HK1115609B - Method for large scale production of virus antigen - Google Patents

Description

Method for large scale production of viral antigens

The present application is a divisional application of "method for large-scale production of viral antigen" in chinese patent application 02827977.8 filed on 10/12/2002.

Technical Field

The present invention relates to an improved method for producing viral antigens on adherent cell cultures bound to microcarriers, wherein the method provides increased production of viral antigen per unit volume of culture medium.

Background

Efficient vaccine production requires the growth of large quantities of virus produced in high yields from the host system. Thus, to maximize the yield of the desired virus, both the system and the culture conditions must be specifically adapted to provide an environment that is advantageous for producing the desired virus. Thus, in order to obtain an acceptably high yield of multiple virus strains, a system is needed that provides optimal growth conditions for a large number of different viruses.

The only economically viable process is the reactor process, as scale-up can be made to suit market size and required vaccine dose. For adherent cells, the vector method using conventional microcarriers is currently the best choice for large-scale culture of cells required for virus propagation (Van Wezel et al, 1967, Nature 216: 64-65; Van Wezel et al, 1978, Process biochem. 3: 6-8). Methods for large-scale production of poliovirus, hepatitis A virus, HSV or Marek's disease virus on microcarriers have been described (US4,525,349; Widell et al, 1984, J.virological meth, 8: 63-71; Fiofentine et al, 1985, Defelop.biol.Standard, 60: 421-. Current microcarrier culture-based methods allow the use of fermentor sizes up to 1200L for virus production.

Caij et al (1989, Arch. Virol., 105: 113-118) compared the production of viral titer of CSFV on microcarrier cultures and conventional monolayer cultures and found that higher virus production per unit volume of medium could be obtained using microcarrier systems.

Griffiths et al (1982, Deverop. biol. Standard., 50: 103-110) investigated the effect of microcarrier concentration on cell growth and HSV production it has been found that optimal microcarrier concentration is necessary to achieve high cell density, which also affects the yield of virus obtained. However, higher microcarrier concentrations in the perfusion system lead to cell loss due to the shedding of the cell layer from the beads.

The productivity of the virus production process on microcarrier systems depends on the virus, the cell, the type of microcarrier and the cell density obtained in the system. Higher microcarrier concentrations in cell culture allow for higher total cell numbers. However, microcarriers are expensive and under these conditions cell loss can occur due to the cell layer being subjected to shear forces in the system and being detached from the beads. This means that a larger volume of microcarrier cell culture is required for higher virus production, but this increases the effort that must be made to handle and purify such large volumes.

For virus propagation, it is important to achieve optimal cell density to achieve maximum virus yield. It is also important to allow efficient adsorption of the virus to the cells. Thus, in conventional methods, the volume of growth medium is reduced prior to infection in order to allow adsorption of the virus onto the cells with a minimum culture volume and with a better virus to cell ratio. However, to obtain optimal virus propagation, the culture medium volume is again increased after a suitable adsorption time in order to maintain cell viability and/or growth. However, this increases the volume of the medium containing the cells and/or virus, which has the disadvantage that large volumes have to be processed to further purify the virus from the cells or the cell culture medium.

In addition, there is a need for methods to obtain viral propagation that use materials that are already available and require minimal time consuming operations, such as handling reduced volumes of cell culture media and facilitating purification and downstream processing for vaccine production.

Disclosure of Invention

It is an object of the present invention to provide a method for producing a virus or a viral antigen in a cell culture of adherent cells bound to a microcarrier.

It is also an object of the present invention to provide a method for producing viruses in small cell culture volumes.

It is also an object of the present invention to provide a cell culture of adherent cells having a higher cell density compared to the original cell culture grown to confluence.

It is an object of the present invention to provide cell cultures of adherent cells bound to microcarriers, which have a higher cell density than the original cell culture grown to confluence, wherein the cells are infected with a virus.

Detailed Description

In accordance with these and other objects, the present invention provides a method for producing a virus or virus antigen comprising the steps of providing a culture of adherent cells bound to a microcarrier, growing the cell culture to confluence, infecting the cells with the virus, wherein the cell density in the cell culture is increased (i) prior to infection with the virus or (ii) after infection with the virus, and incubating the cell culture infected with the virus to propagate the virus. The increase in cell density in the cell culture is performed by concentrating the cell culture, which includes an increase in the concentration of microcarriers in the cell culture.

In general, adherent cells bound to microcarriers require an optimal ratio of microcarrier concentration to cells in order to achieve high cell density. However, the concentration of the vector in the cell culture system is limited to a specific concentration due to the physiological stress of the cells caused by shearing effects, reduction of the nutrient source in the medium and increased concentration of the microcarriers (see also Griffiths et al, 1982, supra).

The method of the invention allows cells to be grown under optimal growth conditions, including microcarrier concentration, feeding and minimal physiological stress, in order to reach the maximum cell density for the system used.

It was found in the present invention that the medium volume was reduced before or after infection with the virus, whereby the cell density and the concentration of microcarriers in the cell culture biomass were increased, which did not affect the productivity of the cells. Conversely, it has also been surprisingly found that the yield of virus obtained per cell is increased compared to cells maintained at the same cell density as the original confluent cell culture. This is highly undesirable when physiological stress is caused by decreased cell viability due to increased microcarrier concentration in cell culture, shedding of cells from microcarriers and higher cell density, and is desirable during virus production.

The method of the invention allows to reduce the volume of medium that has to be processed during further virus purification process, while the virus productivity per cell is similar or even increased compared to the original cell culture the system can be scaled up to 6000L fermentor volumes, which volume makes the virus production process for vaccines more efficient and time consuming.

According to one embodiment of the method, the anchorage-dependent cells are selected from adherent cells such as diploid monolayer cells described in VERO, BHK, CHO, RK44, RK13, MRC-5, MDCK, CEF or Reuveny et al (1985, Deverop. biol. Standard., 60: 243-.

Adherent cells bound to microcarriers can be grown in conventional serum-containing cell culture media. According to a preferred embodiment of the invention, the cells can be grown in serum-free or serum and protein-free medium as described by Kistner et al (1998, Vaccine, 16: 960) -968), Merten et al (1994, Cytotech, 14: 47-59), Cinatl et al (1993, Cell Biology lnt, 17: 885-895), Kessler et al (1999, Dev. biol. Stand, 98: 13-21), WO96/15231, US6,100,061, or in any other serum-free or serum and protein-free medium known in the art. The cells are preferably grown into biomass from ampoules to large scale in serum-free or serum and protein-free medium.

According to one embodiment of the invention, a culture of adherent cells bound to microcarriers is grown to confluence and infected with a virus after the cell density of the cellular biomass of the confluent cell culture and the microcarrier concentration are increased.

According to one embodiment of the invention, a culture of adherent cells bound to microcarriers is grown to confluence and infected with a virus before the cell density and microcarrier concentration of the confluent biomass is increased. In any case, even if a cell culture with higher cell density and microcarrier concentration per unit volume is infected before or after the culture is concentrated, the cell density and microcarrier concentration in the biomass remains unchanged during the virus propagation and production process, while at the same time the volume of the culture medium is no longer increased. The method for increasing cell density and microcarrier concentration in the cell culture biomass that is not infected or infected with a virus can be any method known in the art for concentrating cell cultures. This can be done by e.g. sedimentation, centrifugation, filtration, concentration with a perfusion device such as a sieve, which may result in a reduction of the working volume, or pooling of 2 or more bioreactor systems.

Increasing the cell culture density and microcarrier concentration of the cell culture that is grown to confluence, wherein the increase should be at least 1.3 fold compared to the original biomass that is grown to confluence. The cell density of the original starting cell culture grown to confluence may be about 0.6X 10⁶-about 7.0X 10⁶Cells/ml. In this case, the biomass having an increased cell density compared to the starting culture biomass may have at least 0.8 × 10⁶-at least 9.0 x 10⁶Cell density of cells/ml.

The microcarrier concentration in the starting cell culture is preferably between about 0.5g/L and about 0.7 g/L. After concentrating the pooled biomass, the microcarrier concentration is preferably between about 0.65g/L and about 21 g/L.

The microcarriers used according to the method of the invention are preferably selected from microcarriers based on dextran, collagen, polystyrene, polyacrylamide, gelatin, glass, cellulose, polyethylene and plastic, and the microcarriers described by Miller et al (1989, Advances In Biochem Eng./Biotechnology., 39: 73-95) and Butler (1988, In: Spier & Griffiths, Animal cell Biotechnology, 3: 283-.

According to one embodiment of the method of the invention, the virus is selected from the group consisting of influenza virus, Ross river virus, hepatitis A virus, vaccinia virus and recombinant vaccinia virus, herpes simplex virus, Japanese encephalitis virus, West Nile virus, yellow fever virus and chimeras thereof, as well as rhinovirus and reovirus. It is within the knowledge of the skilled person to select adherent host cells and viruses susceptible to infection of the host, and to use the method of the invention to obtain an increased viral yield of the desired virus.

It is also within the knowledge of the person skilled in the art to select the respective microcarrier type, the concentration of microcarriers in the starting culture, the adherent cells and the culture medium which are susceptible to viruses, and to select the optimal growth conditions such as oxygen concentration, medium supplements, temperature, pH, pressure, rotation speed and feed control, so as to obtain a confluent cell culture biomass which can be used to obtain a cellular biomass with increased cell density and microcarrier concentration according to the method Volumetric viral yield.

The maximum virus yield that can be achieved in each system can be determined by standard methods. Thus, the method of the invention further comprises the step of harvesting the propagated and produced virus.

In another aspect of the invention, there is provided a method for producing a purified virus or virus antigen, the method comprising the steps of providing a culture of adherent cells bound to a microcarrier, growing the cell culture to confluence, infecting the cell culture with a virus, wherein the cell density in the cell culture is increased (i) prior to infection with the virus or (ii) after infection with the virus, and then incubating the cell culture infected with the virus to propagate the virus, (f) harvesting the virus produced and (g) purifying the harvested virus.

Lytic viruses, such as influenza viruses, lyse cells after an appropriate time after infection, and are thus released into the cell culture medium. The virus produced and released into the cell culture medium can be separated from the cellular biomass or other cellular debris by conventional methods, such as centrifugation (including ultracentrifugation, density gradient centrifugation), microfiltration, ultrafiltration, ion exchange chromatography, and the like, and purified.

The non-lytic virus proliferates within the cells and remains associated with the cells of the biomass. These viruses can be harvested by harvesting the biomass, lysing the cells by conventional methods, such as treating the cells with detergent, heat, freeze/thaw, sonication, french press, or other cell lysis methods. The virus released from the cells is harvested, concentrated and purified. Purification of the virus may be carried out by any method known in the art, such as ultrafiltration, ion exchange chromatography or isopycnic centrifugation, and the like.

Influenza viruses can be propagated on cell lines including the most potent MDCK cells, as well as on cell lines that have been approved for the production of human vaccines, such as VERO cells. The large-scale production of influenza viruses in serum-free or serum and protein-free medium on mammalian cell cultures on microcarrier beads in bioreactors has been described, as well as the development of influenza virus vaccines (Merten et al, 1999, Dev. biol. stand., 98: 23-37; Kistner et al, 1998, Vaccine, 16: 960-.

According to one aspect, the present invention provides a method of producing influenza virus comprising the steps of providing a culture of adherent cells bound to a microcarrier, growing the cell culture to confluence, infecting the cells with influenza virus, wherein the cell density in the cell culture is increased (i) prior to infection with the virus or (ii) after infection with the virus, and then incubating the cell culture infected with the influenza virus to propagate the virus. The cells infected with influenza virus may be VERO or MDCK cells or any cells susceptible to influenza virus. According to a preferred embodiment of the invention, VERO cells are used and infected with influenza virus. According to a preferred embodiment, the VERO cells are grown from the initial ampoule to the biomass in serum-free or serum and protein-free medium. VERO cells bound to microcarriers were grown to confluence in the respective culture media, with an increase in cell density and microcarrier concentration of at least 1.3-fold. The cells may be infected with influenza virus before or after the cell density of the culture volume is increased. Following incubation of the infected high cell density biomass and virus production, the produced influenza virus or influenza virus antigen is harvested. The harvested virus is further purified by methods known in the art, for example as described in Kistner et al 1998 (supra) or in U.S. Pat. No. 6,048,537.

Another aspect of the invention provides a cell culture biomass of microcarrier-bound adherent cells having a high cell density, wherein the cell density biomass of the cells in the cell culture is at least 1.3 times higher compared to a cell culture grown to confluence, the culture of microcarrier-bound adherent cells being cells selected from the group consisting of VERO, BHK, CHO, RK44, RK13, MRC-5, MDCK, CEF or diploid monolayer cells. The cell culture biomass having a high cell density is preferably a culture of VERO cells.

According to a preferred embodiment of the invention, the cell culture biomass is grown in serum-free medium, which does not contain any substances or agents derived from serum. According to another preferred embodiment, the biomass is free of serum and proteins, and does not contain any substances from serum or proteins added to the culture medium. Preferably, the cells are grown from the initial ampoule to the biomass in serum-free or serum and protein-free medium. During the virus propagation and production process, biomass with high cell density is maintained in serum-free or serum and protein-free medium.

According to another embodiment of the invention, biomass cells having a higher cell density than cell cultures grown to confluence are infected with a virus. The cell density and media volume of the high cell density biomass infected with the virus is maintained during the virus propagation process.

Another aspect of the invention provides a cell culture biomass of VERO cells bound to a microcarrier, wherein the biomass and cell density of the VERO cells in the cell culture is at least 1.3 fold compared to a VERO cell culture grown to confluence. The cell culture also has a higher microcarrier concentration than cells grown to confluence.

According to a preferred embodiment of the invention, the cell culture biomass having the higher cell density is a biomass of VERO cells. Preferably, the cells are grown in serum-free medium and the biomass is also serum-free. According to another preferred embodiment of the invention, the biomass culture is serum and protein free.

Another aspect of the invention provides a cell culture biomass of adherent cells bound to a microcarrier and infected with a virus, wherein the biomass of infected cells in the cell culture is at least 1.3 times higher than the biomass of a cell culture grown to confluence prior to infection, the biomass having a higher cell density. According to one embodiment, the cell culture biomass of the cells is serum free. According to another preferred embodiment of the invention the cell culture biomass is serum and protein free. The cell density of the high cell density biomass infected with the virus does not decrease during the virus propagation process.

Another aspect of the invention provides a cell culture biomass of VERO cells bound to a microcarrier, which is bound to the microcarrier at a high cell density, wherein the biomass of the VERO cells in the cell culture is at least 1.3 times higher compared to a VERO cell culture grown to confluence, wherein the VERO cells are infected with a virus. The VERO cells are infected with a virus selected from the group consisting of influenza virus, Ross river virus, hepatitis A virus, vaccinia virus and recombinant derivatives thereof, herpes simplex virus, Japanese encephalitis virus, West Nile virus, yellow fever virus and chimeras thereof, rhinovirus and reovirus.

According to a further aspect, the invention provides a cell culture biomass of VERO cells bound to microcarriers, comprising a high cell density of the cells infected with influenza virus, wherein the VERO cell biomass in the cell culture is at least 1.3 fold compared to a VERO cell culture grown to confluence.

According to a further aspect, the invention provides a cell culture biomass of VERO cells bound to microcarriers, comprising a high cell density of the cells infected with ross river virus, wherein the biomass of VERO cells in the cell culture is at least 1.3 fold compared to a VERO cell culture grown to confluence.

According to a further aspect, the invention provides a cell culture biomass of VERO cells bound to microcarriers, which contains a high cell density of the cells infected with hepatitis a virus, whereby the VERO cell biomass in the cell culture is at least 1.3 fold compared to a VERO cell culture grown to confluence.

Having now generally described the invention, the same will be understood by reference to the following examples, which are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified.

Example 1:

production of viral antigens on concentrated VERO cell biomass

a) Growth of cell cultures

VERO cells (African green monkey, Cercopthecus aethiops, kidney) were used as production cell lines. The cells have been obtained from the American type culture Collection under accession number ATCCCL 81 and passage number 124. these cells are suitable for growth in serum-free or serum and protein-free medium as described by Kistner et al, 1998 (supra), WO96/15231 or US6,100,061 for growth in serum-free medium, basic DMEM HAM's F12 medium supplemented with inorganic salts, amino acids, sodium bicarbonate (2g/L) and yeast or soy extract (0.1-10g/L) is used. Working cell banks were prepared, but without using any animal-derived media components.

Cells of the working cell bank were expanded in a division ratio of 1:6 or 1:8 in T-flasks and roller bottles. In stirred tank bioreactorsThe microcarriers serve as attachment material for further cell proliferation and the cells are grown at 37 ℃ for 6-8 days. The culture conditions were kept constant at 20% + -10% oxygen saturation and pH7.25 + -0.35. At the end of biomass production, when the cells have reached confluent growth, a portion of the biomass reactor volume is concentrated 2-fold by sedimentation, and then the cell density of the unconcentrated and concentrated cell culture is determined.

b) Determination of cell density of biomass

The cell number of the biomass of the cell culture is determined at the end of the biomass production by a method in whichEt al (1988, Biotechnology in LaborPraxis, 10: 1096-1103) describe the digestion of cells with trypsin and the use ofCell counts were performed by either a cell counter (method a) or treatment with citric acid and crystal violet followed by counting by a hemocytometer as described by Sanford et al (1951, j. natl. cancer inst., 11: 773-. The data are shown in table 1.

TABLE 1

Determination of cell number in confluent cell cultures at the end of biomass production and after concentration of confluent cell cultures

	Biomass production	Concentrated biomass
			Concentration of carrier g/L	5.0	10.0
Cell Density cells/ml (method A)	4.6×106	9.2×106
			Cell Density cells/ml (method B)	5.6×106	11.2×106

Example 2:

comparison of viral antigen production of pooled biomass with concentrated pooled biomass

VERO cells with the indicated passage number were thawed from liquid nitrogen and then passaged in flat and roller bottles to generate enough cells for seeding into a 1.5 liter bioreactor. After reaching a final cell density of 1.5X 10⁶After confluence of cells/ml, the cells were trypsinized and transferred to a 10 liter bioreactor. This was then used as inoculum for a 100 liter bioreactor having a microcarrier concentration of 1.5g/L from a solution containing 10⁷Individual cell working cell bank ampoules began, requiring approximately 30 passages to reach the final confluent VERO cell biomass. These cultures were grown until confluence was reached, with 1.9X 10⁶Final cell density per ml. Prior to viral infection, 2 10 liter bioreactors were loaded with cell culture biomass, which had different total cell numbers fermenter A was used at 1.9X 10¹⁰The total cell count of the fermenter B was 3.8X 10¹⁰To obtain higher biomass and carrier concentrationsFermentor a contained 100% of the cellular biomass of the original cell culture grown to confluence and fermentor B contained 200% of the cellular biomass of the original cell culture grown to confluence.

a) Production of influenza virus

Cell cultures in fermentors A and B were infected with influenza strain H3N2A/Sydney/5/97 at 0.01 m.o.i.. The same operating parameters, namely 32 ℃ 20% pO, were applied₂And pH 7.1. To activate influenza virus for virus propagation, a protease such as trypsin, pronase or a trypsin-like part thereof is added.

The viral antigen productivity of two different cell cultures in fermenters a and B containing different biomass concentrations was determined and compared according to influenza virus titer (HAU/ml) and antigen content (density gradient purified antigen). The peak region corresponds to the final total antigen concentration in the lysis cycle at day 3 post infection. The data are shown in table 2.

TABLE 2

Determination of influenza virus titer and antigen in confluent VERO cell cultures and concentrated confluent VERO cell biomass

Fermentation tank	A	B
			Concentration of carrier	1.5g/L	3.0g/L
Cell Density cells/ml (method B)	1.90×106	3.80×106
			HAU/ml	640	2560

Peak region (relative unit)

83.3(100％)

412.3(495％)

b) Production of Ross river Virus

The VERO cells were propagated to confluence as described above, with a final density of 1.6X 10⁶Cells/ml. Prior to viral infection, 2 50 liter bioreactor systems were loaded with cell culture biomass having different total cell numbers fermentor A cell density 1.6X 10⁶Cell/ml, cell density of fermenter B2.3X 10⁶Cells/ml, which is 1.5 times the concentration of confluent cell culture biomass. Fermentors a and B were infected with ross river virus and the viral antigen productivity of fermentors a and B was determined as described above. Table 3 shows the results of the virus yields obtained using different concentrations of biomass for virus propagation.

TABLE 3

Determination of Ross river Virus Titers and antigen production

Fermentation tank	A	B
			Concentration of carrier g/L	1.5	2.25
Cell density (. times.10)6Cells/ml)	1.6	2.3
			Viral titer (log TCID)50)	8.71	8.95
Viral titer pfu/106Cells (× 10)6)	321	388
			Yield (%)	100	121

Example 3:

viral antigen production on concentrated RK cell biomass

a) Growth of cell cultures

Rabbit kidney cells RK-13 or its complementary derivative RK-D4R-44 described by Holzer et al (1997, J.Virol., 71: 4997-5002) were used as producer cell lines. Cells were grown in conventional medium containing 2% serum.

Cells from the working cell bank were expanded in T-flasks and roller bottles at a division ratio of 1: 6. In a 10L stirred tank bioreactor(Pharmacia) the microcarriers serve as attachment material for further proliferation of cells.

b) Defective vaccinia virus production

After the cells of RK-13 or RK-D4R-44 reached confluence and final cell density in the tank bioreactor, the biomass was infected at an m.o.i. of 0.01 with either vaccinia virus WR or defective vaccinia virus vD4-ZG #2 as described by Holzer et al, 1997 (supra). after infection, 2 10 liter bioreactor systems were loaded with infected cell culture biomass with different total cell numbers.fermenter A was used at 1.2X 10¹⁰For fermenter B, 2.4X 10¹⁰To achieve higher biomass and carrier concentrations for fermentor B, the infected cell culture grown to confluence is concentrated by biomass precipitation to achieve higher concentrations fermentor A contains 100% of the original fine particles grown to confluenceCell biomass of the cell culture, fermentor B contained 200% of the cell biomass of the original cell culture grown to confluence. Two different cell culture fermentors A and B were tested for viral antigen productivity, which contained different biomass concentrations per unit volume of medium of infected cells the results are summarized in Table 4.

TABLE 4

Determination of vaccinia Virus Titers on RK cells

Fermentation tank	A	B
			Concentration of carrier g/L	1.5	2.5
Cell density (. times.10)6Cells/ml)	1.2	2.4
			Viral titer pfu/106Cells (× 10)6)	0.8	1.3
Yield (%)	100	162

The above examples are provided to illustrate the invention, but are not intended to limit its scope. All publications, patents and patent applications cited herein are hereby incorporated by reference for all purposes.

Claims (11)

1. A cell culture biomass prepared by growing a cell culture of adherent cells bound to a microcarrier to confluence and then concentrating the cell culture, wherein the concentrated cell culture biomass has a cell density and a microcarrier concentration that is between 1.3-fold and 10-fold greater than the cell density and microcarrier concentration of the cell culture biomass grown to confluence.

2. The cell culture biomass of claim 1 wherein the cells are VERO cells.

3. The cell culture biomass of claim 1 or 2, wherein the culture is serum free.

4. The cell culture biomass of claim 3, wherein the culture is serum and protein free.

5. The cell culture biomass of claim 1, wherein the cells are infected with a virus after growth to confluence.

6. The cell culture biomass of claim 5, wherein cell density and microcarrier concentration is increased after infection of cells with a virus.

7. The cell culture biomass of claim 5 infected with a virus selected from the group consisting of influenza virus, Ross river virus, hepatitis A virus, vaccinia virus, herpes simplex virus, Japanese encephalitis virus, West Nile virus, yellow fever virus, and reovirus.

8. The cell culture biomass of claim 5 infected with a rhinovirus.

9. The cell culture biomass of claim 7, wherein the virus is an influenza virus.

10. The cell culture biomass of claim 7 wherein the virus is Ross river virus.

11. The cell culture biomass of claim 7, wherein the virus is a vaccinia virus.