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    PROCESSES FOR THE PRODUCTION OF POLIOVIRUS, AND TO PRODUCE A VACCINE AGAINST POLIO. The invention provides a process for the production of poliovirus, which comprises the steps of: a) providing a culture of serum-free suspension cells, which are PER.C6 cells, b) infecting said cells with poliovirus, at a cell density between 2 x 106 cells / ml and 150 x 106 cells / ml and c) cultivate poliovirus in a period between 12 and 48 hours after infection.
    公开号:BR112012000942B1
    申请号:R112012000942-7
    申请日:2010-07-08
    公开日:2020-10-27
    发明作者:John Alfred Lewis
    申请人:Janssen Vaccines & Prevention B.V；
    IPC主号:
    专利说明:

    [0001] The invention relates to the field of cell culture and poliovirus production. More particularly, this refers to improved methods for culturing cells and producing poliovirus from them for producing polio vaccines. Fundamentals of the invention
    [0002] Polioviruses are members of the Enterovirus genus of the Picornaviridae family. Polioviruses are small, non-enveloped viruses with capsids including a single-strand positive sense RNA genome. There are three types of poliovirus: types 1, 2 and 3. Infections from individuals susceptible to poliovirus can result in paralytic polio. Polio is highly contagious. Two different polio vaccines have been developed, Salk's inactivated poliovirus vaccine (IPV) and Sabin's live attenuated oral poliovirus vaccine (OPV). Both vaccines are safe and effective. Each has its particular advantages and disadvantages and both played an important role in controlling polio. For a review of poliovirus and poliovacins see, for example, Kew et al, 2005.
    [0003] The oral polio vaccine (OPV) is cheap and convenient and has been used massively. However, occasional recipients suffer from vaccine-associated paralytic polio (VAPP) due to reversals in the vaccine. In addition, this has been observed in populations that have not been fully immunized that the attenuated Sabin polio strains have undergone sufficient mutational changes to cause outbreaks of paralytic disease that are clinically and epidemiologically indistinguishable from naturally occurring wild-type polio disease; these mutants are called circulating vaccine-derived polioviruses or cVDPVs (see, for example, Kew et al, 2005; Wright and Modlin, 2008; Yakovenko et al, 2009).
    [0004] There is a growing consensus that inactivated poliovirus (IPV) vaccine can contribute to faster eradication of wild-type polio and control of emerging cVDPV when used in conjunction with existing OPV strategies (Wright and Modlin, 2008; John, 2009).
    [0005] However, the production of BTI is more expensive (see, for example, John, 2009) and can still be prohibitively expensive for less developed countries, when a strong need for polio vaccines still exists. Culture systems for the production of bulky poliovirus material that can be used in a vaccine, in particular, non-attenuated poliovirus, contribute to a large extent at relatively high cost.
    [0006] Still, there remains a need in the art for efficient culture systems for the production of poliovirus for use in vaccines.
    [0007] Poliovirus propagation in HEK293 cells has been described as a system for studying poliovirus neuron-specific replication phenotypes and it has been described that attenuated forms of poliovirus, such as poliovirus containing spot mutations in an IRES element when present in Sabin strains, they showed reduced propagation in HEK293 cells (Campbell et al, 2005).
    [0008] Embryonic retinal cells immortalized by E1 (HER), in particular PER.C6 cells, have been described when suitable for the spread of various viruses, with an emphasis on the influenza virus (Pau et al, 2001; WO 01/38362 ). Although WO 01/38362 describes examples of work for the spread of various strains of influenza virus and Herpes Simplex Virus (HSV) types 1 and 2, measles virus and rotavirus in PER.C6 cells, the spread of poliovirus has not been exemplified in WO 01/38362. In addition, the conditions for poliovirus replication in such cells have not been described and cannot be easily predicted based on the replication of unrelated viruses in these cells. As a result, it has so far been unknown whether it should be feasible to produce poliovirus economically on an industrial scale for the production of vaccine purposes in these cells.
    [0009] For the large-scale manufacture of inactivated polio vaccines, poliovirus is generally propagated in Vero cells, which are derived from monkeys. Vero cells are widely used for vaccine production, including inactivated polio vaccines, as well as live attenuated and, thus, these are the continuous cell lines most widely accepted by regulatory authorities for the manufacture of viral vaccines and use of these cells for vaccine production is expected to increase by experts in the field (Barrett et al, 2009).
    [00010] Large-scale microcarrier culture of Vero cells for the inactivated poliovirus vaccine was described by Montagnon et al, 1982 and 1984. A process for the large-scale production of a polio vaccine using Vero cells and the resulting vaccine is also described in US Patent 4,525,349.
    [00011] The production of high content of poliovirus titers (Sabin type 1) (almost 2 x 109TCIDso / ml) have been described by (Merten et al, 1997) for conditions when Vero cells in microcarriers are cultured in the medium containing serum before of the virus production phase in the serum-free medium, but in view of the disadvantages of using the serum of these authors already indicate that a completely serum-free process is desired and in such an optimized serum-free process, these authors were able to obtain a titration of 6.3 x 108 TCIDδo / ml.
    [00012] Kreeftenberg et al (2006), involved in the production of poliovirus for the production of vaccine on an industrial scale, also mention the yields of several wild-type and Sabin strains of poliovirus in Vero cells developed in micro-carriers, whose yields are similar for the different strains, the log titers being between 8.1 and 8.6. These authors also describe that the amount of virus required for the production of the final vaccine is significantly higher for IPV than for OPV, which results in a significantly higher production cost per dose for IPV than for OPV.
    [00013] Poliovirus serum-free production using Vero cells grown in microcarriers has also been described by (Card et al, 2005) and although the level of productivity has been lower than in static cultures, microcarrier cultures have been described as easier in increasing.
    [00014] Despite the efficacy and industrial applicability of these microcarrier based Vero cell cultures, the production of large quantities of poliovirus remains expensive and therefore remains a necessity for alternative production systems for poliovirus that suffer less from this disadvantage.
    [00015] Poliovirus production using suspended Vero cells has been described, resulting in lower virus titers (10log CCIDso / ml between 6.5 and 7.9) than those observed and routine microcarrier Vero cells (van Eikenhorst et al, 2009).
    [00016] It is an objective of the invention to provide suitable processes that can be used for large-scale and economical production of poliovirus for use in vaccines. This can assist in providing access to productive polyvacin in developing countries on a sustainable basis. Summary of the invention
    [00017] The invention is based on the demonstration of very efficient poliovirus propagation in PER.C6 cells, in which high titers not preceded by poliovirus are obtained according to the methods described here. Obtaining such high titrations, which provide a significant economic advantage over poliovirus production in Vero cells, cannot be predicted based on the replication of other viruses in such cells, nor can the conditions for an industrially feasible process be predicted, given that the conditions and the advantages obtainable can vary widely for several different types of viruses that have vastly different properties.
    [00018] In this way, the invention provides a process for the production of poliovirus, which comprises the steps of: a) providing a culture of serum-free suspension cells, which are PER.C6 cells as deposited under ECACC no. 96022940, b) infect said cells with poliovirus, at a cell density between 2 x 106 cells / ml and 150 x 106 cells / ml and c) cultivate poliovirus in a period between 12 and 48 hours after infection.
    [00019] In certain embodiments, said infection is carried out at a cell density between about 5 x 106 cells / ml and 20 x 106 cells / ml, for example, between about 8 x 106 cells / ml and 15 x 106 cells / ml, for example, around 10 x 106 cells / ml.
    [00020] In certain embodiments, said poliovirus cultivation is carried out in a period between about 18 and 30 hours after infection, for example, around 24 hours after infection.
    [00021] The conditions allow to obtain very high titrations (around 1010 / ml, which is significantly greater than 10 times the titrations typically obtained using microcarrier-based Vero cells for wild type polio strains) of poliovirus in relatively short processing, which therefore have significant economic advantages in the processes currently used to produce poliovirus for vaccine preparation. This has been demonstrated for all three types of poliovirus: type 1 (Brunenders strain), type 2 (MEF strain) and type 3 (Saukett strain).
    [00022] In certain embodiments, therefore, said virulent wild-type poliovirus, for example, poliovirus type 1, poliovirus type 2 or poliovirus type 3. In certain embodiments, said poliovirus is poliovirus type 1 strain Mahoney or Brunenders , poliovirus type 2 strain MEF (or MEF-1), or poliovirus type 3 strain Saukett. In other embodiments, said poliovirus is an attenuated poliovirus (being less neurovirulent), for example, a Sabin strain (which can also be of type 1, 2 or 3).
    [00023] The invention further provides a process for the production of a polio vaccine, which comprises a process for the production of poliovirus according to the invention, further comprises the purification, optionally inactivation and formulation of the cultured poliovirus to obtain a vaccine against polio. For a BTI, inactivation by formalin or other means is performed. For an OPV, the inactivation step is not required.
    [00024] It is also described here to provide a volume of poliovirus useful for the preparation of a polio vaccine, said load of poliovirus being obtainable by a process for the production of poliovirus according to the invention and comprises culture medium and a titration poliovirus of at least 1094 CCIDso / ml, for example, between about 1095 and 1011 CCIDso / ml, for example, between about 1098 and 10108 CCIDso / ml. In certain embodiments, said charge has a volume between 1 and 1000 liters. In additional embodiments, said charge contains cells and / or cell debris, from cells used according to the processes of the invention. In certain embodiments, said charge is present in a bioreactor. In other embodiments, the charge has been removed from the bioreactor and is present in a suitable container.
    [00025] Also, the poliovirus and the polio vaccine obtainable according to the methods of the invention. Said poliovirus and / or said vaccine is free of monkey proteins, preferably free of non-human proteins. It will also be free of other non-human host cell residues. In contrast, poliovirus that was produced according to conventional methods will contain residual non-human protein and / or other non-human residues, from the host cells used and / or from the serum used during cell culture. In this way, the poliovirus produced in accordance with the present invention suffers from less contamination from non-human impurities resulting from the production process than the poliovirus produced using conventional processes.
    [00026] The invention also provides a process for obtaining a poliovirus preparation in cell culture at a titration of at least about 1094, preferably at least 1098, more preferably at least 1010, for example, between 10105 and 1011 CCID / ml, which comprises the steps of: a) providing a culture of serum-free suspension cells, which are PER.C6 cells, b) infecting said cells with poliovirus, at a cell density between 2 x 106 cells / ml and 150 x 106 cells / ml and c) cultivate poliovirus in a period between 12 and 48 hours after infection to obtain the preparation of poliovirus having said concentration. Preferred embodiments are the same as described above for the process for producing poliovirus according to the invention. Brief Description of the Figures
    [00027] FIG. 1. Production of poliovirus in adherent PER.C6 and Vero cells.
    [00028] FIG. 2. Production of poliovirus type 1 in PER.C6 cells suspended in different serum-free medium, in different MOIs and in different cell densities in infection.
    [00029] FIG. 3. Effect of temperature and cultivation time on the production of poliovirus 1, 2 and 3 in PER.C6 cells suspended in serum-free medium.
    [00030] FIG. 4. Effect of cell density on infection, temperature and cultivation time in the production of poliovirus type I in PER.C6 cells suspended in serum-free medium.
    [00031] FIG. 5. Efficient production of poliovirus types 1, 2 and 3 in PER.C6 cells in serum-free suspension at high cell densities. Detailed Description of the Invention
    [00032] The cells used in the process are PER.C6 cells, which are immortalized cells, are also known in the art as continuous cell lines and as such have the potential for an infinite total life (see, for example, Barrett et al, 2009). PER.C6 cells for the purpose of this application should mean cells as deposited under ECACC No. 96022940 on February 29, 1996. it will be clear to the qualified person that this definition will include an upstream or downstream passage or a descendant of an upstream or downstream passage of these deposited cells. PER.C6 cells are described in US Patent 5,994,128 and in (Pollux et al, 1998). These cells are very suitable for the production of influenza viruses for the production of cell-based influenza vaccines, since they can be infected and spread the virus with high efficiency, as, for example, described in (Pau et al, 2001) and WO 01/38362. PER.C6 cells are able to develop in suspension in the absence of serum, as, for example, described in (Yallop et al, 2005). Here, it is shown that these cells are also very suitable for the production of poliovirus at high levels in serum free suspension cultures.
    [00033] In addition, the conditions used are advantageous economic and regulatory conditions.
    [00034] The use of microcarriers is not required for the present invention, in contrast to the processes used widely with Vero cells. Microcarriers contribute to the high cost of poliovirus produced using conventional Vero cell-based processes.
    [00035] Serum free according to the present invention means that the medium used for cell development and infection needs total serum as an ingredient. It may not be entirely free of serum derived products, such as, for example, bovine serum albumin (BSA), however, in preferred embodiments, such components are also present or have been recombinantly produced in the absence of any components derived from animal. In preferred embodiments, the complete method is carried out in the absence of any components that were directly derived from animals, such as serum or serum components, etc. In a preferred embodiment, the method of producing a vaccine is carried out under conditions free of animal component. This means that the medium used for cell development and infection is devoid of any animal-derived components. In addition, any additives supplemented with the medium during the vaccine production process are also free of animal-derived components. The absence of animal components in the process of making said polio vaccine offers a process that is more controlled and safe. For this reason, PER.C6 cells, which are fully characterized human cells and which have been developed in accordance with GLP / GMP, are very well adapted for use in the manufacture of vaccines. Different culture media can be used and the choice of culture medium optimal for the cells and circumstances used and part of the routine tasks of the person skilled in the art in this field. Culture media suitable for the purpose of the present invention are thus well known to the person skilled in the art and can, in general, be well known to the person skilled in the art and can, in general, be obtained from commercial sources in large quantities or made to order according to standard protocols. The culture can be carried out, for example, in plates, rotary bottles or in bioreactors using batch, fed batch or continuous systems and others. In order to achieve large-scale (continuous) virus production through cell culture, it is preferred in the art to have cells capable of growing in suspension and it is preferred to have cells capable of culturing in the absence of animal or human derived serum or components of serum derived from animal or human. The appropriate conditions for culturing the cells (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, editors (1973) and Rl Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Liss Inc ., 2000, ISBN 0-471- 34889-9). The serum-free culture medium can be used according to the processes of the invention include but are not limited to the standard medium that can be ordered from the catalogs of medium, including CDM4PERMAb ™ (Thermo Scientific HyClone, cat. No. SH30871, SH30872). In addition, the usual ordered medium such as Permexcis (Lonza) are suitable. Examples of other serum-free media that may be suitable for use in the processes of the invention are AEM (Invitrogen, cat. no. 12582-011), EX-CELL ™ VPRO medium (JRH Biosciences, catalog number 14561) and CDM4Retino ™ (HyClone, cat. No. SH30520, SH30519) .
    [00036] In certain optional and non-limiting embodiments, it is possible to supplement the serum-free medium in the processes of the present invention with lipids and / or hydrolysates and / or other supplements, to further improve productivity.
    [00037] The term ‘about’ or ‘around’ for numerical values as used in this description means ± 10%.
    [00038] Infecting cells with poliovirus and / or spreading viruses in the processes according to the present invention is, for example, suitably carried out at a temperature between about 33 ° C and 38 ° C. In preferred embodiments, said virus infection and / or spread is carried out at a temperature between about 34 ° C and 36 ° C, in certain embodiments between about 34.5 ° C and 35.5 ° C , for example, around 35 ° C.
    [00039] Infecting the cells with poliovirus in the processes according to the present invention can, for example, be adequately carried out in a multiplicity of infection (MOI) between 0.001 and 10. In certain embodiments, said infection is carried out in a MOI between about 1 and 3, for example, at a MOI of about 2. infecting at a relatively high MOI (> 0.1, preferably about 1 or higher) can still increase the process of high efficiency yield high.
    [00040] In accordance with the present invention, cells are infected with poliovirus, preferably at a high cell density. In certain respects, poliovirus infection occurs when the cells have a density between 1 x 106 cells / ml and 150 x 106 cells / ml, preferably between 2 x 106 cells / ml and 150 x 106 cells / ml. In certain preferred embodiments, said infection is carried out at a cell density between about 5 x 106 cells / ml and 20 x 106 cells / ml, for example, between about 8 x 106 cells / ml and 15 x 106 cells / ml, for example, around 10 x 10 6 cells / ml. As far as we are aware, the processes of the invention provide the highest cell concentrations at which non-adenoviral virus vaccines are manufactured. The advantages of these processes according to the invention are that very high titers of poliovirus can be obtained, that is, at least an order of magnitude higher than with conventional Vero processes of the prior art.
    [00041] In the processes according to the invention, poliovirus is cultured between 12 and 48 hours after infection. In certain embodiments, said poliovirus cultivation takes place between about 18 and 30 hours after infection, for example, between about 20 and 28 hours, between about 22 and 26 hours after infection, for example example, around 24 hours after infection. In this way, the processes according to the invention can be used to obtain extremely fast high titers of poliovirus, which also help in making the processes of the invention extremely economically attractive compared to the much longer processes that are conventional in the art.
    [00042] Most large-scale suspension cultures are operated as batch or fed batch processes because these are the most direct to operate and classify and such processes are, in principle, suitable for the processes of the present invention. However, continuous processes based on perfusion principles are becoming more common. In certain embodiments of the present invention, the producer cells are cultured in an infusion system.
    [00043] The batch production, fed batch and perfusion processes were used with PER.C6 cells, for example, for the production of recombinant antibody. In batch cultures, the concentration of viable cells greater than 12 x 10 6 cells / ml is routinely achieved. The viable cell concentration of up to 40 x 106 cells / ml was demonstrated multiple times using powered batch. In perfusion processes, peak cell concentrations at 150 x 106 cells / ml are routinely achieved (Kral et al, 2009).
    [00044] Cell perfusion culture has its conventional significance in the art, that is, meaning that during culture, cells are retained by a separation device in which there is an effusion of liquid having a lower cell density than before separation and in which there is an influx of cell culture medium. The use of perfused cultures is in response to the challenge of developing cells at high densities (for example, 10 to 50 x 106 viable cells / ml). In order to increase densities, the medium is constantly or intermittently replaced by a fresh supply in order to make up for nutritional deficiencies and to remove toxic products. Perfusion also allows for better control of the culture environment (pH, dÜ2, nutrient levels, etc.). Perfusion of fresh medium through culture can be achieved by retaining the cells with a variety of separation devices (for example, rotary fine-mesh filter, hollow fiber membrane or flat plate filters, sedimentation tubes). In certain embodiments, the separating device is a filter module comprising hollow fibers, that is, tubular membranes. The internal diameter of the tube is usually between 0.3 and 6.0 mm, for example, between 0.5 and 2.0 mm. In certain embodiments, the size of the web (pore size) in the membrane is chosen such that the size of the pores in the web is close to the diameter of the cells, ensuring high cell retention while cell debris can pass through the filter. In other embodiments, the size of the web is significantly smaller than the diameter of the cells. Preferably, the frame size is between 0.1 and 30 pm, for example, between 0.1 and 3 pm, for example, about 0.2 pm. Filter modules comprising hollow fibers are commercially available from, for example, General Electric (formerly Amersham).
    [00045] Perfusion is used in order to maintain the desired levels of certain metabolites and to remove and thereby reduce impurities in the culture medium. This is typically the case that the infusion is not performed at all times during culture and is, in general, performed only from the period to the period during culture when desired. For example, the infusion is not typically started until after certain components of the medium, such as glucose have become worn out and need to be replaced.
    [00046] Various perfusion systems are known in the art and are, in principle, suitable for use in the processes of the present invention. The term "perfusion system" means the combination of a bioreactor connected to a separation device. The separation device can be incorporated in the bioreactor (for example, rotary fine-mesh filter) or remains outside the bioreactor (for example, hollow fiber). In both cases, as explained above, the separation device prevents the washing of the cell mass from the reactor and allows the medium to rest. In certain embodiments, bioreactors are operated with (connected to) an alternating tangential flow (ATF) perfusion system (e.g., ATF System, Refine Technology, Co., East Hanover, NJ). The tangential flow can be achieved according to the methods known to the person skilled in the art as described, for example, in US 6,544,424. The operation of the ATF infusion system has been described and is classifiable (Furey, 2002). ATF systems allow cells to be cultured over a long period of time and to reach high cell densities without having a blocked filter. In fact, extremely high cell densities of more than 100 x 106 viable cells / ml can be achieved using an ATF perfusion system, for example, with PER.C6 cells (see, for example, Yallop et al, 2005 and WO 2005/095578). However, in those previous reports, PER.C6 cells in the perfusion systems were used for recombinant antibody production, that is, a completely different purpose and not infected with poliovirus.
    [00047] In certain embodiments, perfusion with, for example, an ATF system is advantageous during the pre-culture phase (ie, before poliovirus infection), as this allows to obtain very high cell densities and the cells are in good condition for subsequent poliovirus infection. In order to achieve said high cell densities, the culture medium is, in certain embodiments, at least partially perfused over a period of time during cell development. In certain embodiments, the infusion is initiated once the cell density between about 2 x 10 6 viable cells / ml and 8 x 10 6 viable cells / ml is reached.
    [00048] In the processes of the invention, cells are infected with poliovirus.
    [00049] Typically, the virus will be exposed to the appropriate producer cell under optimal conditions, allowing for virus absorption. The person skilled in the art knows how to observe the optimal additional conditions, that is, for agitation, pH, dissolved oxygen (dÜ2 or DO). Usually, the optimum agitation is between about 50 and 300 rpm, typically about 100 to 200, for example, about 150, typical OD is 5 to 60%, the optimum pH is between 6.7 and 7.7. Typically, the poliovirus infects the cells of the invention spontaneously and leads the cells in contact with the poliovirus particles sufficient for infection of the cells. Generally, a seed stock of the poliovirus is added to the culture to initiate the infection and subsequently the poliovirus spreads in the cells.
    [00050] It was advantageously possible to infect a cell culture according to the invention with poliovirus at high cell densities, i.e. around 10 x 106 cells / ml and very high titers (greater than 1010 CCIDso / ml) of the poliovirus were obtained.
    [00051] In certain embodiments, the viability of the cell culture before infection remains greater then 75%, meaning that at least 75% of the total amount of cells in the culture is viable at the time of infection. In certain embodiments, the viability of cell culture in infection is at least 80%, yet in embodiments at least 85%. Viability can be measured using the methods available to that qualified person, for example, trypan blue exclusion, Casy cell count and others.
    [00052] In a certain embodiment, the cell density in the infection is between about 10 x 106 and 50 x 106 viable cells / ml, for example, between about 10 x 106 and 20 x 106 viable cells / ml, for example for example, between about 10 x 10 6 and 15 x 10 6 viable cells / ml. these cell densities allow for high virus productivity with limited accumulation of cell debris and host cell DNA, which gives an advantage of these embodiments in processing downstream of poliovirus cultivation. In this way, the present invention provides an optimized process for poliovirus production, producing high poliovirus titrations, while in the same period it provides a cultivation material that is still controllable for the purposes of downstream processing.
    [00053] In other embodiments, the cell density in the infection is between about 15 x 106 and 150 x 106 cells / ml, for example, between about 15 x 106 and 80 x 106 cells / ml, for example, between about 20 x 106 and 50 x 106 cells / mL Infections of these cell densities can produce even higher concentrations of the virus.
    [00054] In certain embodiments of the invention, a method is provided for volume production of the poliovirus at a titration of at least 1010CCIDso / ml.
    [00055] The titration is expressed as CCIDso, which is 50% of the infectious cell culture dosage. This is sometimes also referred to as TCIDso (50% of the tissue culture infectious dosage), but since it is determined by cell culture, the term CCIDso is used in this.
    [00056] The virus is placed in contact with the cells to allow the virus to infect said cells and spread. For example, the viral seed batch is added to the cell culture and allowed to absorb in the cells, for example, for about 30 minutes with gentle agitation (for example, about 30 rpm), after which the additional culture medium can be added and pH adjusted if desired, stirring speed can be adjusted and the culture maintained. After the infection stage, the amplification of the number of virus particles occurs. Of course, this step is also preferably performed on PER.C6 cells that are cultured in the suspension in the absence of serum and more preferably under conditions that are completely free of components directly derived from the animals. This step can be adequately performed in bioreactors, for example, in the scales between 1 and 20,000 liters, for example, between 10 and 2000 liters, for example, between 50 and 1000 liters in which the scale can be easily adjusted to the demand for the vaccine . In certain embodiments, the bioreactor is a simple-to-use bioreactor (SUB).
    [00057] After the poliovirus has spread in cells, the virus or its components are grown from cell culture. These can be done by routine methods, which are as such known to that skilled person. The virus produced and released in the cell culture medium can be separated from the cell biomass by conventional methods, such as centrifugation or ultrafiltration and culture in the supernatant. In such a case, centrifugation or filtration is the cultivation step. Conventional methods for culturing the virus can be used, for example, those described in U.S. 4,525,349. Soon, the liquid medium suspension containing the virus is typically removed, filtered and concentrated by, for example, ultrafiltration. For example, at the end of the culture, cultivation is performed by cultivating the culture medium containing the viral suspension. The culture can be filtered, for example, using a 0.22 pm filter and optionally stored at 4 ° C.
    [00058] The filtered culture can optionally be ultrafiltered to concentrate the viral suspension and subsequently, the poliovirus can be purified, for example, using gel filtration and / or ion exchange chromatography, for example, following the procedures as described in US 4,525,349, or in (Van Wezel et al, 1978). The suspension of the resulting concentrated virus can be optionally diluted and to prepare IPV the poliovirus in it will be inactivated, by which conventional methods can be used.
    [00059] Methods for culturing and purifying the poliovirus or viral components and producing the vaccines therein have been used in the art for decades and are thus well known and have been widely described, for example, in (Van Wezel et al, 1978; Montagnon et al, 1984; WO 2007/007344; US 4,525,349), all incorporated by reference herein.
    [00060] Polio vaccines in live virus or inactivated virus. These contain the poliovirus D-antigen, which is the important protective antigen. Virus yields can be measured by standard virus titration techniques while determination of D-antigen concentration is also carried out by routine techniques well known to that person skilled, for example, the D-antigen ELISA assay. Immunogenicity can be, for example, determined by in vivo testing on animals. The potency to be determined using the D-antigen ELISA and by the poliovirus neutralizes the serum cell culture assay from previously immunized rats.
    [00061] In general, each of the poliovirus strains is grown in a separate process and if, for example, a trivalent vaccine containing three types of poliovirus is prepared, the virus (inactivated, by IPV) is mixed and formulated for the preparation of individual dosages. In certain embodiments for example, a final vaccine by dosage (for example, 0.5 ml) may for example comprise the 40 D-antigen (DU) units of poliovirus type 1, 8 DU of poliovirus type 2 and 32 DU of poliovirus type 3, determined by comparing reference preparations.
    [00062] Poliovirus inactivation can be done according to methods known in the art, for example, with formalin or with 13-propiolactone (BPL) (see for example, Jiang et al, 1986). In certain embodiments, inactivation is carried out with formalin, for example, by the following method: the purified viral suspension is filtered on a 0.22 pm membrane, heating at 37 ° C with fixed magnetic stirring for 24 hours, after a formaldehyde solution is added to achieve a concentration of 1 per 4,000. While maintaining the viral suspension at 37 ° C, magnetic stirring is continued for the first four days. On the sixth day, the viral suspension is filtered through a 0.22 micron membrane and inactivation is continued under the suspension at 37 ° C until the twelfth day. The inactivated viral suspension is homogenized and can be stored, for example, at 4 ° C. After this step, the concentrated and / or final batches for administration can be prepared, for example, by mixing the desired preparations.
    [00063] In certain embodiments, the purified poliovirus or viral component is formulated into a pharmaceutical composition. This can be done according to a variety of method and using a variety of plugs all according to routine methods well known to the person skilled in the art. In general, this requires conducting the poliovirus particles in a pharmaceutically acceptable composition, comprising the poliovirus and at least one pharmaceutically acceptable excipient. Such a composition can be prepared under conditions known to that skilled person and in certain embodiments is suitable for administration to humans. In certain embodiments, the composition can comprise the buffered culture medium, which can optionally be Medium M-199, which is used as a formulation buffer by certain registered conventional inactivated poliovirus vaccines. In addition, phosphate buffered saline can be used and final dosage formulations can comprise, for example, 0.5% 2-phenoxyethanol and a maximum of 0.02% formaldehyde per dose as antimicrobial preservatives.
    [00064] Pharmaceutically acceptable carriers or excipients or diluents are well known in the art and used extensively in a wide range of therapeutic products. Preferably, carriers are applied by working well on vaccines. In certain embodiments, vaccines still comprise an adjuvant, for example, alum. Adjuvants are known in the art to further enhance the immune response to an applied antigenic determinant.
    [00065] To administer to humans, the invention can use pharmaceutical compositions comprising the poliovirus and a pharmaceutically acceptable carrier or excipient. In the present context, the term "pharmaceutically acceptable" means that the carrier or excipient, at the dosages and concentrations used, will cause any unwanted or harmful effects on the patients they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]). The purified inactivated poliovirus or immunogenic parts thereof are preferably formulated and administered as a sterile solution. Sterile solutions are prepared by sterile filtration or by other methods known to you in the art. The solutions are then lyophilized or filled into the pharmaceutical dosage containers. The pH of the solution is generally in the range of pH 3.0 to 9.5, for example, pH 5.0 to 7.5. The poliovirus or immunogenic parts thereof are typically in a solution having a suitable pharmaceutically acceptable buffer and the poliovirus solution may also contain a salt. Optionally, a stabilizing agent can be present, such as albumin. In certain embodiments, detergent is added. In certain embodiments, the vaccine can be formulated into an injectable preparation. These formulations contain the effective amounts of poliovirus or immunogenic parts of these, are sterile liquid solutions, liquid suspensions or lyophilized versions and optionally contain stabilizers or excipients.
    [00066] A polio vaccine can be monovalent, containing one type of poliovirus (type 1, 2 or 3), or bivalent (containing two types of poliovirus, for example, types 1 and 2, 1 and 3 or 2 and 3) , or trivalent (containing three types of poliovirus, ie types 1, 2 and 3).
    [00067] It is possible to produce IPV from wild-type polio viruses. Alternatively, IPV can be produced from a live non-virulent poliovirus, for example, from Sabin strains, which should further reduce the risk of reintroducing wild-type poliovirus from an IPV manufacture (see for example, WO 2007/007344 and Doi et al, 2001). The present invention is suitable for the production of wild type poliovirus (types 1, 2 and 3, for example, the Mahoney type 1 strain, type 2 MEF strain, or type 3 Saukett strain) as well as non-virulent types poliovirus (for example, Sabin strains). The invention can therefore be used to produce polioviruses by IPV, as well as by OPV. The processes according to the applied invention to produce BTI can serve to drive the low cost to the point that BTI can become available to less developed countries. Although in general OPV is cheaper than IPV when prepared according to conventional methods, the highly efficient processes of the invention can still reduce the volume costs of material per OPV and since they reduce the costs of these as well.
    [00068] The administration of a polio vaccine can be carried out, for example, intramuscularly, intradermally, or orally, according to the methods known in the art.
    [00069] The poliovirus vaccine obtainable according to the invention can be used as a stand-alone vaccine, but in other embodiments they can be combined with other vaccines in the regular manner, for example, in the form of a combined diphtheria vaccine , whooping cough, tetanus and polio and can optionally also include the vaccine components, for example, against hepatitis B and / or heamophilus influenzae, etc. Thus, the poliovirus is suitable for use in the expanded immunization program (PPE) and can be combined with vaccines in that program. Similarly, conventional poliovirus vaccines, the vaccine according to the invention can be given as a single dosage, or preferably in the initial intensification regimens where multiple doses of the vaccine are administered at the appropriate time intervals, for example, two injections over a period of 1 to 2 months, followed by an intensified dosage of 6 to 12 months later; or for example, an initial oral dosage, followed by a second dosage about 8 weeks later and a third dosage 8 to 12 months after the second dosage; or for example, for children for a first oral dosage at 6 to 12 weeks of age, followed by a second dosage at about 8 weeks after the first dosage and a third dosage at about 6 to 18 months of age; or for example, just a simple dosage by people previously vaccinated at increased risk; etc. The optimal dosage regimen can be determined according to standard medical practice and will generally follow the same schedules as that for the available poliovirus vaccines.
    [00070] The invention is further explained in the following examples. The examples do not limit the invention in any way. These merely serve to clarify the invention. EXAMPLES Example 1: Efficient poliovirus production in adherent PER.C6 cells
    [00071] To test the propagation of poliovirus in adherent PER.C6 cells and generate virus loads, poliovirus type 1 (Brunenders), type 2 (MEF-1) and type 3 (Sauckett), were obtained from SBL (Sweden) . The titrations of these charges, each produced in Vero cells, are around 106CCIDso / ml. PER.C6 cells (Fallaux et al, 1998) were grown in the culture medium (DMEM with 10% FBS and 10 mM MgCh). Three T175 flasks were seeded with 30 x 106 PER.C6 cells / flask in the 25 ml culture medium for each type of poliovirus and inoculated the next day with a multiplicity of infection (MOI) of 0.1 (0.1 CCIDso / cell) at 37 ° C and 10% CO2 in a humidified incubator. Three days later, cells and media were cultured and crude lysates were prepared for two freeze / thaw cycles. Following centrifugation to remove cell debris, the supernatants were aliquoted and stored at -80 ° C. In parallel, a T175 flask was seeded with 6.25 x 106 Vero cells in the 25 ml Vero cell culture medium (Optipro SFM medium supplemented with 4 mM L-glutamine) for each poliovirus strain and infected with the same MOIs. Vero cultures were also grown after 3 days, frozen / thawed twice and aliquoted for storage.
    [00072] Poliovirus production was then quantified by a CCID50 assay using Vero cells. So far, 1.25 x 104 Vero cells have been seeded in each well of a 96 well plate in 100 µl medium and incubated at 37 ° C and 5% CO2. The next day, a series of 15 five-fold dilutions of the poliovirus samples were prepared in the Vero cell culture medium and 100 µl of number of dilutions 5 to 15 were added to column 1 to 11 on the 96 well plate eight times. Column 12 serves as an uninfected control column. Seven days later, the reservoirs were analyzed for the occurrence of the cytopathogenic effect (CPE) and titrations were calculated using the Spearman-Karber method: End point titration (logw) = Xo- d / 2 + d / n * ZXi
    [00073] Where Xo is the log ™ value of the largest dilution in which all inoculations are still positive, d is the logw value of the dilution factor used, n is the number of replicates in each dilution and ZXi is the sum of all reservoirs that are positive including the Xo dilution.
    [00074] The results of the titration experiment are described in Fig. 1 and shows that in the adherent PER.C6 cells the titrations were> 5 times greater than in Vero cells by type 1 poliovirus and> 10 times greater in the case of type 2 and 3. Differences in virus particle production per cell are expected to be smaller since more PER.C6 cells have been seeded. For both PER.C6 and Vero the confluence of the cell monolayer was estimated to be ~ 80%.
    [00075] From these experiments we concluded that the production of poliovirus in the adherent monolayers of PER.C6 cells was at least as good as in Vero cells. Example 2: Efficient poliovirus production in PER cells. C6 in suspension
    [00076] To investigate the propagation of poliovirus in suspended PER.C6 cells, smaller scale experiments were performed to test the different culture medium, multiplicity of infections (MOI) and cultivation time (TOH). So far, PER.C6 cells have been grown in three different media: AEM (Invitrogen), BMIVg (commercially available as Permexcis ™, from Lonza) and CDM4PERMAb (Hyclone). On the day of infection, cells grown in one type of medium were counted and seeded again in the same type of medium at different cell densities (1.5, 2.5, 3.5 or 5 million cells / ml) and infected with Different MOIs (0.01, 0.05 or 0.1 CCIDso / cell) at 37 ° C in a humidified incubator on a shaking platform. The platform (IKA KS 260) has an orbital diameter of 10 mm and was used at 100 rpm to shake the 125 or 250 ml bottles filled with the 15 to 20 ml medium. For the AEM medium, the cells were seeded at 1.5 or 2.5 million cells / ml since AEM does not support higher cell densities. In this manner multiple cultures were prepared which were grown 2, 3 or 4 days after infection. All samples were frozen / thawed twice and kept at -20 ° C or less until further analysis.
    [00077] Fig. 2 describes the results of the titration of these samples for day 2 and samples for day 4 (data for day 3 not shown). PER.C6 cells developed and infected in all three media capable of producing high titers of poliovirus type 1, although the BMIVg medium gives a little lower titers compared to the PERMAb and AEM media. Furthermore, larger incubation does not result in larger titrations. In contrast, the cultivation of day 2 given in many cases the higher titrations compared to the cultivations of day 3 and 4. A consistent effect of the variation of MOIs should not be seen in this experiment. Importantly, the use of higher cell densities in infection does not result in higher volumetric titrations showing that a suspension culture process using high cell densities is beneficial for the yield of the infectious poliovirus.
    [00078] In a next experiment, the period of cultivation and temperature during infection was compared by all three strains of poliovirus. So far, PER.C6 cells were seeded in the AEM medium at 2.5 x 106 cells / ml in 15 ml volumes in the shaker flasks and infected with an MOI of 0.1 to 37 ° C and at 35 ° C of each strain of poliovirus. The cells and medium were cultured on days 2, 3 and 4 after infection and processed as described above. Analysis of virus production under the different conditions was done by determining the CCIDso values as described above and shows an increase in yield at 35 ° C compared to 37 ° C for all three types of poliovirus (Fig. 3). In addition, it was confirmed and extended to poliovirus types 2 and 3 that in many cases the higher titers were measured when the cultures were taken on day 2. Example 3: Performance of poliovirus in the suspension of PER cells. Increased C6 in higher cell density
    [00079] To study whether an additional increase in cell density leads to an increase in virus titer, productions with 2.5 x 10 6 cells / ml were compared to 10 x 10 6 cells / ml. So far, the PER.C6 cells in the PERMAb medium were seeded in 15 ml volume by shaking the flasks at the indicated cell densities and infected with 2 CCID50 / poliovirus type 1 cells in triplicate. After 24 and 48 hours the cells and medium were cultured and clean lysates were prepared by freezing / thawing and centrifuging as described above. In addition to the previously tested temperatures 35 and 37 ° C, the experiment was also carried out at 33 ° C.
    [00080] Analysis of the titrations by the CCID50 assay (Fig. 4) confirms that the yield was improved when the cells were infected at a density of 10 x 106 cells / ml compared to 2.5 x 106 cells / ml. The best titrations were obtained at 35 ° C without considering cell density or cultivation day. In addition and indicative for the efficient propagation of poliovirus in PER.C6 cells, it was shown that the cultures can also be taken after 24 hours since the yield in the samples of 24 hours or 48 hours were completely comparable.
    [00081] In a next experiment, these conditions were also tested for other types of poliovirus. PER.C6 cells were seeded in PERMAb medium at 10 x 106 cells / ml and infected with 2 CCID50 / cells at 35 ° C in the flasks shaken in triplicate with different poliovirus loads. Cultures were performed after 24 and 48 hours and cells and media were processed to clean lysates as described above. The titration for the CCIDso assay showed that the use of high cell densities also results in high virus yields for types 2 and 3 (Fig. 5).
    [00082] This clearly shows that cultures of higher densities of PER.C6 cells in the suspension provide an excellent platform for the production of wild-type poliovirus. Since the cell density of PER.C6 cells and size / volumes of the culture can be increased by using bioreactor systems, wave bags or other types of systems classifiable for culture, poliovirus production can be significantly improved compared to the microcarrier system with Vero cell cultures.
    [00083] The produced poliovirus is cultured and purified according to methods known in the art and used by the poliovirus propagated in Vero cells, inactivated by formalin according to known methods and subsequently immunogenicity is tested using a standard rat immunogenicity assay , according to methods well known in the art (for example, Bevilacqua et al, 1996). The poliovirus thus produced is expected to have an immunogenicity comparable to the poliovirus produced with conventional processes using Vero cells. Example 4: Poliovirus production in PER cells. C6 in a bioreactor
    [00084] The cells are thawed from a PER.C6 useful cell bank and propagated in serum-free culture medium in a humidified incubator at 37 ° C and 10% CO2. Subculture is performed every 3 to 4 days until sufficient cell density is reached to inoculate a 2 L bioreactor at a cell density of 0.2 to 0.4 x 106 cells / mL. The cells are propagated in the 2 L bioreactor at 37 ° C, 40% OD and a pH of 7.3. When a cell density of approximately 2 x 10 6 cells / ml is reached (day 4 post inoculation) an ATF system is started, to allow the cells to be cultured for a longer period of time and to reach high cell densities. After approximately 11 to 12 days at a cell density in the 2 L bioreactor, more than 50 x 10 6 cells / mL is achieved. at this point the cell suspension is transferred in a 10 L bioreactor. The cell suspension from the 2 L bioreactor is diluted 1: 5 with serum-free culture medium. The cell density in the 10 L bioreactor is between 10 and 15 x 106 cells / mL. Subsequently, the 10 L bioreactor is infected with a poliovirus seed stock in a 2 CCIDso / cell MOI. Poliovirus production is carried out at 35 ° C, pH 7.3 and 40% OD. The 10 L bioreactor is sampled at certain time points for cell count and poliovirus production and poliovirus culture is properly performed between 12 and 48 hours after infection. References Barrett PN, Mundt W, Kistner 0, Howard MK. 2009. Vero cell platform in vaccine production: moving towards cell culture-based viral vaccines. Expert Rev. Vaccines 8: 607-618. Bevilacqua JM, Young L, Chiu SW, Sparkes JD, Kreeftenberg JG. 1996. Rat immunogenicity assay of inactivated poliovirus. Dev. Biol. Stand. 86: 121-127. Campbell SA, Lin J, Dobrikova EY, Gromeier M. 2005. Genetic determinants of cell type-specific poliovirus propagation in HEK 293 cells. J. Virol. 79: 6281-6290. Card CJ, Smith T, Hunsaker B, Barnett B. 2005. Serum- free production of poliovirus: A comparative study using microcarriers, roller bottles and stationary cell culture. In: F. Godia and M. Fussenegger (Eds.), Animal Cell Technology meets Genomics, 761-765. Doi Y, Abe S, Yamamoto H, Horie H, et al. 2001. Progress with inactivated poliovirus vaccines derived from the Sabin strains. In: Brown F (ed): Progress in Polio Eradication: Vaccine Strategies for the End Game. Dev. Biol. 105: 163-169. Van Eikenhorst G, Bakker WAM, Thomassen YE, van der Pol LA. 2009. Platform technology for viral vaccine production: comparison between attached and suspension Vero cells. Poster and Abstract P70. In: 21st Meeting of the European Society for Animal Cell Technology, Program and Book of Abstracts. Fallaux FJ, Bout A, van der Velde I, van den Wollenberg DJ, Hehir KM, Keegan J, et al. New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication- competent adenoviruses. Hum GeneTher 1998 Sep 1; 9 (13): 1909-17. Furey J. Scale-up of a cell culture perfusion process - A low-shear filtration system that inhibits filter-membrane fouling. Genetic Engineering News. Vol. 22, No. 7, April 2002. Jiang S, Pye D, Cox JC. 1986. Inactivation of poliovirus with f3-propiolactone. J. Biol. Stand. 14: 103-109. John J. 2009. Role of injectable and oral polio vaccines in polio eradication. Expert Rev. Vaccines 8: 5-8. Kew OM, Sutter RW, from Gourville EM, Dowdle WR, Pallansch MA. 2005. Vaccine-derived polioviruses and the endgame strategy for global polio eradication. Annu. Rev. Microbiol. 59: 587-635. Kral KM, Golden K, Zijlstra G, Swaying J, Nieboer M, Chon JH. 2009. Advances in high yielding platform production processes using the PER.C6® human cell line. Abstract P142. In: 21st Meeting of the European Society for Animal Cell Technology, Program and Book of Abstracts. Merten O.-W., Wu R, Kale E, Crainic R. 1997. Evaluation of the serum-free medium MDSS2 for the production of poliovirus on Vero cells in bioreactors. Cytotechnology 25: 35-44. Montagnon B, Vincent-Falquet JC, Fanget B. 1982. Thousand liter scale microcarrier culture of Vero cells for killed poliovirus vaccine. Promising results. Develop. Biol. Standard. 55: 37- 42. Montagnon BJ, Fanget B, Vincent-Falquet JC. 1984. Industrial-scale production of inactivated poliovirus vaccine prepared by culture of Vero cells on microcarrier. Rev. Infect. Dis. 6 (suppl. 2): S341-S344. Pau MG, Ophorst C, Koldijk MH, Schouten G, Mehtali M, Uytdehaag F. The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines. Vaccine 2001 Mar 21; 19 (17-19): 2716-21. Van Wezel AL, van Steenis G, Hannik CA, Cohen H. 1978. New approach to the production of concentrated and purified inactivated polio and rabies tissue culture vaccines. Develop, biol. Standard. 41: 159-168. Wright PF, Modlin JF. 2008. The demise and rebirth of Polio - The Phoenix modem J. Infect. Dis. 197: 335-336. Yakovenko ML, Korotkova EA, Ivanova OE, Eremeeva TP et al. 2009. Evolution of the Sabin vaccine into pathogenic derivatives without appreciable changes in antigenic properties: need for improvement of current poliovirus surveillance. J. Virol. 83: 3402- 3406. Yallop C, Crowley J, Cote J, Hegmans-Brouwer K, Lagerwerf F, Gagne R, Martin JC, Oosterhuis N, Opstelten DJ, Bout A. Per.C6 cells for the manufacture of biopharmaceutical proteins. Modem Biopharmaceuticals - Design, Development and Optimization. Vol. 3, 2005.
    权利要求:
    Claims (10)
    [0001]
    1. Process for the production of poliovirus, characterized by the fact that it comprises the steps of: a) providing a culture of serum-free suspension cells, which are PER.C6 cells as deposited under ECACC no. 96022940, b) infect said cells with poliovirus, at a cell density between 2 x 106 cells / ml and 150 x 106 cells / ml and c) cultivate poliovirus in a period between 12 and 48 hours after infection.
    [0002]
    2. Process according to claim 1, characterized by the fact that said virus infection and / or spread is carried out at a temperature between 34 ° C and 36 ° C.
    [0003]
    Process according to claim 1 or 2, characterized by the fact that said infection is carried out at a cell density between 5 x 106 cells / ml and 20 x 106 cells / ml.
    [0004]
    Process according to any one of claims 1 to 3, characterized by the fact that said infection is carried out at said cell density around 10 x 10 6 cells / ml.
    [0005]
    Process according to any one of claims 1 to 4, characterized by the fact that said infection is carried out in a multiplicity of infection (MOI) between 1 and 3, for example, around 2.
    [0006]
    Process according to any one of claims 1 to 5, characterized by the fact that said poliovirus cultivation is carried out between 18 and 30 hours after infection, for example, around 24 hours after infection.
    [0007]
    Process according to any one of claims 1 to 6, characterized in that said poliovirus is poliovirus type 1, poliovirus type 2 or poliovirus type 3.
    [0008]
    8. Process according to claim 7, characterized in that said poliovirus is poliovirus type 1 strain Mahoney, poliovirus type 2 strain MEF or poliovirus type 3 strain Saukett.
    [0009]
    Process according to claim 7, characterized in that said poliovirus is an attenuated poliovirus, such as a Sabin strain.
    [0010]
    10. Process for producing a polio vaccine, characterized in that it comprises a process as defined in any of claims 1 to 9, further comprising purifying, optionally inactivating and formulating the cultured poliovirus to obtain a polio vaccine.
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    法律状态:
    2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2018-08-21| B25D| Requested change of name of applicant approved|Owner name: JANSSEN VACCINES AND PREVENTION B.V (NL) |
    2019-01-29| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |
    2019-08-20| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-04-14| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-10-27| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 27/10/2020, OBSERVADAS AS CONDICOES LEGAIS. |
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    优先权:
    申请号 | 申请日 | 专利标题
    EP09165620.7|2009-07-06|
    US27103809P| true| 2009-07-16|2009-07-16|
    US61/271,038|2009-07-16|
    EP09165620|2009-07-16|
    PCT/EP2010/059796|WO2011006823A1|2009-07-16|2010-07-08|Production of polio virus at high titers for vaccine production|
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