 T-flask using a pipette. Then, the cells were washed with 1 ml of phosphate buffered saline (PBS). After that, 1 ml of acutase was added into the flask. Trypsin may be used instead of acutase. Next, the cell culture was incubated in a CO ⁇ incubator for 5 minutes. When the cells become rounded, 5ml of cell culture medium was added. The cells were mixed thoroughly and the base of the flask was flushed using a pipette to disperse any clumps.
  • PBS phosphate buffered saline
  • the microcarriers used are Cytodex microcarriers, a type of dextran-based microcarrier.
  • the dry Cytodex microcarriers were added at a concentration of 3g/L to a suitably siliconized glass vessel. They were swollen using Ca 2+ and Mg 2+ -free phosphate buffered saline (PBS) for at least 3 hours at room temperature with ' occasional gentle agitation. The hydration process was accelerated by incubating at 37°C in a C ⁇ 3 ⁇ 4 incubator.
  • PBS Ca 2+ and Mg 2+ -free phosphate buffered saline
  • the supernatant was decanted and the microcarriers were washed once with gentle agitation fo a few minutes in 30-50 ml fresh Ca 2+ and Mg 2+ -free PBS.
  • the PBS was then discarded and replaced with 30-50 ml fresh Ca 2+ and Mg 2+ -free PBS and the microcarriers were sterilized by autoclaving with steam from purified water (1 15°C, 15 min., 15psi). Tween 80 can be added to avoid sedimentation-.
  • the supernatant was removed while the microcarriers were rinsed in cell culture medium (20-50 mL/g Cytodex). The microcarriers were allowed to settle before the supernatant was removed.
  • the Vera cell lines were cultured in 25 cm 2 T-flask. When the cells were 80-90% confluent, the RPMI-1640 medium in the T-flask was removed. Then, the cells were washed with 2 ml PBS. After that, the 25 cm 2 T-flask was inoculated with 00 yL NDV using passage number 6 (P6) having a HA titre of 128. P6 was taken because after being tested in the T-flask, no virus was detected in the first to fourth passages (P1 - P4), but after the fifth and sixth passages (P5 and P6) the virus was detectable.
  • P6 passage number 6
  • the virus titre was higher (HA titre 256) after P6, at the seventh passage (P7). Therefore, the sixth passage, P6, of the virus was used in this project.
  • the T-flasks containing the virus and Vero cell culture were incubated in a C0 2 incubator for 1 hour. After 1 hour, the cell culture medium was replaced. The virus was then incubated with the cell culture in the T-flask to propagate the virus.
  • Cell culture medium of 150 ml of RPMI-1640 medium supplemented with 10 % fetal bovine serum was added to a spinner vessel. After that, 20 ml of Cytodex-3 microcarriers were added to make it up to 170 ml. The cell culture medium was stirred for 2-3 hours to saturate the microcarriers with C0 2 gas. After that, 30 ml cell suspension containing the Vero cells was added to make it up to the working volume of 200 ml. The cell culture was intermittently stirred for 2 minutes at intervals of 30 minutes to ensure proper cell attachment on the microcarriers. This was done for 2-3 hours.
  • the cell culture medium was removed and washed twice with " 50 ml of PBS. Then, 15 ml of virus (P6) was added into the spinner vessel and incubated for 1 hour with continuous stirring at 30 rpm. After 1 hour, the virus was taken out and 200 ml fresh nutrient medium supplemented with 2 % fetal bovine serum was replaced into the spinner vessel. The virus and cell culture medium was then incubated with stirring at 30 rpm.
  • RPMI-1640 medium 400 ml of RPMI-1640 medium supplemented with 10% fetal bovine serum was added into a stirred tank bioreactor. The cell culture medium was allowed to stabilize for 8 hours. After 8 hours, 150 ml Cytodex-3 microcarriers and 50 ml of cells were added into bioreactor. After that, another 400 ml RPMI-1640 medium was added to make it up to 1000 ml as the working volume. Sampling was after every 8 hours to calculate the total cell number in bioreactor before infecting with the virus.
  • the medium was removed and the cell culture was washed with 100 ml of PBS. Then, 30 ml of virus (P6) with 300 ml medium without serum was added into the bioreactor and stirred for 1 hour. After 1 hour, the excess virus was removed and 700 ml fresh nutrient medium with 2% fetal bovine serum was added into the bioreactor. The virus and cell culture was then incubated under controlled parameters of pH 7.2, agitation speed 70 rpm, temperature 37°C and dissolved oxygen content ( ⁇ 2 ) 30%.
  • the following describes the growth profile, growth rate and doubling, time of Vera cells when cultured as above, as well as the results for haemagglutination and tissue culture infectivit-y tests carried out on the cultures produced- as described above.
  • the growth profile of Vero cells in T-flask is shown in Fig. 2.
  • Total cell number and cell viability were determined by cell counting after every 8 hours. The optimum time to infect the cells with the virus was derived thereof.
  • the highest viable cell number was at day 3 which was 7.55 x 10 5 cells/ml.
  • the virus was infected at hour 56 on day 3.
  • the cell number increased because cells were stable and proliferated.
  • cell number decreased gradually. This was due to cell death as a result of virus infection.
  • the total and viable cell numbers remain same until end of experiment because the dead cells were smaller compared to the viable cells.
  • the highest peak number of viable cells was at day 3 which is 2.03 x 10 6 cells/ml at hour 56. At that day, the virus was infected in spinner vessel. After 56 hours infected, the growth of virus was decreased because the virus was propagated in the cells and consumed all cells. However, according to the previous study, the highest cell density was obtained at hour 88 and was equal to 2.4*10 5 cells/ml for 3g/L Cytodex-3. Cells showed an exponential growth for the period of 88 hour for Cytodex-3 and viable cells number decreased at hour 96. The total cell number and viable cell number were remains same because the total cell death was smaller.
  • Fig.4 shows the growth profile of Vero cells in stirred tank bioreactor.
  • the virus was infected in the cells at day 3 because the higher viable number was obtained at hour 72 on day 3.
  • the viable cell number at hour 72 was 7.40 x 10 5 cells/ml.
  • the growth, of virus was still increasing because the cells were stable and proliferated.
  • the growth of virus was decreased because there were no more hosts- for virus to multiply and replicate into the cell since all the cells had -been killed by the virus. Determination of growth rate ( ⁇ ), and doubting time (t d )
  • Table 1 Comparison of growth rate, u and doubling time, , in three different types of bioreactors
  • Table 1 shows the growth rate, ⁇ , and doubling time, t d , of the cells in T-flask, spinner vessel and stirred tank bioreactor.
  • the doubling time in T-f!ask was highest which is 38.941 h compared to spinner vessel and stirred tank bioreactor.
  • the growth rate in T-flask was the lowest, which is 0.0178h “1 compared to in spinner vessel and stirred tank bioreactor which were 0.0662 h "1 and 0:0323 h " . respectively. This is because incubation in the T-flask was not carried out under controlled parameters. So that, the growth rate was lower because the doubling time needed for cells to proliferate was longer.
  • the shortest doubling time was found in spinner vessel, which was 10.471 h.
  • the shorter time required for the cells to grow is desirable as it provides the highest growth rate of the cells.
  • the spinner vessel had higher growth rate and shorter doubling time because spinner vessel ' does not have environmental control. It only controls the stirrer speed which at 30 rpm.
  • the growth rate and doubling time was measured from the exponential phase. In this phase the cells constantly proliferated at their maximum growth rate. When, the growth rate of the cells increased, the tit e number of virus also increased.
  • the spinner vessel was found here to be the best method to be used com pared to T-flask and stirred tank bioreactor in terms of growing of the cells because it provided the highest growth rate of the cells and the shorter doubling time.
  • HA haemagglutination test was carried out to quantify the virus present in the cell culture system.
  • Fig. 5 shows the HA titre that was obtained from T- flask, spinner vessel, and stirred tank bioreactor.
  • the passage 6 (P6) of NDV virus was used in this project. This is because after tested in T-flask, there was no virus detected in P1 to P3 but, after P4 arid P7, the virus was detected. When the virus was propagated in Vero cells, it became passage 7 (P7).
  • the HA titre for P6 was 64 and when it was propagated in T-flask it became P7 and HA titre obtained was 128. In T-flask, the maximum HA titre of 16 was obtained on day 2 and day 3.
  • HA titre for P7 was 256 and the maximum titre that was obtained was 16, on day 3.
  • the maximum HA titre obtained in stirred tank bioreactor was 32, on day 2 and day 3.
  • the HA titre for P7 in stirred tank bioreactor was only 4. From this, it can be concluded that in order to get higher HA titre of virus, the passage that is being propagated in T-flask, spinner vessel and stirred tank bioreactor should be as low as possible.
  • the stirred tank bioreactor is the best method for propagating NDV viruses in Vero cells because it can produce the highest HA titre compared to T-flask and spinner vessel. This is because parameters affecting the process such as p0 2 , agitation speed, pH and temperature can be more easily controlled when using the stirred tank bioreactor.
  • the stirred tank bioreactor is also provided with baffle and rotating stirrer in order to aid mixing and mass transfer by increasing turbulence, preventing vortex formation and eliminating dead spaces.
  • T-flask the parameters were not totally controlled, whereas in spinner vessel, the speed was controlled at 30 rpm and temperature at 37 °C during incubation.
  • the tissue culture infective dose, TCID 5 0, is the amount of a pathogenic agent that will produce pathological change in 50% of cell cultures inoculated.
  • Fig. 6 shows the tissue culture infective dose, TCID 50 , obtained for T-flask, spinner vessel and stirred tank bioreactor. According to the graph, the highest virus titre was on day 1 from stirred tank bioreactor, which was 3.39 x 10 6 TGID 5 o /ml because the cells were still growing and stable. The culture was stopped on day 4 post-infection, when the virus titre was equal ' to 7.94 x 10 3 TCIDso /ml because the cells had stopped growing. TCID 5 0 for T-flask was maximum on day 3 which was 8. 3 x 10 3 pfu/ml.
  • the highest virus titre of NDV in spinner vessel was on day 3 which was 2.75 x 10 5 pfu/ml. After day 3, the value was decreased relatively because the virus could not multiply and the cells were not growing well and not stable. Thus, the virus could not survive due to cell death.
  • the highest virus titre was obtained from the stirred tank bioreactor. This is due to the ability to control parameters such as pH 7.2, agitation speed 70 rpm and p0 2 30% in the stirred tank bioreactor.
  • the pH and dissolved oxygen content were not controlled.
  • the agitation speed was controlled at 30 rpm and the temperature controlled at 37 °C in the incubator.

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Abstract

The present invention relates to a method for virus propagation in a bioreactor (10) comprising the steps of: firstly, culturing a cell in a bioreactor to produce more cells to form a bioreactor culture (11 ); then, inoculating the cells with virus (12); and subsequently, incubating the virus with the bioreactor culture (13); wherein the virus is Newcastle Disease Virus (NDV) and the cells are Vero cells.

Description

METHOD FOR NEWCASTLE DISEASE VIRUS PROPAGATION IN A
BIOREACTOR
Background of the Invention
Field of the Invention
This invention relates to a method for virus propagation in a bioreactor, and more particularly, to a method for propagating Newcastle Disease Virus in a bioreactor using Vero cells.
Description of Related Arts
Newcastle Disease Virus (NDV) is a virus that causes the Newcastle disease in chickens, turkeys and other birds. The Newcastle disease is highly contagious in birds and is of particular importance in domestic poultry due to their high susceptibility to infection. The NDV virus is therefore a threat to the poultry industry, resulting in a global problem with severe economic implications. However, it is possible to produce a vaccine against the virus. In humans who have been exposed to infected birds, the virus causes mild conjunctivitis and influenza-like symptoms but otherwise poses no hazard to human health. Recently, the NDV virus has also been studied as a potential anticancer agent due to its selective toxicity towards human tumour cells. Therefore, a method for large-scale propagation of the NDV virus, whether for production of vaccines or other therapeutic agents such as anticancer agents, is beneficial.
Currently, most vaccines are produced from egg-based culture, where eggs are used to propagate the virus. The virus will first need to be modified to ensure it can propagate in the egg without killing the embryo. The eggs are then infected with the virus and incubated to propagate the virus. Control of incubation parameters and contamination is difficult, leading to poor production levels. This culture system therefore requires a long production time and the amount of virus vaccine produced is low. A great . number of embryonated hen's eggs are required annually to grow and produce sufficient vaccine to meet global demands. This will be a particular problem in the event of an avian disease pandemic as there will be a shortage of eggs for vaccine production. The cost of the eggs and the cost of isolating and incubating the infected eggs result in high production costs for the vaccine. Furthermore, some people are allergic to egg protein, making egg-based products unacceptable for them. If the NDV virus is to be used as a potential therapeutic agent in humans, it would be preferable to avoid egg-based systems. It is therefore desirable to find an alternative method to large scale production of the NDV. virus that does not involve the use of eggs.
An alternative to the traditional chicken egg-based culture system is the bioreactor based culture system. This system uses cells in bioreactor culture to propagate the virus. Vaccine can be produced in a shorter time, control of process parameters is easier and eggs are not required. The process begins when a cell line is infected with the seed virus. The critical step is the availability of the seed virus. Once the virus is propagated and harvested, the downstream processing parameters for purification, filling, and packaging of the vaccine are similar to current pharmaceutical methodologies with egg-based methodologies. The bioreactor based culture process is suitable for large-scale manufacture of vaccine and the process parameters can be run routinely and more cost effectively.
However, in order to apply bioreactor based cell culture technology to vaccine production, it is necessary to find the right host cell in which to propagate the virus. The cell line used must be able to propagate the virus in large quantities, and must be rapid and efficient in expressing the desired virus. The cell line should also be able to grow in a chemically defined synthetic medium. It should also be scalable for industrial processes. A possible cell line is the Vero cell line. This cell line was derived from the kidney of a normal African green monkey and has been used for the industrial production of viral vaccines in preference to primary monkey kidney cells because of availability and the reduced risk of contamination by endogenous viruses. However, a method for using the Vera ceil line for propagation of Newcastle Disease Virus has not been elucidated to date.
Summary of Invention
It is an object of the present invention to provide a method for propagating Newcastle Disease Virus in bioreactors by using Vero cells.
It is another object of the present invention to provide a method for propagating Newcastle Disease Virus in bioreactors such as to provide a high virus titre.
It is also an object of the present invention to provide a method for propagating Newcastle Disease Virus without requiring the use of egg-based systems.
It is an object of the present invention to provide a method for propagating the Newcastle Disease Virus that is suitable for large-scale industrial production of the virus for therapeutic purposes.
The objectives above can be achieved by using the method for virus propagation in a bioreactor (10) of the invention, which comprises the steps of: firstly, culturing a cell in a bioreactor to produce more cells to form a bioreactor culture (1 1 ); then, inoculating the cells with virus (12); and subsequently, incubating the virus in the bioreactor culture (13); wherein the virus is Newcastle Disease Virus (NDV) and the cells are Vero cells.
Brief Description of the Drawings
The features of the invention will be more readily understood and appreciated from the following detailed description when read in conjunction with the accompanying drawing, in which:
Fig. 1 is a flow chart the method for virus propagation in a bioreactor.
Fig. 2 is a graph showing the growth profile of Vero cells in T-flask.
Fig. 3 is a graph showing the growth profile of Vera cells in spinner vessel.
Fig. 4 is a graph showing the growth profile of Vero cells in stirred tank bioreactor. Fig. 5 is a graph showing the haemagglutination (HA) titre over time in T-flask, spinner vessel and stirred tank bioreactor.
Fig. 6 is a graph showing the TCID50 over time in T-flask, spinner vessel and stirred tank bioreactor.
Detailed Description of the invention
As required, detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting but merely as a basis for claims. It should be understood that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modification, equivalents and alternatives falling within the spirit and scope of the present invention as defined by the appended claims. As used throughout this application, the word "may" is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Similarly, the words "include," "including," and "includes" mean including, but not limited to. Further, the words "a" or "an" mean "at least one" and the word "plurality" means one or more, unless otherwise mentioned. Additionally, where "NDV" or "NDV virus" are used, this is understood to mean "Newcastle Disease Virus", including all possible- strains or modifications of said virus. The words "Vero cells" or "Vero cell line" are used to refer to the Vero cell line derived from the kidney of the normal African green monkey, including any variants or modifications of said cell line. For ease of reference, common reference numerals will be used throughout the figures when referring to the same or similar features common to the figures. The present invention will now be described with reference to Figs. 1 -6.
The present invention relates to a method for virus propagation in a bioreactor (10) comprising the steps of: firstly, culturing a cell in a bioreactor to produce more cells to form a bioreactor culture (1 ); then, inoculating the cells with virus (12); and subsequently, incubating the virus in the bioreactor culture (13); wherein the virus is Newcastle Disease Virus (NDV) and the cells are Vero cells.
The method of the present invention may be applied using any type of bioreactor. Preferred bioreactors are T-flask, spinner vessel and stirred tank bioreactor. It is further preferred that the bioreactor is provided with means to monitor and control the process parameters such as temperature, agitation speed, pH, and dissolved oxygen content (p02).
According to a preferred embodiment of the method for virus propagation in a bioreactor (10) of the present invention, the step of culturing the cell in the bioreactor to produce more cells to form the bioreactor culture (11 ) comprises the steps of: firstly, adding a cell culture medium into the bioreactor; then, removing cells from a stock cell culture; then, transferring the cells from the stock cell culture into the bioreactor; then, mixing the cells with the cell culture medium in the bioreactor; and subsequently, incubating the cells in the bioreactor until confluence is achieved. The step of culturing the cell in the bioreactor to produce more cells to form the bioreactor culture (11 ) may further include the step of adding, microcarriers to the bioreactor. The necessity of using microcarriers will depend on, for instance, the type of bioreactor used. The cell culture medium used in the present invention preferably comprises- of a nutrient medium and a serum. For example, the nutrient medium may. be Dulbecco's Modified Eagle's Medium (DMEM). More preferably, the nutrient medium is RPMI-1640 medium. The preferred serum is fetal, bovine serum, more particularly at 10% concentration. It will be obvious to persons skilled in the art that other additives common to the art may be added to the. cell culture medium such as pH buffer agents (e.g. sodium bicarbonate) and antimicrobial agents (e.g. antibiotic agents like penicillin with streptomycin). Although various types of microearriers may be used in the present invention, the microcarriers of the preferred embodiment are dextran-based microcarriers.
The next step of inoculating the cells with virus (12) preferably comprises the steps of: firstly, removing the cell culture medium from the bioreactor; then, washing the cells of the cell culture; then, adding the virus to the cell culture; then, incubating the virus with the cell culture for up to one hour; and subsequently, adding a -fresh supply of the cell culture medium to the bioreactor. Preferably, the cells of the cell culture are washed using phosphate buffered saline (PBS). The virus used for inoculating the cells ( 2) has preferably passed through up to six serial passages prior to inoculation of the cells with the virus (12).
Subsequently the virus is incubated in the bioreactor culture (13), preferably for 2-3 days post inoculation. Although the incubation of the virus in the bioreactor culture (13) may be carried out without control of incubation parameters, it is preferred that the incubation is maintained under controlled parameters comprising of temperature, agitation speed, pH, and dissolved oxygen content (p02). In the preferred embodiment, the temperature is 37°C. Where the bioreactor is a spinner vessel, the preferred agitation speed is preferably 30 rpm. Where the bioreactor is a stirred tank bioreactor, the preferred agitation speed is 70 rpm. The preferred pH for the incubation of the virus in the bioreactor culture (13) is pH 7.2. Preferably, the dissolved oxygen content (p02) is 30%. It would be obvious to persons skilled in the art that the method for virus propagation in a bioreactor (10) of the present invention should be carried out using appropriate aseptic techniques to avoid risk of contamination.
The following non-limiting examples are provided to illustrafe the present invention and in no way limits the scope thereof.
Examples
Preparation of Medium for Vero Cell Culture
The cultures described below were prepared using RPMI-1640 nutrient medium supplemented with 10% fetal bovine serum (FBS).
Subculture of Vero Cells
Vero cells were sub-cultured by removing the cell culture medium from the stock cell culture in a 25 cm2 T-flask using a pipette. Then, the cells were washed with 1 ml of phosphate buffered saline (PBS). After that, 1 ml of acutase was added into the flask. Trypsin may be used instead of acutase. Next, the cell culture was incubated in a CO≥ incubator for 5 minutes. When the cells become rounded, 5ml of cell culture medium was added. The cells were mixed thoroughly and the base of the flask was flushed using a pipette to disperse any clumps.
Preparation of Microcarriers for Vero Cell Culture
In the following examples, the microcarriers used are Cytodex microcarriers, a type of dextran-based microcarrier. The dry Cytodex microcarriers were added at a concentration of 3g/L to a suitably siliconized glass vessel. They were swollen using Ca2+and Mg2+-free phosphate buffered saline (PBS) for at least 3 hours at room temperature with 'occasional gentle agitation. The hydration process was accelerated by incubating at 37°C in a C<¾ incubator. Following hydration, the supernatant was decanted and the microcarriers were washed once with gentle agitation fo a few minutes in 30-50 ml fresh Ca2+and Mg2+-free PBS. The PBS was then discarded and replaced with 30-50 ml fresh Ca2+and Mg2+-free PBS and the microcarriers were sterilized by autoclaving with steam from purified water (1 15°C, 15 min., 15psi). Tween 80 can be added to avoid sedimentation-. After 15 minutes, the supernatant was removed while the microcarriers were rinsed in cell culture medium (20-50 mL/g Cytodex). The microcarriers were allowed to settle before the supernatant was removed.
Virus Propagation in a T-Flask
Firstly, the Vera cell lines were cultured in 25 cm2 T-flask. When the cells were 80-90% confluent, the RPMI-1640 medium in the T-flask was removed. Then, the cells were washed with 2 ml PBS. After that, the 25 cm2 T-flask was inoculated with 00 yL NDV using passage number 6 (P6) having a HA titre of 128. P6 was taken because after being tested in the T-flask, no virus was detected in the first to fourth passages (P1 - P4), but after the fifth and sixth passages (P5 and P6) the virus was detectable. It was found that the virus titre was higher (HA titre 256) after P6, at the seventh passage (P7). Therefore, the sixth passage, P6, of the virus was used in this project. The T-flasks containing the virus and Vero cell culture were incubated in a C02 incubator for 1 hour. After 1 hour, the cell culture medium was replaced. The virus was then incubated with the cell culture in the T-flask to propagate the virus.
Virus Propagation in a Spinner Vessel
Cell culture medium of 150 ml of RPMI-1640 medium supplemented with 10 % fetal bovine serum was added to a spinner vessel. After that, 20 ml of Cytodex-3 microcarriers were added to make it up to 170 ml. The cell culture medium was stirred for 2-3 hours to saturate the microcarriers with C02 gas. After that, 30 ml cell suspension containing the Vero cells was added to make it up to the working volume of 200 ml. The cell culture was intermittently stirred for 2 minutes at intervals of 30 minutes to ensure proper cell attachment on the microcarriers. This was done for 2-3 hours.
When the cells were confluent, the cell culture medium was removed and washed twice with" 50 ml of PBS. Then, 15 ml of virus (P6) was added into the spinner vessel and incubated for 1 hour with continuous stirring at 30 rpm. After 1 hour, the virus was taken out and 200 ml fresh nutrient medium supplemented with 2 % fetal bovine serum was replaced into the spinner vessel. The virus and cell culture medium was then incubated with stirring at 30 rpm.
Virus Propagation in a Stirred Tank Bioreactor
400 ml of RPMI-1640 medium supplemented with 10% fetal bovine serum was added into a stirred tank bioreactor. The cell culture medium was allowed to stabilize for 8 hours. After 8 hours, 150 ml Cytodex-3 microcarriers and 50 ml of cells were added into bioreactor. After that, another 400 ml RPMI-1640 medium was added to make it up to 1000 ml as the working volume. Sampling was after every 8 hours to calculate the total cell number in bioreactor before infecting with the virus.
When the cells were confluent, the medium was removed and the cell culture was washed with 100 ml of PBS. Then, 30 ml of virus (P6) with 300 ml medium without serum was added into the bioreactor and stirred for 1 hour. After 1 hour, the excess virus was removed and 700 ml fresh nutrient medium with 2% fetal bovine serum was added into the bioreactor. The virus and cell culture was then incubated under controlled parameters of pH 7.2, agitation speed 70 rpm, temperature 37°C and dissolved oxygen content ( Ο2) 30%.
Results and Discussion
The following describes the growth profile, growth rate and doubling, time of Vera cells when cultured as above, as well as the results for haemagglutination and tissue culture infectivit-y tests carried out on the cultures produced- as described above.
Growth profile of Vero cells in T-Flask
The growth profile of Vero cells in T-flask is shown in Fig. 2. Total cell number and cell viability were determined by cell counting after every 8 hours. The optimum time to infect the cells with the virus was derived thereof. In T-flask, the highest viable cell number was at day 3 which was 7.55 x 105 cells/ml. Thus, the virus was infected at hour 56 on day 3. However, after infection at hour 72, the cell number increased because cells were stable and proliferated. Following cell infection after hour 72, cell number decreased gradually. This was due to cell death as a result of virus infection. The total and viable cell numbers remain same until end of experiment because the dead cells were smaller compared to the viable cells.
Growth profile of Vero ceiis in Spinrier Vessel
As shown in Fig 3, the highest peak number of viable cells was at day 3 which is 2.03 x 106 cells/ml at hour 56. At that day, the virus was infected in spinner vessel. After 56 hours infected, the growth of virus was decreased because the virus was propagated in the cells and consumed all cells. However, according to the previous study, the highest cell density was obtained at hour 88 and was equal to 2.4*105cells/ml for 3g/L Cytodex-3. Cells showed an exponential growth for the period of 88 hour for Cytodex-3 and viable cells number decreased at hour 96. The total cell number and viable cell number were remains same because the total cell death was smaller.
Growth profile of Vero cells in Stirred Tank Bioreactor
Fig.4 shows the growth profile of Vero cells in stirred tank bioreactor. The virus was infected in the cells at day 3 because the higher viable number was obtained at hour 72 on day 3. The viable cell number at hour 72 was 7.40 x 105 cells/ml. After 72 hours, the growth, of virus was still increasing because the cells were stable and proliferated. However, after 80 hours, the growth of virus was decreased because there were no more hosts- for virus to multiply and replicate into the cell since all the cells had -been killed by the virus. Determination of growth rate (μ), and doubting time (td)
The growth rates and doubling time for the three p referred, bioreactors are shown below in Table 1.
Table 1 : Comparison of growth rate, u and doubling time, , in three different types of bioreactors
Figure imgf000012_0001
Table 1 shows the growth rate, μ, and doubling time, td, of the cells in T-flask, spinner vessel and stirred tank bioreactor. The doubling time in T-f!ask was highest which is 38.941 h compared to spinner vessel and stirred tank bioreactor. The growth rate in T-flask was the lowest, which is 0.0178h"1 compared to in spinner vessel and stirred tank bioreactor which were 0.0662 h"1 and 0:0323 h" . respectively. This is because incubation in the T-flask was not carried out under controlled parameters. So that, the growth rate was lower because the doubling time needed for cells to proliferate was longer. The shortest doubling time was found in spinner vessel, which was 10.471 h. The shorter time required for the cells to grow is desirable as it provides the highest growth rate of the cells. The spinner vessel had higher growth rate and shorter doubling time because spinner vessel' does not have environmental control. It only controls the stirrer speed which at 30 rpm. The growth rate and doubling time was measured from the exponential phase. In this phase the cells constantly proliferated at their maximum growth rate. When, the growth rate of the cells increased, the tit e number of virus also increased. Thus, the spinner vessel .was found here to be the best method to be used com pared to T-flask and stirred tank bioreactor in terms of growing of the cells because it provided the highest growth rate of the cells and the shorter doubling time.
Haemagglutination (HA) Test in T-Flask, Spinner Vessel, and Stirred Tank Bioreactor
The haemagglutination (HA) test was carried out to quantify the virus present in the cell culture system. Fig. 5 shows the HA titre that was obtained from T- flask, spinner vessel, and stirred tank bioreactor. The passage 6 (P6) of NDV virus was used in this project. This is because after tested in T-flask, there was no virus detected in P1 to P3 but, after P4 arid P7, the virus was detected. When the virus was propagated in Vero cells, it became passage 7 (P7). The HA titre for P6 was 64 and when it was propagated in T-flask it became P7 and HA titre obtained was 128. In T-flask, the maximum HA titre of 16 was obtained on day 2 and day 3. However, in spinner vessel HA titre for P7 was 256 and the maximum titre that was obtained was 16, on day 3. The maximum HA titre obtained in stirred tank bioreactor was 32, on day 2 and day 3. The HA titre for P7 in stirred tank bioreactor was only 4. From this, it can be concluded that in order to get higher HA titre of virus, the passage that is being propagated in T-flask, spinner vessel and stirred tank bioreactor should be as low as possible.
According to these results, the stirred tank bioreactor is the best method for propagating NDV viruses in Vero cells because it can produce the highest HA titre compared to T-flask and spinner vessel. This is because parameters affecting the process such as p02, agitation speed, pH and temperature can be more easily controlled when using the stirred tank bioreactor. The stirred tank bioreactor is also provided with baffle and rotating stirrer in order to aid mixing and mass transfer by increasing turbulence, preventing vortex formation and eliminating dead spaces. However, in T-flask the parameters were not totally controlled, whereas in spinner vessel, the speed was controlled at 30 rpm and temperature at 37 °C during incubation. Tissue culture infective dose (TCID50) in T-Fiask, Spinner Vessel, and Stirred Tank Bioreactor
The tissue culture infective dose, TCID50, is the amount of a pathogenic agent that will produce pathological change in 50% of cell cultures inoculated. Fig. 6 shows the tissue culture infective dose, TCID50, obtained for T-flask, spinner vessel and stirred tank bioreactor. According to the graph, the highest virus titre was on day 1 from stirred tank bioreactor, which was 3.39 x 106 TGID5o /ml because the cells were still growing and stable. The culture was stopped on day 4 post-infection, when the virus titre was equal' to 7.94 x 103 TCIDso /ml because the cells had stopped growing. TCID50 for T-flask was maximum on day 3 which was 8. 3 x 103 pfu/ml.
In this study, the highest virus titre of NDV in spinner vessel was on day 3 which was 2.75 x 105 pfu/ml. After day 3, the value was decreased relatively because the virus could not multiply and the cells were not growing well and not stable. Thus, the virus could not survive due to cell death.
Among the three types of bioreactors studied, the highest virus titre was obtained from the stirred tank bioreactor. This is due to the ability to control parameters such as pH 7.2, agitation speed 70 rpm and p0230% in the stirred tank bioreactor. For T-flask and spinner vessel, the pH and dissolved oxygen content were not controlled. For spinner vessel, the agitation speed was controlled at 30 rpm and the temperature controlled at 37 °C in the incubator.

Claims

. A method for virus propagation in a bioreactor (10) comprising the steps of:
firstly,. culturing a cell in a bioreactor to. produce more cells to form a bioreactor culture (1 1 );
then, inoculating the cells with virus (12); and
subsequently, incubating the virus with the bioreactor culture (13); wherein the virus is Newcastle Disease Virus (NDV) and the cells are Vero cells.
A method for virus propagation in a bioreactor (10) as in claim 1 , wherein the bioreactor is a T-flask.
3. A method for virus propagation in a bioreactor (1 0) as in claim 1 , wherein the bioreactor is a spinner vessel.
4. A method for virus propagation in a bioreactor (10) as in claim 1 , wherein the bioreactor is a stirred tank bioreactor.
5. A method for virus propagation in a bioreactor (10) as in claim 1 , wherein culturing a cell in a bioreactor to produce more cells to form a bioreactor culture (1 1 ) comprises the steps of:
firstly, adding a cell culture medium into the bioreactor; then, removing cells from a stock cell culture;
then, transferring the cells from the stock cell culture into the bioreactor;
then, mixing the cells with the cell culture medium in the bioreactor; and subsequently, incubating the cells in the bioreactor until confluence is achieved:
6. A method for virus propagation in a bioreactor ( 0) as in claim 1 , wherein culturing a cell in a- bioreactor to produce more cells to form a bioreactor culture (1 1 ) further includes the step of adding microcarriers to the bioreactor.
7. A method for virus propagation in a bioreactor (10) as in claim 5, wherein the cell culture medium comprises of a. nutrient medium and a serum.
8. A method for virus propagation in a bioreactor (10) as in claim 7, wherein the nutrient medium is Dulbecco's Modified Eagle's Medium (DMEM) or
■ RPMI-1640 medium.
9. A method for virus propagation in a bioreactor (10) as in claim 7, wherein the serum is fetal bovine serum, preferably 10%.
10. A method for virus propagation in a bioreactor (10) as in claim 6, wherein the microcarriers are dextran-based microcarriers.
1 . A method for virus propagation in a bioreactor (10) as in claim 1 , wherein inoculating the cells with virus (12) comprises the steps of:
firstly, removing the cell culture medium from the bioreactor;
then, washing the cells of the cell culture;
then, adding the virus to the cell culture;
then, incubating the virus with the cell culture for up to one hour; and subsequently, adding a fresh supply of the cell culture medium to the bioreactor.
12. A method for virus propagation in a bioreactor (10) as in claim 1 , wherein the virus used for inoculating the cells (12) has passed through up to six serial passages prior to inoculation of the cells with the virus (12).
13. A method for virus propagation in.a bioreactor (10) as in claim 1 , wherein , incubating the virus with the bioreactor culture (13) is maintained for 2-3 days post inoculation.
14. A method'for virus propagation in a bioreactor (1-0) as in claim 1 , wherein , incubating the virus with the bioreactor culture (13) is maintained under controlled parameters comprising of temperature, agitation speed, pH, and dissolved oxygen content- (· θ2).
15. A method for virus propagation in a bioreactor (10) as in claim 1 , wherein , incubating the virus with the bioreactor culture (13) is maintained at temperature of 37°C.
16. A method for virus propagation in a bioreactor (10) as in claim 3, wherein , incubating the virus with the bioreactor culture (13) is maintained with agitation speed of 30 rpm.
17. A method for virus propagation in a bioreactor (10) as in claim 4, wherein , incubating the virus with the bioreactor culture (13) is maintained with agitation speed of 70 rpm.
18. A method for virus propagation in a bioreactor (10) as in claim 4, wherein , incubating the virus with the bioreactor culture (13) is maintained at pH 7.2.
19. A method for virus propagation in a bioreactor (10) as in claim 4, wherein , incubating th virus with the bioreactor culture (13) is maintained with dissolved oxygen content (PO2) of 30%.
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Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
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FRAZZATI-GALLINA N M ET AL: "Higher production of rabies virus in serum-free medium cell cultures on microcarriers", JOURNAL OF BIOTECHNOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 92, no. 1, 15 November 2001 (2001-11-15), pages 67 - 72, XP027295856, ISSN: 0168-1656, [retrieved on 20011115] *
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