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    • 专利摘要:
    A method of purifying a virus comprising at least the following steps: a) obtaining a fluid containing a virus, b) providing a preformed linear density gradient of a given range X% -Y% ( mass / volume) (X being the lower limit and Y being the upper limit) in a centrifugal rotor having a capacity of at least 4L, at least 6L, at least 8L, or at least 10L, c) the addition of fluid at the preformed linear density gradient, d) centrifugation to separate the virus from contaminating impurities, and e) collection of fractions comprising the purified virus, wherein the linear portion of the gradient comprises the percentage of density corresponding to the percentage at which the virus to be purified will migrate.
    公开号:BE1023903B1
    申请号:E2015/5818
    申请日:2015-12-14
    公开日:2017-09-08
    发明作者:Pascal Charles Louis Gerkens；Jean-François Jose Alain Marie Ghislain Leruse
    申请人:Glaxosmithkline Biologicals Sa；The Chemo-Sero-Therapeutic Research Institute；
    IPC主号:
    专利说明:

    NEW PROCESS
    STATEMENT ON SPONSORED RESEARCH AT FEDERAL LEVEL
    The present invention has been carried out with the support of the United States Government under Contract No. HHS0100200600011C granted by the Department of Health and Human Services; the United States Government has certain rights in the present invention.
    TECHNICAL AREA
    The present invention relates to the field of virus purification.
    BACKGROUND
    The development of cell culture technologies as an alternative to traditional egg-based production systems for the manufacture of antiviral vaccines seems to be the fastest and most promising solution to overcome the disadvantages and constraints associated with traditional drug-based systems. eggs. Commercial production of viral vaccines requires large amounts of virus as a source of antigen. However, the egg-based process is vulnerable because of the variable biological quality of the eggs and lacks flexibility because of logistical problems due to the unavailability of large quantities of suitable eggs.
    However, after production, whether it is produced on eggs or on a cell culture, the virus produced must be recovered from the cell culture and, where appropriate, it must be purified. Virus purification methods are known in the art. A typical method employs sucrose gradient centrifugation. For example, WO 2009/007608, WO 2008/135229 and WO 2010/089339 describe the purification of viruses by sucrose gradient centrifugations using a self-generating gradient.
    Effective vaccine production requires the development of large-scale quantities of virus produced in high yields from a host system. The culture conditions in which a virus is grown are extremely significant in achieving an acceptable high yield of the virus. Therefore, in order to optimize the desired virus yield, the system and culture conditions must be tailored specifically to provide an environment that is advantageous for the production of the desired virus, suitable for large scale production. One way is to improve the specific productivity of the cell, for example, by improving the culture medium or by increasing the cell density and / or reducing the loss of virus material that occurs as the different purification steps progress. . An additional level of complexity is introduced at the manufacturing level, when large volumes or quantities have to be processed. When scaling up, the risk of losing more product quality (eg product purity) and / or product yield is significant. Such occurrence requires further optimization of a process developed satisfactorily on a small scale. Therefore, it remains necessary to provide methods for producing and purifying viruses having an adequate level of purity and good performance on a large scale.
    SUMMARY OF THE INVENTION
    In a first aspect of the present invention, there is provided a method for purifying a virus comprising at least the following steps: a) obtaining a fluid containing a virus, b) providing a preformed linear density gradient a given range X% -Y% (v / w) (where X is the lower limit and Y is the upper limit) in a centrifugal rotor having a capacity of at least 4L, at least 6L, at least 8L, or at least 10L, c) adding the fluid to the preformed linear density gradient, d) centrifuging to separate the virus from contaminating impurities, and e) collecting fractions comprising the purified virus, wherein the linear portion of the gradient comprises the percentage of density corresponding to the percentage at which the purified virus will migrate.
    In a second aspect of the present invention, there is provided a method for the preparation of a vaccine comprising at least the step of purifying the virus for use as an antigen in the vaccine according to the method of the invention and formulating said virus. purified in a vaccine.
    In a third aspect of the present invention there is provided a method for preparing a vaccine comprising at least the following steps: A) obtaining a fluid containing a virus, B) providing a preformed linear density gradient a given range X% -Y% (v / w) (where X is the lower limit and Y is the upper limit) in a centrifugal rotor having a capacity of at least 4L, at least 6L, at least 8L, or at least 10L, wherein the linear portion of the gradient comprises the percentage by density corresponding to the percentage at which the purified virus will migrate, C) the addition of the fluid to the preformed linear density gradient, D) the centrifugation so as to separate the virus from the contaminant impurities, and E) the collection of fractions comprising the purified virus, F) the formulation of purified virus in a vaccine.
    DESCRIPTION OF THE DRAWINGS
    Figure 1: Form of a self-generating sucrose gradient obtained in a rotor of 3.2L. Successive fractions of 100 ml of the gradient formed were collected and analyzed for their sucrose content by refractometry.
    Figure 2: Form of the self-generating saccharose gradient obtained in an 8L rotor. Successive fractions of 250 ml of the gradient formed were collected and analyzed for their sucrose content by refractometry.
    Figure 3: Form of a preformed sucrose gradient obtained in an 8L rotor. Figure 3A shows a diagram representing the method of forming the preformed linear gradient - 3 containers comprising 4L of 55% sucrose (Container 1) and 1L of PBS / Citrate (Containers 2 and 3) are successively connected to each other, the third container being connected to an 8L rotor. Fig. 3B shows the form of the preformed sucrose obtained by following the method shown in Fig. 3A. Successive fractions of 250 ml of the gradient formed were collected and analyzed for their sucrose content by refractometry.
    Figure 4: Form of a preformed sucrose gradient obtained in an 8L rotor. Figure 4A shows a diagram showing the process of forming the preformed linear gradient - 2 containers comprising 4L of 55% sucrose (Container 1) and 1L of PBS / Citrate (Container 2) are successively connected to each other, the second being connected to an 8L rotor. Figure 3B shows the form of the preformed sucrose obtained by following the method shown in Figure 3A. Successive fractions of 250 ml of the gradient formed were collected and analyzed for their sucrose content by refractometry.
    DETAILED DESCRIPTION
    The present invention relates to an improved method for purifying viruses, whether produced in eggs or in cell cultures, which is particularly useful for large scale production. A typical step used during virus purification is a density gradient ultracentrifugation step, such as a sucrose gradient for example. Ultracentrifugation in density gradient is a technique commonly used to purify viruses, especially enveloped viruses. It is particularly used in the field of vaccine manufacturing. Generally, separation of particles, such as a virus and possible contaminating impurities, by density gradient ultracentrifugation can be effected by zone, that is, based on differences in particle size, or it can be isopycnic, that it relies on differences in particle density, or it can rely on a combination of both. To achieve zone separation, the density of the particles to be separated must be greater than the density of the gradient in any position of the gradient. In isopycnic methods, the density gradient includes the entire range of densities of the particles to be separated. Depending on their density and the density gradient range, after centrifugation, the particles migrate to a position in the gradient where the density is equal to the particle density (the particles float in this position and remain there), or the particles are put into pellets in the bottom of the gradient. After centrifugation, only gradient fractions containing the virus will be collected. The preformed linear gradient of the invention can be suitably used for zone separation or isopycnic separation. In one embodiment, the separation of the virus from contaminating impurities in the method of purifying a virus according to the invention is isopycnic. Generally, a density gradient can be linear or discontinuous. In particular, a linear gradient offers the advantages of allowing a better separation between virus particles and residual contaminating impurities from the host used to produce virus particles and obtaining a purified virus which is concentrated in a small volume. When scaling up the production process of a virus, and in particular when considering the use of a rotor having a high capacity to record a number of centrifuges required to process large volumes, the inventors observed that the known method they used for small rotors, consisting of the creation of a self-generator gradient from a simple mother solution did not provide a linear gradient. In order to obtain a linear gradient in rotors having a high capacity, the inventors observed that the gradient had to be preformed as linear, namely formed before starting the centrifugation.
    By "preformed" is meant, in the context of the present invention, that the gradient is created, and therefore formed in the rotor, before starting the centrifugation, as opposed to a self-generating gradient.
    By "self-generator" is meant in the context of the present invention that the gradient is created, and therefore formed during centrifugation.
    By "linear" is meant in the context of the present invention, a density gradient in which a plot of concentration against volume of the gradient gives a substantially straight line, as opposed to a gradient "discontinuous" or step, which is composed of layers, with sudden changes in concentration from one layer to another.
    By "plateau" is meant in the context of the present invention a significant gradient zone, namely a significant portion of the gradient volume, over which the concentration value, or percentage, remains approximately constant. For example, a tray may have 5%, 10%, 15%, or 20% or more of the total gradient volume. The trays may be present, in particular, at the ends of the gradient.
    By "an approximately constant value" is meant in the context of the present invention, relative to a given value, values having a variation of 5% or less.
    For clarity and simplicity, the preformed linear gradient implemented in the process of the invention will be described below with respect to a sugar gradient. However, such a preformed linear gradient is not limited to sugar gradients. All the detailed disclosure below is applicable to other types of gradients.
    The inventors have observed that when loading on the rotor a total of 4 and up to 7 different layers of sugar with an increased density, they could obtain a gradient with acceptable linearity after centrifugation. Alternatively, in order to avoid manually changing the sugar bottle for each new layer of sugar to be loaded, the inventors have developed an automatic method of forming a large-scale preformed linear gradient based on progressive dilution.
    By "dilution" is meant that a mother solution forming a gradient at an appropriate percentage (mass / volume) is diluted in a single dilution solution, or in successive dilution solutions, before being injected into the rotor . As described in more detail below, the extent of dilution, the percentage of the stock solution, the volume of the stock solution, and the volume of the dilution solution (s) may vary depending, for example, on the targeted range and / or the targeted inclination of the linear gradient to be preformed.
    By "progressive dilution" is meant a drip system in which each drop of a concentrated solution is diluted in a dilution solution. For example, the concentrated solution may be the stock solution, or the diluted stock solution to be further diluted, when more than one dilution is contemplated as described below.
    By a "large capacity rotor" is meant a rotor having a capacity of at least 4L, suitably at least 6L, suitably at least 8L, more suitably at least 10L or more.
    By "rotor capacity" is meant the maximum volume of liquid that a rotor can carry.
    By "load capacity" is meant the amount of fluid containing a virus that is charged per liter of rotor.
    The method according to the present invention is particularly advantageous when it is necessary to purify a virus produced on a large scale. For example, the method of the invention is suitably used when 1L, 50L, 100L, 200L, 500L, 1000L, or higher volumes of virus-containing fluids are to be processed and purified.
    The method of the present invention is applicable to the purification of any type of virus and is independent of the types of host substrate used to produce viruses. For example, the method according to the invention is applicable to viruses produced on cell cultures or eggs.
    The method of the invention is capable of use in a wide range of viruses, any virus that is generally purified by density gradient ultracentrifugation. By way of example, the method of the invention contemplates the purification of enveloped viruses, in particular hepnavirus, herpes virus, orthomyxovirus, such as, for example, influenza virus, paramyxovirus such as the virus. measles, togaviruses such as the rubella virus, rhabdoviruses such as, for example, rabies virus, poxviruses and retroviruses. In one embodiment, the purified viruses produced by the method of the invention belong to the Orthomyxovirus family, in particular the influenza virus.
    The present invention does not rely on the use of a specific density gradient type and can therefore be applied to any type of density gradient. The choice of density gradient medium depends on the virus to be purified and the application that is provided for the purified virus obtained. For example, enveloped viruses are less dense than non-enveloped viruses. This is known in the art. Specifically, depending on the purpose for which the virus produced by the method is prepared, a medium that does not affect the integrity of the virus or its biological activity will be used. For example, when the virus prepared according to the method of the invention is intended for vaccination, the medium is chosen so as to maintain the immunogenicity of said virus, or the corresponding viral antigen. Sugar solutions, in particular sucrose, can be used to generate density gradients for use in the process according to the invention. Sucrose is particularly useful for purifying enveloped viruses, such as, but not limited to, influenza viruses. It is also a sugar frequently used in the field of vaccine manufacturing.
    In one embodiment, the sugar used to create a preformed linear density gradient according to the method of the present invention is sucrose. This is advantageous for the purification of products for human use when safety considerations are to be taken into account. However, the invention also contemplates the use of other sugars, including alcohol sugars, such as, for example, sorbitol or hydrogenated sugars, linear sugars and modified sugars or any other sugar provided that said sugar has a solubility in water sufficient to produce solutions having specified densities according to the type of virus to be purified.
    Preformed linear density gradients used in the process of the present invention are not limited to sugar gradients. The present invention also contemplates, as other illustrative examples, the use of salt gradients, such as, for example, cesium chloride, which is suitable for the purification of enveloped and non-enveloped viruses. Alternatively, the gradient can also be prepared with potassium tartrate, which has the advantage of reaching a gradient having a higher density, relative to the sucrose gradient. Therefore, potassium tartrate gradients are, in particular, suitably employed to purify unsheathed viruses.
    The preformed linear gradient to be provided in a rotor having a high capacity according to the method of the invention may be formed by gradually diluting a stock solution in one or more dilution solutions prior to injection into the rotor.
    linearity
    The linearity of the preformed gradient used in the process of the invention can vary in two ways. On the one hand, the slope that defines the inclination of the gradient can vary. The appropriate inclination may be chosen, for example, depending on the desired level of virus purification and the desired number of gradient fractions from which the purified virus is drawn, i.e. the concentration of the virus fluid after purification on a density gradient ultracentrifugation. Generally, the more the linear gradient is inclined, the more the purified virus will be concentrated because it will be present in a number of gradient fractions corresponding to a smaller volume. Conversely, the more a linear gradient is progressive, the less the concentrated virus will be concentrated, as this will be present in a number of gradient fractions corresponding to a higher volume. Similarly, the slope of the linear gradient will impact the ability to separate the virus from residual contaminating impurities. It is incumbent upon one skilled in the art to determine the appropriate slope of the linear gradient, if necessary, taking into account the two elements above, namely the virus concentration and the virus separation quality of residual contaminating impurities. On the other hand, the extent of the linearity, namely the gradient volume over which said gradient is linear, may vary. The minimum requirement is that the preformed linear gradient used in the method of the invention be linear over the density range of the gradient comprising the percentage of density corresponding to the percentage at which the virus to be purified will migrate. Suitably, the preformed linear gradient used in the process of the invention is linear over at least 30%, suitably at least 50%, suitably at least 60%, suitably at least 70%, suitably at least 80%, suitably at least 90%. % of its total volume or the gradient is suitably linear over its entire volume. In particular embodiments, the preformed linear density gradient used in the process of the invention is linear over 50% to 90%, over 60% to 80% of its total volume. In particular subsequent embodiments, the preformed linear density gradient used in the method of the invention is linear over at least 70%. Therefore, the preformed linear gradient to be used in the method of the invention may have trays at the ends of the gradient, either at one end only, or at both ends. However, the added volume corresponding to trays, if multiple trays are present, does not adequately exceed 70%, suitably 50%, suitably 40%, suitably 30%, suitably 20%, suitably 10%, or 5% of the total gradient volume. .
    The linearity, as well as the magnitude of the linearity, of a density gradient can be verified using any method known in the art for plotting the percentage of the gradient against the volume of the gradient. Linearity, as defined above, is established when such a pattern gives a substantially straight line. Similarly, from such a plot, the slope can be calculated, ie the inclination can be monitored and adapted, if necessary. For example, when sugar is the medium used for the density gradient, refractometric analysis such as Brix analysis, which is well known in the art, can be used to measure the percentage of sugar (expressed in grams to 100 ml) in successive fractions of a given volume of the gradient.
    Gradient concentration range
    The present invention is not limited to a particular percentage range of density gradients. Said range should be determined according to the virus to be purified, in particular, depending on the density of the virus, the densities of the residual contaminating impurities provided and the type of separation to be used, whether isopycnic or by zone. Those skilled in the art will know how to associate with a given virus (having a given density) the appropriate percentage range of a density gradient, ensuring that the linear part of the preformed gradient comprises the percentage of density corresponding to the percentage at which the virus to be purified will migrate. The presence of a specific virus or a corresponding viral antigen at a certain range in a gradient can be monitored by standard protein detection techniques, such as Western-blot analysis using antigen specific antibody. viral. In the particular case of the influenza virus, the content of one of its surface antigens, the HA antigen (hemagglutinin) can be monitored by the SRID test (Single Radial Immuno Diffusion) which is a known technique. Those skilled in the art (JM Wood et al .: An improved single radial immunodiffusion technique for the assay of influenza haemagglutinin antigen: adaptation for potency determination of inactivated whole virus and subunit vaccines J. Biol Stand 5 (1977) 237-247, JM Wood et al .: International collaborative study of single radial diffusion and immunoelectrophoresis techniques for the assay of haemagglutinin antigen of influenza viruses, J. Biol Stand 9 (1981) 317-330). For a given percentage range, those skilled in the art will determine the appropriate dilution of a stock solution of a suitable percentage (mass / volume) necessary to create a preformed linear gradient with appropriate linearity to be used in the process. method of the present invention. A typical gradient range for purifying viruses is 0-55% (w / v). In one embodiment, the preformed linear density gradient for use in the method of the invention, such as the sucrose gradient, has a percent range of 0-55% (w / v).
    The stock solutions and the dilution solution (s) can be prepared in water or added with a salt at physiological concentrations (eg NaCl) and with a buffer (eg Tris or sodium phosphate). Stock solutions and dilution solutions can be conveniently prepared in a buffered solution comprising a salt at a physiological concentration, such as TBS or PBS. In one embodiment of the invention, stock solutions, such as, for example, stock solutions of sugar, in particular sucrose, and dilution solutions are prepared in a phosphate buffered saline (PBS) solution containing citrate, because this type of solution advantageously prevents the aggregation of the virus during centrifugation.
    Mother solution
    By "mother solution" is meant the solution forming the gradient which will be diluted to provide a preformed linear density gradient in a high capacity rotor according to the method of the invention. The parent solution forming the gradient to be used to prepare a preformed linear gradient of a given range X% -Y% (mass / volume) (where X is the lower limit and Y is the upper limit), such as a sugar gradient , is suitably a percentage that equals Y, or is greater. A typical non-limiting sugar gradient range for purifying viruses is 0-55% (w / v). For example, if a sugar gradient of 0-55% (w / v) is targeted, the mother sugar solution may suitably be a 55% (w / v) or greater sugar solution.
    The volume of the stock solution is chosen so as to obtain a preformed linear gradient reaching the upper limit of the expected range. For a given range X% -Y% (where X is the lower limit and Y is the upper limit) (mass / volume), the volume of the stock solution is chosen so as to obtain a preformed linear gradient reaching the upper limit Y % from the beach. The inventors have observed that the ratio "volume of the stock solution / volume of the dilution solution" is a factor to be taken into account. The mother solution suitably has a volume greater than the dilution solution. For example, such a ratio is suitably 2: 1.3: 1.4: 1, 5: 1 or 6: 1.
    The volume of the stock solution is also chosen according to the capacity of the rotor. Suitably, the volume of the stock solution does not exceed the capacity of the rotor. In some embodiments, in which batch centrifugation, the volume of the stock solution is the same as the capacity of the rotor. When centrifugation is performed in a continuous mode, the volume of the stock solution can have a volume of 90% or less rotor capacity, suitably 80% or less. In embodiments, in which the centrifugation is performed in a continuous mode, the volume of the stock solution forming the gradient is between 40% and 80% of the rotor capacity, between 50% and 60% of the rotor capacity , or represents 50% of the rotor capacity. In particular embodiments, in which the rotor is an 8L rotor, the parent solution forming the gradient has a volume of between 3.2L and 6.4L, or between 4L and 4.8L. In a particular further embodiment, wherein the rotor is an 8L rotor, the mother solution forming the gradient has a volume of 4L.
    Dilution solutions
    The dilution solution may not include sugar at all. Alternatively, it may also include some sugar, usually at a percentage lower than the percentage of the stock solution. Generally, a stock solution of a given percentage is diluted in a dilution solution (s) before being injected into a rotor of a given capacity. The inclination of the preformed linear gradient to be used in the process according to the invention can be adjusted by adjusting the dilution ratio. Generally, the more a gradient is inclined, the more likely it is to observe the presence of trays at the ends of the gradient.
    Decreasing the dilution ratio helps to reduce the trays at the ends of the gradient, to increase the magnitude of the linearity, namely to increase the volume of the gradient on which the gradient is linear and to make the linear gradient more gradual, namely with a softer slope. The decrease in the dilution ratio can be obtained for example by adding sugar or increasing its percentage in the dilution solution. An alternative option to lowering the dilution ratio is to perform a multiple dilution.
    By "multiple dilution" is meant successive dilutions. For example, the stock solution forming the gradient may be gradually diluted two or more times, ie diluted in a first dilution solution, said diluted stock solution being in turn gradually diluted in a second dilution solution, and so on before to be injected into the rotor. In one embodiment, the preformed linear gradient is formed by progressively diluting a stock solution given twice, i.e. diluted in a given first dilution solution, said diluted stock solution being progressively diluted in a second given dilution solution before be injected into the rotor. In particular embodiments, where such multiple dilutions are made, the dilution solutions do not include any sugar and optionally have the same volume.
    The volume of the dilution solutions used to create a preformed linear gradient for use in the process according to the invention may vary. In particular, the volume is dependent on the volume of the gradient-forming stock solution, and the target dilution ratio, ie, on the predicted inclination of the preformed linear gradient. For example, a given dilution solution may suitably have a volume of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 50% of the stock solution.
    In one embodiment, for an expected gradient of a range X% -Y% (mass / volume) (where X is the lower limit and Y is the upper limit), a stock solution having a percentage Y% is all the same. firstly diluted in a dilution solution having a volume of 25% of the volume of the stock solution, said diluted stock solution being gradually diluted in a second dilution solution having a volume of 25% of the volume of the stock solution. Generally, the different containers each comprising the mother solution forming a gradient and the dilution solution (s) and the rotor which will receive the preformed linear gradient are successfully connected to each other, the last container being connected to the rotor by means of 'a pump. Said pump, once activated, creates a vacuum causing suction of the stock solution in the first dilution solution, which is in turn sucked, and thus gradually diluted in the next dilution solution, depending on the number of solutions of dilutions used before the final diluted solution is injected into the rotor. The flow rate of the pump is chosen in a suitable range so as not to disturb the gradient obtained and formed by a progressive dilution. A non-limiting example of a pump flow rate to be suitably used in the process according to the invention is between 100 and 200 ml / min, and is suitably 160 ml / min.
    centrifugation
    Centrifugation conditions are standard conditions. It is incumbent upon those skilled in the art to determine appropriate conditions such as, for example, centrifugal force, operating time, flow rate, or strapping time, if any. The determination of said conditions will have to take into account the type of fluid containing the virus to be purified, the type of virus, the type of contaminating impurities, the mode of centrifugation, by batch or continuously, the type of separation, by zone or isopycnique. In particular, information provided in the manufacturer's instructions on how to use the rotors and centrifuges may guide those skilled in the art in the selection of appropriate centrifugation conditions. As described above, those skilled in the art can use any standard protein detection technique, such as a Western blot using an antibody specific for a viral antigen, or in the particular case of influenza, the SRID test to detect the presence of the virus and / or monitor whether acceptable virus yield and / or virus purity is obtained after centrifugation on a preformed linear density gradient of the invention.
    The centrifugation can be carried out in continuous mode, in semi-continuous mode or in successive batches. In one embodiment, the centrifugation during the method of purifying a virus according to the invention is carried out in continuous mode. Continuous mode centrifugation is advantageously used on a large scale when large volumes of virus-containing fluids need to be processed and purified.
    Depending on whether centrifugation is performed batchwise or in continuous mode, the loading of the preformed linear gradient may vary. In batch mode, the preformed linear gradient can be loaded into an empty rotor at rest. In these embodiments, the charged volume may be the same as the capacity of the rotor. Alternatively, when the centrifugation is carried out in continuous mode, the rotor may be pre-filled with a suitable buffer solution at rest, and during the injection of the preformed linear gradient into the rotor still at rest, said buffer solution is progressively replaced by the preformed linear gradient. Such pre-filling has the advantage of avoiding the formation of bubbles. In some embodiments, wherein the centrifugation is performed in a continuous mode, the volume of the preformed linear gradient that is injected into the rotor corresponds to the volume of the stock solution forming the gradient, i.e. the injection is stopped when the container comprising the stock solution is empty.
    Once the preformed linear gradient is loaded into the rotor, the fluid containing the virus can be loaded onto the gradient at rest, and the centrifugation starts after the fluid containing the virus has been loaded onto the gradient. These embodiments are conveniently performed when the centrifugation is performed batchwise. Alternatively, the centrifugation can be started after the preformed linear gradient is loaded into the rotor, and once the appropriate centrifugal force is reached, the virus-containing fluid can be loaded onto the gradient. Such alternative embodiments are suitably implemented when the centrifugation is performed in continuous mode.
    The appropriate centrifugal force may be chosen by those skilled in the art and may depend on the type of rotor and centrifuge used. By way of nonlimiting examples, a centrifugal force of between at least 20,000 g and 200,000 g, suitably between 90,000 g and 150,000 g, suitably between 100,000 g and 120,000 g, is applied to the rotor in the centrifuge. Once all the fluid containing the virus has been treated, and after an adequate run time, the centrifugation is stopped and the gradient fractions containing the virus are collected.
    Fluid containing the virus
    The fluid containing the virus to be purified within the scope of the invention is not limited to crude fluids, but also contemplates fluids that include partially purified viruses. For example, the purification may include a number of different filtration, concentration and / or other separation steps such as ultrafiltration, centrifugation, chromatography (such as ion exchange chromatography) and adsorption in a variety of combinations. A clarification step may be required to separate the virus from the cellular material contaminant, in particular floating cells or cell debris, or contaminants derived from eggs from allantoic fluids.
    The term "crude" in the context of the present invention means that no purification has been carried out on the fluid containing the virus after its collection, and therefore it may contain any kind of contaminants in varying magnitudes. By way of non-limiting example, when the virus has been produced by inoculating cells with said virus and when, after infection, newly formed viruses are released into the cell culture medium or supernatant, then said culture medium, which includes the virus, refers to an example of a crude fluid. Another example of a crude fluid that can be cited is the allantoic fluid harvested after virus inoculation on embryonated eggs and culture of the virus. Therefore, the term "partially purified" includes any intermediate purification state, namely a fluid that has been subjected to any purification step, for example, any of the steps mentioned above, individually, or in any combination.
    In one embodiment, the fluid containing the virus according to the invention is the culture medium collected after the infection of the cells with the virus of interest, such as an influenza virus for example, and its release in the medium of culture. In another embodiment, the fluid containing the virus according to the invention is the allantoic fluid collected after the eggs are inoculated with a virus of interest, such as the influenza virus.
    In another suitable embodiment, the fluid containing the virus according to the invention has been clarified before being added to a preformed linear density gradient according to the invention. This clarification can be done by filtration. Alternatively, centrifugation and / or filtration may be combined together, in any order, to achieve the desired level of clarification of the virus preparation. Although not required, a multiple filtration process may be performed, such as a two or three phase process consisting of, for example, sequential and progressive removal of impurities depending on their size, using filters having a size Suitable pore size, in particular, filters having a decreasing nominal pore size, to start removing large precipitates and cell debris. In addition, single operations employing a relatively narrow filter or centrifugation can also be used for clarification. More generally, any clarification approach, including, but not limited to, frontal filtration, depth filtration, micro-filtration or centrifugation, which provides a filtrate of clarity adapted to not soil the membrane and or the resins in successive steps, will be acceptable for use in the clarification step of the present invention.
    Although not required, it may be convenient to concentrate the fluid containing the virus before loading it onto the preformed linear density gradient of the invention to reduce the volume of fluid to be loaded. Therefore, the present invention also contemplates a virus-containing fluid that has been concentrated, prior to loading on the density gradient. Thus, the fluid containing the virus can be subjected to ultrafiltration (sometimes called diafiltration when used for buffer exchange), for example on a 750 kD membrane, to concentrate the virus and / or the exchange of buffer. This step is particularly advantageous when the virus to be purified is diluted, as is the case when grouping viral crops collected by infusion over a few days after inoculation. The method used to concentrate the virus may include any filtration method when the concentration of the virus is increased by forcing the diluent to pass through a filter so that the diluent is removed from the virus suspension while the virus is unable to pass through the filter and thus remains in concentrated form in the virus preparation. Ultrafiltration may include diafiltration which is an ideal way of removing and exchanging salts, sugars, non-aqueous solvents, removing low molecular weight materials, rapid ionic environments and / or pH changes. Microsolutes are removed more efficiently by adding solvent to the ultrafiltered solution at a rate equal to the ultrafiltration rate. This makes it possible to wash the micro-species of the solution at a constant volume, isolating the retained virus. Diafiltration is particularly advantageous when a downstream step requires that a specific buffer be used in order to obtain an optimal reaction. Concentration and diafiltration can be applied at any suitable stage of the purification process, when it is desired to remove undesirable compounds, such as sucrose, after a sucrose gradient ultracentrifugation, or as a formaldehyde, after a step of inactivating the virus with formaldehyde. The system consists of three distinct process streams: the feed solution (including the virus), the permeate and the retentate. Depending on the application, filters with different pore sizes can be used. The filter composition may be, but is not limited to, regenerated cellulose, polyethersulfone, polysulfone or the corresponding derivatives. The membranes can be flat sheets (also called flat screens) or hollow fibers.
    In one embodiment, the virus-containing fluid has been concentrated by ultrafiltration / diafiltration prior to loading it onto the preformed linear density gradient used in the process of the present invention.
    The purification method of the present invention may include additional steps in addition to the density gradient ultracentrifugation step as claimed herein. These steps can be implemented before or after the density gradient ultracentrifugation step. Specifically, the virus preparation obtained after using the density gradient ultracentrifugation step of the present invention may be further purified by applying any of the aforementioned virus purification techniques, such as filtration. , ultracentrifugation (including gradient ultracentrifugation), chromatography (such as ion-exchange chromatography), and adsorption in a variety of combinations.
    In one embodiment, the method of the present invention further comprises at least one step selected from the group consisting of: filtration, ultrafiltration / diafiltration, ultracentrifugation and chromatography, or any combination thereof. Depending on the level of purity desired, the above steps can be combined in any way.
    Alternatively, it is also possible to further purify viruses by chromatography, including ion exchange, anionic or cationic chromatography, size exclusion chromatography, such as gel filtration or gel permeation, hydrophobic interaction, hydroxyapatite or any combination thereof. As mentioned above, the chromatography steps can be carried out in combination with other purification steps, such as density gradient ultracentrifugation. eggs
    Fluids containing a virus, such as the influenza virus, according to the invention can be derived from the conventional method of embryonated eggs, by developing an influenza virus in eggs and by purifying the harvested allantoic fluid.
    cells
    The virus to be purified according to the method of the invention may be produced on a cell culture. The method according to the present invention is applicable to any type of cells, whether they are adherent cells grown on microcarriers or suspension cells.
    The cells can be developed in different ways, such as, for example, using batch, batch, or continuous systems, such as infusion systems. An infusion is particularly advantageous when a high cell density is desired. A high cell density may be particularly advantageous for optimizing the amount of virus that can be produced from a given cell type.
    The cells used to produce a virus-containing fluid to be purified according to the method of the invention can in principle be any desired type of animal cells that can be cultured in the cell culture and can support virus replication. It can be either primary cells or continuous cell lines. Genetically stable cell lines are preferred. Mammalian cells are particularly suitable, for example, human, hamster, cattle, monkey or dog cells. Alternatively, insect cells are also suitable, such as for example SF9 cells or Hi-5 cells.
    A number of mammalian cell lines are known in the art and include PER.C6, HEK, human embryonic kidney cells (293 cells), HeLa cells, CHO cells, Vero cells and MDCK cells.
    Suitable monkey cells are, for example, African green monkey cells, such as renal cells as in the Vero cell line. Suitable dog cells are, for example, renal cells as in the MDCK cell line.
    Mammalian cell lines for developing an influenza virus include MDCK cells, Vero cells, or PER.C6 cells. These cell lines are all widely available, for example, from the American Type Cell Culture (ATCC) cell culture bank.
    Alternatively, cell lines for use in the invention may be derived from avian sources, such as chickens, ducks, geese, quails or pheasants. Avian cell lines can be derived from a variety of developmental stages including embryos, juveniles and adults. In particular, cell lines can be derived from embryonic cells, such as embryonic fibroblasts, germ cells, or from individual organs, including neuronal tissues, brain, retina, kidneys, liver, heart, cells, and cells. muscles or extraembryonic tissues and membranes protecting the embryo. Embryo embryo fibroblasts (CEF) can be used. Examples of avian cell lines include avian embryonic stem cells (WO01 / 85938) and duck retinal cells (WO05 / 042728). In particular, the EB66® cell line derived from duck embryonic stem cells is contemplated in the present invention. Other suitable avian embryonic stem cells include the EBx® cell line derived from chicken embryonic stem cells EB45, EB14 and EB14-074 (WO2006 / 108846). This EBx cell line has the advantage of being a stable cell line whose establishment was naturally produced and did not require genetic, chemical or viral modification. These avian cells are particularly suitable for the development of influenza viruses.
    According to a particular embodiment, the method of the invention, when the virus to be purified is produced on a cell culture, uses EB66® cells.
    The cell culture conditions (temperature, cell density, pH value, etc.) are variable over a very wide range due to the adaptability of the cells used and they can be adapted to the conditions required of the particular conditions for the development of a cell. virus in particular. It is the responsibility of those skilled in the art to determine appropriate culture conditions, since cell culture is widely documented in the art (see for example Tissue Culture, Academie Press, Kruse and Paterson, editors (1973), and Rl Freshney , Culture of animal cells: A manual of technical basic, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9).
    Before harvest, a cell infection can last from 2 to 10 days. The optimal pre-harvest time of the virus produced by the cell is usually based on the determination of the peak of infection. For example, the CEP (CytoPathic Effect) is measured by monitoring the morphological changes occurring in host cells after virus inoculation, including cell rounding, disorientation, swelling or shrinkage, death, detachment of the surface. Detection of a specific viral antigen can also be monitored by standard protein detection techniques, such as Western blot analysis. Harvesting can then be performed when the desired detection level is reached. In the particular case of the influenza virus, the HA content can be monitored at any time after inoculation of the cells with the virus, by SRD analysis (Wood, JM, et al (1977) J. Biol Standard 5, 237-247), which is a technique known to those skilled in the art. In addition, the SRD test can also be used to determine the optimal cell density range required to obtain optimized virus yield.
    Immunogenic Compositions At the completion of the purification, the virus preparation obtained according to the method of the present invention can be conveniently subjected to sterile filtration, as is commonly done in processes for pharmaceutical grade materials, such as immunogenic compositions. or vaccines, and known to those skilled in the art. This sterile filtration may, for example, be conveniently accomplished by filtering the preparation through a 0.22 μm filter. After preparation, the virus or viral antigens are ready for clinical use, if desired. The purified virus according to the method of the invention may be suitably formulated to be included in an immunogenic composition, such as in a vaccine. Therefore, a method for preparing a vaccine, such as an influenza vaccine, comprising at least the step of purifying a virus for use as an antigen in the vaccine according to the method of the invention and formulating said purified virus in a vaccine is also contemplated in the present invention.
    Immunogenic compositions, in particular vaccines, can generally be formulated in a sub-virion form, for example as a split-virion virus, where the lipid shell has been dissolved or disrupted, or as a one or more purified viral proteins (subunit vaccine). Alternatively, the immunogenic compositions may include a complete virus, for example a live attenuated whole virus or an inactivated whole virus virus.
    Virus fragmentation methods, such as influenza viruses, are well known in the art (WO02 / 28422). Virus fragmentation is accomplished by interrupting or fragmenting the entire virus whether it is infectious (wild type or attenuated) or non-infectious (inactivated) with an interruption concentration of a fragmentation agent. The fragmentation agents generally comprise agents capable of breaking and dissolving the lipid membranes. Traditionally, a fragmented virion influenza virus has been produced using a solvent / detergent treatment, such as tri-n-butyl phosphate, or diethyl ether in combination with Tween ™ (known as "Tween-" fragmentation). ether) and this process is still used in some production facilities. Other fragmentation agents now used include detergents or proteolytic enzymes or bile salts, for example sodium deoxycholate. Detergents which can be used as fragmentation agents include cationic detergents, for example cetyl trimethyl ammonium bromide (CTAB), other ionic detergents, for example sodium lauryl sulphate (SLS), taurodeoxycholate, or nonionic detergents, such as Tween or Triton X-100, or a combination of any two or more detergents.
    The fragmentation process can be performed as a batch, continuous or semi-continuous process. When applied batchwise, the fractionated virus may require an additional purification step, such as a chromatography step. It is not necessary to implement a fractionation step as such, since it is not possible to perform the fractionation simultaneously with a purification step. For example, a detergent may be added to the preformed linear gradient for purifying the viral proteins by ultracentrifugation, as described above. In one embodiment, the method according to the invention further comprises a fragmentation step carried out batchwise with a detergent, in particular Triton X-100.
    Alternatively, fragmentation may occur before or after the density gradient centrifugation step as claimed herein.
    For vaccine safety, it may be necessary to reduce infectivity of the virus suspension at different stages of the purification process. The infectivity of a virus is determined by its ability to replicate on a cell line. Therefore, the method according to the present invention optionally comprises at least one step of inactivation of the virus. Inactivation can be performed using, for example, beta-propiolactone (BPL), formaldehyde, or UV, or any corresponding combination that can take place either before or after the density gradient centrifugation step. of the invention. In one embodiment, the method according to the invention further comprises an inactivation step, carried out with GLP for example, said step having suitably taken place after density gradient centrifugation of the invention. Viral inactivation conditions may vary and will be determined, in particular, by evaluating infectivity by measuring the Infectious Tissue Culture Dose (TCID 50 / ml).
    Immunogenic compositions of the present invention, including vaccines, may optionally contain the usual additives for vaccines, particularly substances that increase the immune response caused in a patient receiving the composition, that is, adjuvants.
    In one embodiment, immunogenic compositions are contemplated which comprise a viral virus or antigen of the present invention mixed with a suitable pharmaceutical excipient. In a specific embodiment, they comprise an adjuvant.
    Flu virus
    Viral viruses or antigens can be derived from an Orthomyxovirus, such as the influenza virus. Orthomyxovirus antigens can be selected from one or more of the viral proteins, including hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), protein matrix (M1), membrane protein (M2), one or more of the transcriptases (PB1, PB2 and PA). Particularly suitable antigens include HA and NA, the two surface glycoproteins that determine the antigenic specificity of influenza subtypes.
    The influenza virus can be selected from the group of human influenza virus, avian influenza virus, equine influenza virus, swine flu virus, feline influenza virus. The influenza virus is more particularly selected in strains A, B and C, preferably from strains A and B.
    Influenza antigens can be derived from inter-pandemic influenza strains (annual or seasonal). Alternatively, influenza antigens can be derived from strains with the potential to cause a pandemic focus (ie influenza strains with new hemagglutinin relative to hemagglutinin in currently circulating strains, or strains of influenza that are pathogenic to avian subjects and have the potential to be transmitted horizontally in the human population, or strains of influenza that are pathogenic to humans). Depending on the particular season and the nature of the antigen included in the vaccine, influenza antigens can be derived from one or more of the following hemagglutinin subtypes: H1, H2, H3, H4, H5 , H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16. Preferably, the influenza virus or corresponding antigens come from subtypes H1, H2, H3, H5, H7 or H9. In one embodiment, the influenza viruses are derived from the subtypes H2, H5, H6, H7 or H9. In an alternative embodiment, influenza viruses are derived from H1, H3 or B subtypes.
    Immunogenic compositions of the influenza virus
    The purification method according to the invention is particularly suitable for preparing immunogenic compositions of the influenza virus, including vaccines. Different forms of influenza viruses are currently available. They are usually based on either a live virus or an inactivated virus. Inactivated vaccines can be based on whole virions, fragmented virions, or purified surface antigens (including HA). Influenza antigens can also be presented as virosomes (liposomal particles similar to viruses without nucleic acids).
    Virus inactivation methods and fragmentation methods have been described above and are applicable to the influenza virus.
    Influenza virus strains for use in vaccines change from season to season. In the current pandemic period, vaccines generally include two strains of influenza A and one strain of influenza B. Trivalent vaccines are typical but a higher valency, such as a quadrivalence, is also contemplated in the present invention. The invention may also utilize ΙΉΑ from pandemic strains (i.e. strains to which the vaccine recipient and the general human population are immunologically naive), and influenza vaccines for pandemic strains may be monovalent or may be based on a normal trivalent vaccine supplemented with a pandemic strain.
    Compositions of the invention may include antigen (s) from one or more strains of the influenza virus, including influenza A virus and / or influenza B virus. a trivalent vaccine comprising antigens from two strains of influenza A virus and a strain of influenza B virus is contemplated by the present invention. Alternatively, a quadrivalent vaccine comprising antigens from two strains of influenza A virus and two strains of influenza B virus is also within the scope of the present invention.
    The compositions of the invention are not limited to monovalent compositions, ie which comprise only one type of strain, namely only seasonal strains or only pandemic strains. The invention also includes polyvalent compositions comprising a combination of seasonal strains and / or pandemic strains. In particular, a quadrivalent composition, which may be adjuvanted, comprising three seasonal strains and one pandemic strain, is within the scope of the invention. Other compositions falling within the scope of the invention are a trivalent composition comprising two strains A and one strain B, such as strains H1N1, H3N2 and B, and a quadrivalent composition comprising two strains A and two strains B of a line different, like H1N1, H3N2, B / Victoria and B / Yamagata. HA is the major immunogen in current inactivated influenza vaccines, and vaccine doses are standardized by reference to HA levels, usually measured by SRD. Existing vaccines generally contain about 15 μg of HA per strain, although lower doses may be used, for example for children, or in pandemic situations, or when using an adjuvant. Split doses such as one half (ie 7.5 μg HA per strain) or one quarter were used, as well as higher doses, particularly 3x or 9x doses. Thus, immunogenic compositions of the present invention may include between 0.1 and 150 μg of HA per influenza strain, in particular between 0.1 and 50 μg, for example 0.1-20 μg, 0.1- 15 μg, 0.1-10 μg, 0.1-7.5 μg, 0.5-5 μg, etc. Particular dosages include about 15, about 10, about 7.5, about 5 μg per strain, about 3.8 μg per strain, and about 1.9 μg per strain.
    Once an influenza virus has been purified for a particular strain, it can be combined with viruses of other strains to make a trivalent vaccine, for example, as described above. It is more suitable to treat each strain separately and to mix monovalent sets to give a final multipurpose blend, rather than mixing viruses and degrading the DNA and purifying it of a polyvalent mixture.
    Adjuvant compositions may include an oil-in-water emulsion which comprises a metabolizable oil and an emulsifier. In order for an oil-in-water composition to be suitable for human administration, the oily phase of the emulsion system must include a metabolizable oil. The meaning of the term metabolizable oil is well known in the art. Metabolizable can be defined as "able to be metabolized" (Dorland's Illustrated Medical Dictionary, W. B. Sanders Company, 25th Edition (1974)). The oil can be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and is able to be metabolized. Peanuts, plants and seeds are common sources of vegetable oils. Synthetic oils are also part of the present invention and may include commercially available oils such as NEOBEE® and others. The oil-in-water emulsion further comprises an emulsifying agent. The emulsifier may suitably be a polyoxyethylene sorbitan monooleate. In addition, said emulsifier is suitably present in the vaccine or immunogenic composition at 0.125 to 4% (w / v) of the total volume of the composition. The oil-in-water emulsion optionally comprises a tocol. Tocols are well known in the art and are described in EP0382271. A suitable tocol is alpha-tocopherol or a corresponding derivative such as alpha-tocopherol succinate (also known as vitamin E succinate). The tocol is suitably present in the adjuvant composition in an amount of 0.25% to 10% (w / v) of the total volume of the immunogenic composition. The invention will be described below with reference to the following nonlimiting examples.
    Example I: Gradient Formation for a 3.2L Self-Generating Gradient Rotor The continuous flow ultracentrifuge was a KM centrifuge using a K3 rotor (3.2L capacity) from Alfa Wasserman. The K3 / 3.2L rotor was filled with a 125mM PBS / Citrate buffer solution. Once filled, the centrifuge was started while keeping the circulating buffer solution in closed circulation to remove all air from the rotor. The centrifuge was then stopped and half the volume (1.6 L) of the 125 mM PBS / Citrate buffer solution was replaced with a 125mM PBS / Citrate solution containing 55% sucrose. After replacement, the rotor was first slowly accelerated to 4000 rpm and then to a final speed of 35000 rpm. As the rotor was progressively accelerated to 4000 rpm, a linear gradient from 0% to 55% sucrose was formed in the rotor. After the formation, successive fractions of 100 ml of the gradient were collected and analyzed for their sucrose content by refractometry (% Brix) in order to check the linearity of the gradient. The results are shown in Figure 1. Results - Conclusions
    The gradient obtained was substantially linear, ie linear over most of its volume.
    Example II: Gradient Formation for an 8L Rotor - Self-Generating Gradient
    The same process as that described in Example I was applied for the formation of a gradient in an 8L rotor. The continuous flow ultracentrifuge was a K11 centrifuge using a K10 rotor (8L capacity) from Alfa Wasserman. The K10 / 8L rotor was filled with a 125mM PBS / Citrate buffer solution. Once filled, the centrifuge was started while keeping the circulating buffer solution in closed circulation to remove all air from the rotor. The centrifuge was then stopped and half the volume (4L) of the 125mM PBS / Citrate buffer solution was replaced with a 125mM PBS / Citrate solution containing 55% sucrose. After replacement, the rotor was slowly accelerated to 4000 rpm and then subsequently to a final speed of 35000 rpm. After the formation, successive fractions of 250 ml of the gradient were collected and analyzed for their sucrose content by refractometry (% Brix) in order to check the linearity of the gradient. The results are shown in Figure 2. Results - Conclusions
    No linear gradient was obtained. The gradient obtained was a graded gradient - a first plateau around 55% sucrose was observed on the first 3.5 liters of the gradient and a second plateau was observed at around 5% sucrose on the last 3 liters of the gradient. The linear part of the gradient extended only from 3.5L to 5L, ie the gradient was linear over 1.5L, i.e., only about 20% of its total volume.
    Example III: Gradient Formation for an 8L Rotor - Preformed Gradient 111.1 The same centrifuge and rotor as in Example II were used. The first (container 1) contained 4L of 125mM PBS / Citrate buffer comprising 55% sucrose. The second (container 2) and the third (container 3) each contained 1L of PBS / 125mM citrate buffer. The container 1 was connected to the container 2 which was connected to the container 3. The liquids in the containers 2 and 3 were mixed by a magnetic stirrer at a speed of 90 rpm. The vessel 3 was connected through a pump to the bottom of the K10 / 8L rotor which was filled with 125mM PBS / Citrate buffer. When the pump was turned on at a flow rate of 160 ml / min, a vacuum was created in the containers 2 and 3 and the sucrose from the container 1 was first diluted in the container 2, whose contents was subsequently diluted in the container 3, the contents of which were injected into the rotor (FIG. 3A). Once empty 1, the gradient was formed in the rotor and the centrifuge was started. Centrifugation was performed at 35000 rpm. After the formation, successive fractions of 250 ml of the gradient were collected and analyzed for their sucrose content by refractometry (% Brix) in order to check the linearity of the gradient. The results are shown in Figure 3B. Results - Conclusions
    Using the above method which relies on the successive dilution of a single mother solution of 55% sucrose prior to injection into an 8L rotor, a gradient was obtained which is substantially linear, i.e. linear on most of its volume. In particular, no significant plateau at each end of the gradient was observed (at around 55% sucrose and 5% sucrose), as opposed to the two plateaux observed in Figure 2 at these percentages of sucrose. The linear portion of the gradient extended from about 1L to about 7L, ie the gradient was linear over 6L, ie about 75% of its total volume. The inventors also checked the linearity of the gradient before centrifugation and observed a curve similar to that shown in FIG. 3B. III.2 The same centrifuge and the rotor as in Example II were used. The first (container 1) contained 4L of 125mM PBS / Citrate buffer comprising 55% sucrose. The second (container 2) contained 1 L of 125mM PBS / Citrate buffer. The container 1 was connected to the container 2 which was connected through a pump to the bottom of the K10 / 8L rotor which was filled with 125mM PBS / Citrate buffer. The liquid in the vessel 2 was mixed by a magnetic stirrer at a speed of 90 rpm. When the pump was turned on at a rate of 160 ml / min, a vacuum was created in the container 2 and the sucrose from the container 1 was diluted in the container 2, the contents of which were injected into the rotor (FIG. 4A). Once empty 1, the gradient was formed in the rotor and the centrifuge was started. Centrifugation was performed at 35000 rpm. After the formation, successive fractions of 250 ml of the gradient were collected and analyzed for their sucrose content by refractometry (% Brix) in order to check the linearity of the gradient. The results are shown in Figure 4B. Results - Conclusions
    Using the above method which relies on the successive dilution of a single mother solution of 55% sucrose before injection into an 8L rotor, a gradient was obtained which is linear over the first 5 liters. of its volume, namely about 62% of its total volume. A plateau with 5% sucrose was observed on the last 3 liters of the gradient, ie representing about 40% of its total volume. The inventors also checked the linearity of the gradient before centrifugation and observed a curve similar to that shown in FIG. 4B.
    Example IV: Yield and purity of the virus
    A sucrose gradient of 0-55% was formed either in a K3 / 3,2L rotor as described in Example I or in a K10 / 8L rotor as described in Example III.1. Once the centrifuge reached a speed of 35000 rpm, a fluid containing a whole cell-derived influenza virus was loaded onto each rotor for purification of the virus. 17L of said fluid per rotor L was loaded onto the rotor of 3.2L and 35L of the same fluid per L rotor were loaded onto the 8L rotor with a strapping time of 1 hour. The purified whole virus was pooled from gradient fractions corresponding to a sucrose percentage ranging from 23% to 50% (3.2L rotor) or 11% to 42% (8L rotor). These ranges were determined on the basis of profiles from SDS-PAGE and from Western Blot analysis using anti-HA antibodies. The HA protein content (hemagglutinin) was evaluated by an SRD test (as described in Example V) before and after centrifugation, so that the percentage of "HA recovery" after the step gradient centrifugation step. sucrose can be calculated. The percentage of "purity" represents the percentage of HA protein on the total protein obtained at the end of the centrifugation. The percentage of "protein suppression" represents the removal of the total proteins following the sucrose gradient centrifugation step. The total protein concentration was evaluated by the Lowry method before and after centrifugation, so that the percentage of "purity" and the percentage of "protein suppression" after the sucrose gradient centrifugation step can be calculated. The results are shown in Table 1.
    Table 1 - Yield and purity of the virus after sucrose gradient ultracentrifugation
    Results - Conclusions
    The method for creating a preformed linear sucrose gradient in a high capacity 8L rotor provides an influenza virus (after purification by centrifugation) with a similar yield and a level of purity similar to that obtained during the use of a smaller rotor of 3.2L. Such a result indicates that with such a process, virus purification by density gradient centrifugation can be increased by reducing the number of equipment / cycles required, without impacting the quality or quantity of the product (yield and purity of the product). virus).
    Example V: SRD method used to measure the HA content
    Glass plates (12.4 - 10 cm) were coated with an agarose gel containing a NIBSC-recommended concentration of anti-flu HA serum. Once the gel cured, 72 sample wells (3 mm in diameter) were punched into the agarose. 10 μl of appropriate dilutions of the reference and sample were loaded into the wells. The plates were incubated for 24 hours at room temperature (20-25 ° C) in a humid chamber. After which, the plates were immersed overnight in NaCl solution and washed briefly in distilled water. The gel was then compressed and dried. When completely dry, the plates were stained on a solution of Coomassie Brilliant Blue for 10 minutes and decolorized twice in a mixture of methanol and acetic acid until clearly defined discolored areas become visible. After drying the plates, the diameter of the stained areas surrounding the antigen wells were measured in two perpendicular directions. Alternatively, equipment for measuring the surface may be used. Dilution-response curves of antigen dilutions against the surface were performed and the results were calculated according to standard slope ratio test methods (Finney, DJ (1952).) Statistical Methods in Biological Assay. Cited in Wood, JM, et al (1977) J. Biol Standard, 5, 237-247).
    权利要求:
    Claims (38)
    [1]
    A method of purifying a virus comprising at least the following steps: a) obtaining a fluid containing a virus; b) providing a preformed linear density gradient of a given range X% -Y% (weight / volume) (where X is the lower limit and Y is the upper limit) in a centrifugal rotor having a capacity of at least 4L, at least 6L, at least 8L, or at least 10L, c) the addition of fluid to the preformed linear density gradient, d) centrifugation to separate the virus from contaminating impurities, and e) collection fractions comprising the purified virus, wherein the linear portion of the gradient comprises the percentage by density corresponding to the percentage at which the virus to be purified will migrate.
    [2]
    2. The method of claim 1, wherein the separation is isopycnic.
    [3]
    The method of claim 1 or claim 2, wherein the preformed linear density gradient of step b) is linear over at least 30%, at least 50%, at least 60%, at least 70%, at least less than 80%, at least 90% of its total volume or is linear over its entire volume.
    [4]
    The method of claim 1 or claim 2, wherein the preformed linear density gradient of step b) comprises trays at its ends whose added volume does not exceed 70%, 50%, 40%, 30%. , 20%, 10%, or 5% of its total volume.
    [5]
    The method of claim 1 to 4, wherein the preformed linear density gradient of step b) is formed by diluting a stock solution forming a gradient prior to injection into the rotor.
    [6]
    The method of claim 5, wherein the gradient forming stock solution is Y% (w / v) solution.
    [7]
    The method of claims 5 and 6, wherein the gradient forming stock solution is diluted at least once in a dilution solution prior to injection into the rotor.
    [8]
    The method of claims 5 and 6, wherein the gradient forming stock solution is diluted in a first dilution solution, said diluted stock solution being diluted in a second dilution solution prior to injection into the rotor.
    [9]
    The method of claims 7 and 8, wherein the dilution solution (s) does not contain any gradient medium.
    [10]
    The process according to claims 7 to 9, wherein the dilution solution a, or the dilution solutions have a volume of at least 10%, at least 15%, at least 20%, at least 25%, at least minus 30%, or at least 50% of the mother solution forming the gradient.
    [11]
    The method according to claims 7 to 9, wherein the ratio between the volume of the stock solution forming the gradient and the volume of the dilution solution (s) is at least 2: 1, at least 3 : 1, at least 4: 1, at least 5: 1 or at least 6: 1.
    [12]
    12. The method of claims 5 to 11, wherein the stock solution forming a gradient and the dilution solution (s) are connected to each other and connected to the rotor.
    [13]
    13. The method of claims 5 to 12, wherein the dilution is a progressive dilution based on a drip system.
    [14]
    14. The method of claim 13, wherein the drip system is obtained by creating a vacuum in the container (s) containing the dilution solution (s).
    [15]
    The method of claims 1 to 14, wherein the preformed linear density gradient of step b) is a sucrose gradient.
    [16]
    The method of claims 1 to 15, wherein the rotor has a capacity of at least 8L.
    [17]
    17. The method of claims 1 to 16, wherein the preformed linear gradient of step b) is between 0 and 55%.
    [18]
    18. The method of claims 1-17, wherein the virus is propagated on eggs.
    [19]
    The method of claim 18, wherein the fluid containing the virus of step a) is the allantoic fluid collected after egg inoculation with the virus.
    [20]
    The method of claims 1 to 17, wherein the virus is propagated on a cell culture.
    [21]
    The method of claim 20, wherein the fluid containing the virus of step a) is the cell culture medium collected after infection of cells with the virus and release of the virus into the cell culture medium.
    [22]
    The method of claims 1 to 18 or claim 20, wherein the fluid containing the virus of step a) is partially purified before being added to the preformed linear density gradient.
    [23]
    23. The method of claim 22, wherein the fluid containing the virus has been clarified.
    [24]
    24. The method of claims 1 to 23, wherein the centrifugation is performed in batch mode.
    [25]
    25. The method of claims 1 to 23, wherein the centrifugation is carried out in continuous mode.
    [26]
    26. The method of claim 25, wherein the centrifuge is started after step b) and before step c).
    [27]
    27. The method of claims 1 to 26, further comprising a step of inactivating the virus.
    [28]
    28. The method of claim 27, wherein the inactivation is performed with beta-propiolactone, UV or both.
    [29]
    The method of claim 27, wherein the inactivation is performed with formaldehyde.
    [30]
    30. The method of claims 1 to 29, further comprising a virus fragmentation step.
    [31]
    31. The method of claims 1 to 30, wherein the virus is an influenza virus.
    [32]
    32. The method of claim 31, wherein the influenza virus is of subtype H2, H5, H6, H7 or H9.
    [33]
    33. The method of claim 31, wherein the influenza virus is of subtype H1, H3 or B.
    [34]
    The method of claims 20 to 33, wherein the virus is propagated on mammalian or avian cells.
    [35]
    The method of claim 34, wherein the virus is propagated on duck embryonic stem cells such as EB66® cells.
    [36]
    36. A process for preparing a vaccine, wherein the virus to be used as an antigen in the purified vaccine according to the process of claims 1 to 35 and the purified virus is formulated in a vaccine.
    [37]
    37. A method for the preparation of a vaccine comprising at least the following steps: A) obtaining a fluid containing a virus, B) providing a preformed linear density gradient of a given range X% - Y% (mass / volume) (X being the lower limit and Y being the upper limit) in a centrifugal rotor having a capacity of at least 4L, at least 6L, at least 8L, or at least 10L, wherein The linear part of the gradient includes the percentage of density corresponding to the percentage at which the virus will migrate. C) the addition of the fluid to the preformed linear density gradient, D) the centrifugation so as to separate the virus from the contaminating impurities, and E) the collection of the fractions comprising the purified virus, F) the formulation of the purified virus in a vaccine .
    [38]
    38. The method of claim 37, comprising one or more features according to any one of claims 2 to 35.
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    EP3234113A1|2017-10-25|
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    法律状态:
    2017-12-13| FG| Patent granted|Effective date: 20170908 |
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    US62092413|2014-12-16|
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