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    method for producing vaccinia virus certain embodiments relate to viral production processes for the large-scale production of vaccinia virus.
    公开号:BR112014000787B1
    申请号:R112014000787-0
    申请日:2012-08-03
    公开日:2021-04-13
    发明作者:David Kirn；John Bell
    申请人:Sillajen Biotherapeutics, Inc；
    IPC主号:
    专利说明:

    DESCRIPTIVE REPORT REMISSIVE REFERENCE TO RELATED REQUESTS
    [0001] This application claims the benefit of United States Provisional Application No. 61 / 515,724, filed on August 5, 2011, the content of which is incorporated into this document by reference in its entirety. BACKGROUND OF THE INVENTION I. FIELD OF THE INVENTION
    [0002] In general terms, the embodiments of the present invention refer to virology, medicine and viral therapy. Certain embodiments refer to viral production processes. II. BACKGROUND
    [0003] Oncolytic viruses (VOs) are therapeutic replicating viruses designed or selected specifically to develop within and kill tumor cells. Several groups are in the early stages of commercialization based on pre-clinical animal models and initial clinical data in humans. However, a challenge that the field still faces is to produce pharmaceutical-grade viruses on a large scale for administration to patients. Viruses are a biological entity rather than a synthesized drug, so the manufacturing process involves the production of the virus inside cells and the subsequent removal of contaminating cellular waste without losing the infective potential. To date, most, if not all, of the commercial manufacture of vaccinia virus products for human use has been in the form of vaccines in non-human cell cultures. In general, for the application of a vaccine, patients receive small doses of the virus at a local site (usually intramuscular) where contaminating cellular proteins can actually serve as adjuvants to increase the immunogenicity of the preparation. In contrast, with therapeutic oncolytic viruses, the required virus doses are up to a million times higher for the application of the vaccine and can be administered intravenously, intracranial, intraperitoneal or by direct injections into the tumor. In the case of oncolytic viruses, it is essential to be able to manufacture large quantities of viruses free of contaminating non-human cell residues. In addition, vaccinia viruses are enveloped viruses that incorporate host cell proteins into the viral membrane. The incorporation of human proteins in the virus envelope increases its ability to move around undetected by the host human immune system.
    [0004] Therefore, the need for new methods and compositions for the production of oncolytic viruses on a large scale persists. SUMMARY OF THE INVENTION
    [0005] The vaccinia virus is produced in HeLa cells grown in a suspension culture. However, suspension culture conditions are currently not suitable for large-scale virus production. Thus, the production of the vaccinia virus on a large scale or on a commercial scale has not yet been successfully achieved using HeLa cells. Furthermore, adhering HeLa cell lines were not expected to produce enough cell numbers to guarantee the use of adherent HeLa cells in the production of the vaccinia virus in quantities necessary for large-scale production. The present application describes a large-scale process for the production of the vaccinia virus using adherent HeLa cells.
    [0006] Certain embodiments relate to methods for producing a vaccinia virus that include one or more of the following steps.
    [0007] In certain respects, the methods include infecting HeLa cells adhered to a surface with a vaccinia virus by bringing said adherent HeLa cells into contact with said vaccinia virus. Preferably, the vaccinia virus is a recombinant vaccinia virus, such as those described in paragraphs 28 through 52 in United States Patent Application Publication No. 2009/0004723, incorporated herein by reference in its entirety. In certain embodiments, HeLa cells adhered to a Petri dish are specifically excluded from the scope of the Claim. In various embodiments, the present invention proposes a method for producing a vaccinia virus that comprises (a) infecting HeLa cells adhered to a surface with the vaccinia virus, (b) cultivating the infected cells under conditions favorable to the production of progeny viruses, and ( c) collect the vaccinia virus from the culture.
    [0008] In some embodiments, methods are proposed to produce a vaccinia virus comprising (a) infecting HeLa cells adhered to a surface with a recombinant vaccinia virus by placing at least 1 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011, 5 x 1011, 1 x 1012, 5 x 1012, 1 x 1013, 5 x 1013 or more adherent HeLa cells, including all values and ranges between these, in contact with a vaccinia virus composition; and (b) culturing the infected cells under conditions favorable to the production of 50, 60, 70, 80, 90, 100 or more recombinant vaccinia viruses per cell, including all values and ranges between them. Preferably, according to the methods, at least 50 plaque forming units 50 (pfu) of vaccinia virus are produced per HeLa cell; more preferably, at least 75 pfu of vaccinia virus per HeLa cell.
    [0009] In other embodiments, HeLa cells adhered to a surface are infected with the vaccinia virus at a multiplicity of infection (moi) between 0.001 and 1.0, preferably between 0.005 and 0.5 and, more preferably, between 0.01 and 0.1. In a particularly preferred embodiment, m.o.i. is between 0.01 and 0.05, including all values and ranges between them. In other preferred embodiments, m.o.i. is between 0.015 and 0.03. The term "multiplicity of infection", or "m.o.i.", refers to the ratio of plaque-forming units (pfu) of the virus added per HeLa cell during infection.
    [0010] In related embodiments, HeLa cells adhered to a density of about 104 to about 106 HeLa cells / cm2, preferably about 105 HeLa cells / cm2, are infected with the vaccinia virus according to the methods, the concentration of the vaccinia virus being between 103 and 105 pfu / ml, preferably 104 pfu / ml.
    [0011] Cultivation of HeLa Adherent Cells. Adherent HeLa cells for infection with the vaccinia virus according to the methods can be cultured by any suitable method, including (among others) cultivation in glass and plastic containers, for example, culture flasks, bioreactors, including, among others, factories cell phones, such as Nunclon® Cell Factories ™, cell cubes, such as the CELLCUBE® system from Corning Life Sciences, malleable plastic bags (such as infusion bags) filled with a cell support matrix, such as Gibco® or Lampire® cell culture bags ; rotating bottles; spinner bottles, such as Magna-Flex® Spinner Bottles; fermenters; or hollow fibers of various materials, such as Cellex Biosciences cell culture systems (AcuSyst® hollow fiber reactor), BioYest (Cell-Pharm® 100 or 2500 system) or Fibercell ™. Cultivation in bioreactors is preferred because they allow precise monitoring and control of variables such as temperature and pH. In some ways, it is possible to use CellBind ™ plastic items (www.sigmaaldrich.com). Cell cultures can be in the form of cell clusters, such as spheroids on a microcarrier bead, or cells in a structure (i.e., biodegradable structures), tissue or tissue organoids. In some respects, adherent HeLa cells are grown in a microcarrier, cell cube, cell factory, T flask or rotary flask. In a preferred embodiment, the adherent HeLa cells are grown in one or more rotating bottles, for example, using a RollerCell 40 apparatus (Cellon S.A., Luxembourg). The container used can comprise, among others, a cumulative culture area of about at least, or at most, 1,700, 5,000, 10,000, 25,000, 50,000, 100,000, 150,000, 200,000 or 500,000 cm2, including all ranges and values between Those. In some respects, the container has a cumulative culture area of at least 168,000 cm2.
    [0012] Adherent HeLa cells that will be infected with the vaccinia virus can be grown in any suitable culture medium that stimulates growth, maintenance and attachment to the cell support surface, including, among others, BME (Eagle basal medium) ), MEM (minimal Eagle medium), 199 medium, DMEM (Dulbocco modified Eagle medium), GMEM (Glasgow modified Eagle medium), DMEM-HamF12 and Ham-F10, Isocove modified Dulbecco medium, 5A medium MacCoy or RPMI 1640. Preferably, the culture medium is a defined culture medium. In one aspect, the culture medium is substantially serum-free. Examples of serum-free media optimized for use with HeLa cells include, but are not limited to, VP-SFM (Gibco BRL / Life Technologies), HeLa Ex-Cell® serum-free medium (SFM) (Sigma Aldrich) and Quantum 101 (GE Healthcare ). In other embodiments, the culture medium may also comprise animal serum, such as fetal bovine serum (FBS). For example, the culture medium can comprise 5% to 10% serum (for example, FBS) or it can contain less than 5% serum (for example, FBS), or any range or value between them. In one embodiment, the culture medium is DMEM supplemented with 5% to 10% FBS, such as 10% FBS.
    [0013] The term "microcarrier" means a small, distinct particle for use in cultured cells and to which the cells bind. The microcarriers can be in any suitable format, such as sticks, spheres and the like. In many embodiments, a microcarrier includes a microcarrier base, which is coated to provide a surface suitable for cell culture. A polypeptide can be attached, grafted, or otherwise connected to the surface coating. Microcarriers are used in cell cultures to provide large productions of binding-dependent cells. Microcarriers are usually agitated or stirred in cell culture media and provide a ratio of the surface area of attachment and growth to very large volume in relation to more traditional culture equipment. Refer to United States Patent Publication 2011/0027889, which is incorporated by reference in its entirety by reference, for a detailed example of a micro charger.
    [0014] In certain embodiments, the vaccinia virus is a strain of the vaccinia virus from IHD-J, Wyeth, Western Reserve or Copenhagen. In some respects, the vaccinia virus is a recombinant vaccinia virus. The recombinant vaccinia virus can comprise a heterologous coding region. As used in this document, a heterologous coding region is a coding region that is not found naturally in the vaccinia virus genome. In certain aspects, the heterologous coding region encodes an immunostimulatory polypeptide. The immunostimulatory polypeptide can be a cytokine, such as, among others, the granulocyte and macrophage colony stimulating factor (GM-CSF).
    [0015] In certain respects, the recombinant vaccinia virus replicates selectively in a tumor cell. The recombinant vaccinia virus can comprise a mutation in an endogenous gene. The mutation can be the deletion of a nucleic acid sequence, the replacement of nucleic acid residues or the introduction of one or more nucleic acid residues that affect the function or expression of the vaccinia virus. The mutation can result in selective growth in a tumor cell, attenuated growth in a non-tumor cell, enhanced growth in a tumor cell, or a combination of these. A recombinant vaccinia virus with mutation in an endogenous gene can be a vaccinia virus with thymidine kinase (TK) deficiency (i.e., a vaccinia virus with mutation in a gene encoding thymidine kinase making the encoded polypeptide non-functional). In certain respects, the recombinant vaccinia virus with mutation in an endogenous gene is a strain of the vaccinia virus from IHD-J, Wyeth, Western Reserve or Copenhagen and / or comprises a heterologous coding region. In another embodiment, a vaccinia virus with mutation in an endogenous gene also comprises a heterologous coding region, such as, among others, a coding region for granulocyte and macrophage colony stimulating factor polypeptide.
    [0016] Cultivation of infected HeLa cells. Adhered HeLa cells submitted to contact with the vaccinia virus are grown under conditions favorable to the production of progeny vaccinia virus. Culture conditions may comprise, but are not limited to, a defined infection medium, defined pH, defined culture vessel, defined temperature or temperature range, defined cell number, defined cell confluence, defined physical manipulation (eg, enzymatic treatment or chemical, rotation frequency, agitation speed, etc.), defined gas phase conditions, defined culture time, defined cell seeding density and defined number of cell passages. In one aspect, the virus can simply be added to the HeLa cell culture medium used to prepare the adherent HeLa cells, in which case the culture medium and the infection medium are the same. In other respects, the culture medium used to prepare the adherent HeLa cells is removed at the infection stage and the HeLa cells subjected to contact with the vaccinia virus are grown in an infection medium that can comprise or be substantially serum-free until it produces the progeny virus. Suitable infection media include, but are not limited to, BME, MEM, medium 199, DMEM, GMEM, DMEM-HamF12 and Ham-F10, Isocove modified Dulbecco medium, MacCoy 5A medium or RPMI 1640, any of which can be optionally supplemented with serum. Serum-free media, such as VP-SFM, HeLa Ex-Cell® serum-free media (SFM) and Quantum 101 are also suitable for use as an infection medium.
    [0017] In certain respects, infected cells are cultured in an infection medium with 5% to 10% serum (for example, fetal bovine serum (FBS)) at a pH of about 7, 7.2, 7, 3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, preferably at a pH greater than 7.1, more preferably at a pH greater than 7 , 2 and, even more preferably, at a pH of about 7.3. In other respects, cells are cultured in an infection medium with less than 5% serum (eg, FBS), such as 0% serum (eg, FBS) at a pH of about 7, 7.2 , 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, preferably at a pH greater than 7.1, more preferably at a pH greater than 7.2 and, even more preferably, at a pH of about 7.3. In related aspects, the cells are grown at a pH of about 7.3 in an infection medium comprising between 0% and 10% serum (eg, FBS). In other embodiments, the cells are cultured at a pH greater than 7.2 and less than 7.6 in an infection medium comprising between 0% and 10% serum (for example, FBS). In a preferred embodiment, the infection medium comprises DMEM and, optionally, 100 to 300 mM glutamine. The cells can be grown at a temperature of about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 ° C. In a preferred embodiment, the cells are grown at a temperature between 36 ° C and 37.5 ° C, preferably at 37 ° C. In certain respects, the infection medium will specifically exclude one or more from dextran sulfate, Pluronic F-68, Tween-80 and / or soy protein hydrolyzate. In preferred embodiments, the infection medium specifically excludes dextrate sulfate and at least one of the following components: Pluronic F-68, Tween-80 and soy protein hydrolyzate. In other preferred embodiments, the infection medium is substantially free of all such components.
    [0018] Methods may further include expanding HeLa cell culture before, during or after infection. HeLa cells can pass through at least 1, 2, 3, 4, 5 or more culture vessels, for example, rotating bottles. In certain aspects, cells are detached from a surface by treating them with a detached solution comprising proteases and / or collagenolytic enzymes, for example, HyQtase®. Each container can have a larger culture area compared to the previous one. In some respects, a culture vessel has a total culture area of at least 84,000 to 1,000,000 cm2. In certain aspects, the final passage is to a culture vessel with a culture area of at least 168,000 cm2.
    [0019] In certain embodiments, the time interval between sowing a culture vessel and infection is 12, 24, 48, 72, 96, 120, 144 or more hours, including all values and ranges between them. In certain respects, adherent HeLa cells are subjected to contact with 1 x 101, 1 x 102, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108 or 1 x 109 pfu / ml of vaccinia virus, including all values and ranges between them. In a preferred embodiment, HeLa cells adhering to a density of about 104 to about 106 HeLa cells / cm2, preferably about 105 HeLa cells / cm2, are contacted with between 103 and 105 pfu / ml of the virus vaccinia, more preferably, about 104 pfu / ml of the vaccinia virus.
    [0020] In certain respects, methods include isolating the vaccinia virus produced by adherent HeLa cells. The harvested vaccinia virus can be concentrated and purified according to methods known to those skilled in the art. Other embodiments of the invention relate to a vaccinia virus composition produced by the methods described in this document, such as those described in paragraphs 295 to 303 of United States Patent Application Publication No. 2009/0004723, incorporated herein by reference. in full.
    [0021] Other embodiments of the invention are discussed throughout this application. Any embodiment described with reference to one aspect of the invention applies to other aspects of the invention and vice versa. The embodiments in the Examples section are seen as embodiments of the invention applicable to all aspects of the invention.
    [0022] The use of the indefinite article "one" together with the term "understand" in the Claims and / or in the specific Report can mean the numeral "one", but it is also consistent with the meaning of "one or more", "when minus one ”and“ one or more than one ”.
    [0023] It is contemplated that any embodiment discussed in this document is implemented with respect to any method or composition of the invention and vice versa. In addition, the compositions and kits of the invention can be used to carry out methods of the invention.
    [0024] Throughout this specification, the term “about” is used to indicate that a value includes the standard error margin of the device or method used to determine the value.
    [0025] The use of the term "or" in the Claims is such that it means "and / or", unless explicitly stated otherwise to refer to alternatives or if the alternatives are excluded from each other, although the disclosure allows a definition that is refer to both exclusive and “and / or” alternatives. It is also contemplated that any item listed using the term "or" is also specifically excluded.
    [0026] As used in this specification and in the Claims, the words "understand" (and any derivation of understand, such as "comprising" "understand" and "understand") "possess" (and any derivation of possess, as "possessing" , “Own” and “own”), “include” (and any derivation of include, such as “including”, “includes” and “include”) or “contain” (and any derivation of contain, such as “containing”, “ contains ”and“ contain ”) are inclusive or open and do not exclude additional or unmentioned elements or method steps.
    [0027] Other objectives, characteristics and advantages of the present invention will be apparent from reading the detailed description below. However, it should be borne in mind that, although they indicate specific embodiments of the invention, the detailed description and specific examples are given for illustrative purposes only, since various changes and modifications within the scope and essence of the invention will be apparent to those skilled in the art. in the technique by reading this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
    [0028] The following drawings are part of this specification and are intended to better demonstrate certain aspects of the present invention. The invention can be better understood with reference to one or more of these drawings together with the detailed description of the specific embodiments presented in this document.
    [0029] FIG. 1 illustrates the production of the JX-594 strain of the recombinant vaccinia virus (pfu / cell) in various human cell lines. The cells were infected at a multiplicity of infection of 0.1 and, 72 hours after infection, the lysates were collected and the titer was determined in the U-2 OS cells.
    [0030] FIG. 2 illustrates the effect of the pH of the infection medium on the productivity of the JX-594 strain of recombinant vaccinia virus in adherent HeLa cells. The cells were infected at a multiplicity of infection of 0.1 in media with the indicated pH and, 72 hours after infection, the lysates were collected and the titer was determined in the U-2 OS cells. DETAILED DESCRIPTION OF THE INVENTION
    [0031] Vaccinia viruses are enveloped viruses that produce four infectious forms that differ in terms of the outer membrane: mature intracellular virion (IMV), intracellular enveloped virion (IEV), cell associated enveloped virion (CEV) and extracellular enveloped virion (EEV) . The EEV form is resistant to complement due to the incorporation of host complement activation regulators in the host cell to the external EEV membrane (Vanderplasschen et al, (1997) PNAS 95: 7,544 to 7,549). The ability to evade complement inactivation and the general ability to evade recognition by the host immune system are important characteristics for the effectiveness in systemic administration to patients.
    [0032] The plasma membrane of IMV incorporates several proteins of the host cell during its formation. Since IMV represents most of the infectious progeny, producer cells of the same species as patients targeted for oncolytic virotherapy may be advantageous for systemic diffusion within the patient since the viruses will be less immunogenic. These host cell proteins in the plasma membranes of IMV may also provide certain advantages in the infection process.
    [0033] Although IMV represents most of the infectious progeny, it has not been definitively established that the EEV form of the virus is not contained in cell culture preparations. Complement activation regulatory proteins of the same species as the targeted patients can play a fundamental role in inhibiting complement activation and, therefore, avoiding immune clearance by the host immune system. Accordingly, the inventors have developed a system for producing large quantities of the vaccinia virus with a human cell component.
    [0034] In the past, the vaccinia virus for use as a vaccine was produced from human wounds, however, later on, it started to be cultivated on the skin of calves, sheep and buffaloes in India, on the chorialantoic membrane of chick embryos or in embryonic fibroblasts of primitive cattle (Ellner, 1998). For global smallpox eradication initiatives, stocks of infection were produced through scarification of calves' skin (Fenner, 1977). Virus stocks were prepared from bovine lymph, this formula being the only licensed and available vaccine, known as DRYVAX® (Wyeth). The virus preparation was concentrated and lyophilized, which accounts for its stability (Fenner, 1977). It was produced at a concentration of at least 108 pfu / ml of lyophilized material. It was reconstituted in 50% glycerin and 0.25% phenol in sterile water for injection and administered to up to 400 vaccines intradermally using a bifurcated needle (Fenner 1977) with a dose of about 2.5 x 105 pfu; that is, at least 104 times less than the standard dose of 109 pfu (or more) of the vaccinia virus as an oncolytic virus.
    [0035] A prominent clinical candidate for the oncolytic vaccinia virus is the vaccinia virus with deactivated thymidine kinase, which expresses GM-CSF (Jennerex, JX-594). However, other variants and strains of the vaccinia virus can also be produced by the methods described in this document. For example, JX-963 is a recombinant vaccinia virus that, along with the removal of the TK gene, performs the additional deletion of the vaccinia growth factor (VGF) gene built into a Western Reserve vaccinia structure. This doubly-deleted vaccinia virus (vvDD) proved to be a tumor-specific virus with antitumor activity in animal models (McCart et al., 2001; Thorne et al., 2007; Naik et al., 2006). A second variant of vvDD is JX-929, which expresses the human somatostain receptor (SSTR2) in infected cells, facilitating the generation of molecular images after systemic administration using 111In pentatreotide (McCart et al. 2004). I. Cell Culture
    [0036] Viral production on a cell basis was examined in search of alternatives for vaccination with DRYVAX® and for the production of the vaccinia virus used in the field of viral immunotherapy and gene therapy. A second generation vaccinia virus strain grown on the simian renal cell line Vero called ACAM200 has been developed. In these studies, the virus was effectively purified from cell cultures. In addition, cancer vaccines that use the vaccinia virus together with tumor-specific antigens (ie, PSA, CEA) and immunostimulatory molecules (ie, B7.1, ICAM-1 and LFA-1) have been produced in cells and administered to patients in Phase I and II clinical trials (Li, et al., 2005; Guo and Bartlett, 2004). However, in both of the above cases, patients received local administration instead of systemic administration of viral preparations and generally received a fraction of the total dose of the virus needed for oncolytic efficiency in vivo.
    [0037] Compared to other viruses reported as candidates for oncolytic viruses, poxviruses, such as vaccinia, pose some specific challenges to large-scale manufacturing and purification. As one of the largest viruses in nature, poxviruses are visible by light microscopy and measure 200 to 400 nm in length (Moss, in Fields' virology BN Fields, DM Knipe, PM Howley, DE Griffin, Eds. (Lippincott Williams & Wilkins, Philadelphia, 2001) pp. 2,849 to 2,883). Due to its large size, the vaccinia virus cannot be sterilized by filtration and, therefore, any manufacturing process is typically closed.
    [0038] As its entire life cycle occurs within the cytoplasm of the host cell, most of the infected particles are not released into the cell medium, but remain within the infected cell, therefore, purification requires lysis of the infected cell and the purification of the viral particles to eliminate cell residue.
    [0039] In poxvirus morphogenesis, they acquire membranes by de novo membrane synthesis at the beginning of morphogenesis and, later, acquire new membranes from the host golgi (Moss, in Fields' virology BN Fields, DM Knipe, PM Howley, DE Griffin, Eds. (Lippincott Williams & Wilkins, Philadelphia, 2001) pp. 2,849 to 2,883).
    [0040] Current methods are for an adherent cell process developed to produce GMP-grade viruses for cancer testing. The adherent cell process can be performed in serum-free conditions or in the presence of serum. The productivity goals for the manufacturing platform described in this document are at least 30, 100, 125, 150, 200 or more pfu / cell - the estimated virus concentration per dose is about 109 pfu / ml.
    [0041] One embodiment of the invention is for procedures for the production of JX-594 with high titer in cell culture. Several human cell lines have been tested and HeLa, a human cervical cancer cell line, has been shown to support robust virus replication in adherent cultures. The present methods are not limited to HeLa cells, it is possible to use other adherent cells in the production process, but, currently, HeLa cells are considered the best cell line for the production of the vaccinia virus.
    [0042] The typical approach for assessing virus productivity is to perform a standard plaque assay, which involves a dilution series, followed by a period of 2 to 3 days waiting for plaques to form, and then calculating the title. As an alternative, a quantitative PCR approach (Q-PR) has been standardized, which allows to determine the number of viral genomes produced during infection. Amplification initiator oligonucleotides have been optimized, which allow quantifying the number of copies of the viral E9L gene in infected cells and correlating these results with the data from the standard plaque assay. The advantage of this approach is that it can be adapted to a 96-well format and data is obtainable in hours instead of days. Thus, the initial standard screening of media and supplements, the time to harvest and the influences of the concentration of the incoming virus can be quickly performed and analyzed in 96-well plates. After this initial screening, all positive results were confirmed with the classic plaque assay.
    [0043] Components found in serum-free commercial media have been tested for their effect on virus productivity. The HeLa S3 cell line was adapted for growth in suspension and, in our hands, these cells grew in a HeLa EX-CELL ™ serum-free suspension medium (SAFC Biosciences) as well as adherent cells grow in media with serum. However, as mentioned above, despite the excellent cell growth characteristics, the inventors observed quite unsatisfactory viral growth in this medium (less than one virus particle per infected cell). Although the exact formula of the HeLa EX-CELL ™ serum-free medium is copyrighted, a known component of these serum-free media is a sulfated polysaccharide, dextrate sulfate, apparently added to prevent cell clumping. However, dextran sulfate is also known to inhibit the replication of a wide variety of enveloped viruses, including retroviruses, herpesviruses, rhabdoviruses and aerenaviruses (De Clercq, 1993). There are about 20 mg / l dextran sulfate in the EX-CELL medium. Various concentrations of dextran sulfate were used and evaluated in the rapid test described above. Dextran sulfate was actually found to have a dose-dependent negative impact on virus productivity, a finding confirmed later using classic title assays. Although the removal of dextran sulfate does improve the virus productivity, it remains below the productivity usually obtained with adherent HeLa cells in media containing serum. Other components of the media that affect viral productivity include Pluronic F-68, a bifunctional copolymeric block surfactant generally non-toxic to cells, Tween-80, a commonly used detergent, and soy hydrolyzate. In certain aspects of the invention, the medium will lack one or more of dextran sulfate, Pluronic F-68, Tween-80 and / or soy protein hydrolyzate. Examples of some of these formulas for the medium and serum supplements are outlined in Table 1. Table 1. Formulas for culture media and supplements

    [0044] Since the vaccinia virus prefers cell-to-cell contact for its efficient diffusion, it is possible that suspension cell cultures are limited by cell transmission to unsatisfactory virus cells. In certain aspects, cell clumping is promoted, cells are grown in microcarriers or the adherent culture is sufficient for viral production.
    [0045] In a non-limiting example, the following conditions were used for the cultivation of JX-594, which consistently resulted in titers of more than 100 pfu / cell. The cells are placed in rotating bottles at 4 x 104 cells / cm2 and cultured for 2 days in culture. After that, the growth medium is removed and a fresh medium containing 104 pfu / ml of JX-594 is added. The cells are kept for another 60 to 70 hours, after which the cells are collected and the viruses are collected. This configuration can produce about 170 doses (109 pfu / dose) of JX-594. During this process, the inventors found that, unexpectedly, certain factors had a significant effect on the production of the virus. For example, the pH of the medium influenced the replication of JX-594. Viral production was optimized in a well-buffered medium at a pH of 7.3. Productivity from adherent cultures was significantly better than all experiments in suspension cultures.
    [0046] To guarantee the production of high quality viral preparations sufficiently free from contaminating products, several assays can be used for the analysis of the purity and / or quality of the virus. These tests will ensure that the viral preparations match other verified batches of the virus preparation and will guarantee the success of the process.

    [0047] In certain embodiments, the vaccinia virus can be isolated and purified. The method for purifying the vaccinia virus may include: a. loading a solid phase matrix with a vaccinia virus contained in a liquid phase; B. washing the matrix, and c. elute the vaccinia virus.
    [0048] In certain respects, the matrix may comprise a ligand that connects to vaccinia. The ligand can connect to the solid phase matrix by attaching or attaching to it. The interaction between the ligand and the virus forms a reversible complex, so the virus is reversibly retained in the matrix. In some respects, glycosaminoglycan (GAG) is used as a binder, in particular heparan sulfate or heparin, or a substance similar to GAG. As used herein, "glycosaminoglycans" (QAGs) are long, unbranched polysaccharides composed of a repeated disaccharide unit.
    [0049] The linker can be a biological molecule such as, for example, a peptide and / or a lectin and / or an antibody and / or, preferably, a carbohydrate. The binder can also comprise or be composed of sulfate. In another embodiment, the binder comprises one or more negatively charged sulfate groups.
    [0050] In certain respects, the linker is a hydrophobic molecule such as, for example, an aromatic phenyl group, a PPG group, a butyl group or a hexyl group.
    [0051] In one aspect, the method comprises purifying the vaccinia virus by hydrophobic interaction chromatography (HIC). In another embodiment, the method comprises purification of the vaccinia virus by HIC along with affinity chromatography. The use of HIC can provide large virus productions with large reductions in DNA and contaminating proteins. The level of DNA contamination can drop to 0.01% of the starting material and the level of protein contamination can drop to less than 0.1%.
    [0052] In some respects, sulfated cellulose can be used with HIC chromatography on a phenyl column to produce virus particles with high yields and high purity levels.
    [0053] In some respects, DNA is degraded or removed before or after purification of the vaccinia virus. DNA can be removed by nuclease treatment, such as treatment with Benzonase®. Nuclease treatment decreases the risk that vaccines or viral vectors contain intact oncogens or other functional DNA sequences.
    [0054] In related aspects, proteins are degraded or removed before or after purification of the vaccinia virus. Proteins can be removed by treatment with protease, such as treatment with TrypLE® Select. Preferably, a combination of nuclease treatment and protease treatment is used to decrease or eliminate contaminating DNA and HeLa proteins.
    [0055] The matrix can be a gel, bead, cavity, membrane, column etc. In a preferred embodiment of the invention, the solid phase is a membrane, in particular a cellulose membrane. A wide range of modified polymers capable of binding to the virus can be used. Examples of such polymers include cellulose derivatives (cellulose esters, cellulose hydrate, cellulose acetate, cellulose nitrate); agarose and its derivatives; polysacerides, such as chitin or chitosan; polyolefins (polypropylene); polysulfone; polyethersulfone; polystyrene; aromatic and aliphatic polyamides; polysulfonamides; halogenated polymers (polyvinyl chloride, polyvinyl fluoride, polyvinylidene fluoride); polyesters; acrylonitrile homopolymers and copolymers.
    [0056] The vaccinia virus can be purified under aseptic conditions in order to obtain an active, stable and highly pure virus preparation. Vaccinia viruses can be native or recombinant.
    [0057] As used herein, “contaminants” includes any unwanted substances that may be derived from the host cells used for the growth of the virus (for example, DNA or proteins in the host cell) or any additives used during the manufacturing process, including upstream (for example, gentamicin) and downstream (for example, benzonase).
    [0058] As used herein, the "industrial scale" or "large scale" manufacture of the vaccinia virus or the recombinant vaccinia virus comprises methods capable of providing a minimum of 50,000 doses of 1.0 x 108 virus particles ( minimum total of 5.0 x 1012 virus particles) per batch (production cycle).
    [0059] As used in this document, the "Purity" of the vaccinia virus preparation or vaccine is investigated for the content of DNA, protein, benzonase and gentamicin impurities. Purity is expressed by specific impurity, which is the amount of each impurity per dose (for example, ng of DNA / dose).
    [0060] As used in this document, “Stability” means a measure of how the quality of the virus preparation (bulk drug substance (BDS) or final drug product (FDP)) varies over time under the influence of a series of environmental factors, such as temperature, humidity and lighting, and which establishes a time interval for the realization of new tests, in the case of a BDS, or the life span, in the case of a FDP, guaranteeing the storage conditions recommended.
    [0061] The ligand allows the elution of the vaccinia virus bound in such mild conditions in which the vaccinia virus retains its biological activity totally. This means that the virus is infectious. The infectivity of the vaccinia virus can be preserved during purification in such a way that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the TCID50 (infectious dose in culture median tissue volume) is maintained during purification. Preferably, at least 30% of the initial TCID50 is maintained during purification.
    [0062] In one embodiment, the solid phase matrix is a gel or membrane with a pore size of 0.25 μm, preferably greater than 0.25 μm, more preferably, 1.0 to 3.0 μm, with a linear flow rate under actual purification conditions from 10 cm / min to 20 cm. The pore size of the matrix can be from 0.25 to 0.5 μm, from 0.5 to 0.75 μm, from 0.75 to 1.0 μm, from 1.0 to 2.0 μm, from 2 , 0 to 3.0 μm or greater than 3.0 μm.
    [0063] The loading of the solid phase with a binder can be carried out by a batch, column or membrane approach.
    [0064] The membrane approach can bring some benefits for the purification of large viruses such as vaccinia viruses. The large pore sizes and the availability of the binder on the membrane surface allow high binding capacities even for large viral particles.
    [0065] In one embodiment, the virus binds to the ligand in ammonium sulfate, for example, at 0.2 M, 0.4 M, 0.6 M, 0.8 M, 1.0 M, 1, 2 M, 1.4 M, 1.6 M, 1.8 M or 2.0 M.
    [0066] When the binding of vaccinia viruses or recombinant viruses with the ligand or matrix has progressed sufficiently, contaminants in the host cell (especially the DNA and proteins in the host cell) that remain in the liquid phase can be removed by washing matrix to which the vaccinia virus has attached itself with a suitable washing medium. In one aspect, the matrix is washed with 0.2 M, 0.4 M, 0.6 M, 0.6 M, 1.0 M, 1.0 M, 1.2 M, 1.4 M, 1 M ammonium sulfate , 6 M, 1.8 M or 2.0 M.
    [0067] Vaccinia viruses or linked recombinant viruses can be eluted from the matrix. The elution of the captured vaccinia viruses can be carried out by agents that specifically break the specific interaction between the ligand or matrix and the vaccinia virus or by agents that do not specifically break the electrostatic interaction between the ligand or matrix and the proteins on the surface. In one aspect, the agent is ammonium sulfate. In another aspect, the vaccinia virus can be eluted with GAG or a GAG-like ligand. In another aspect, the agent is sodium chloride, most preferably, with an increasing concentration gradient of NaCl ranging from 0.15 M to 2.0 M.
    [0068] Before loading into the matrix, a pretreatment of the virus suspension can be performed, specifically to remove contaminants from the vaccinia virus in the liquid phase culture. Pre-treatment can be one or more of the steps to follow separately or together: homogenization of host cells, ultrasound treatment, freeze-thaw cycles, hypo-osmotic lysis, high pressure treatment, centrifugation, filtration, treatment with nuclease, protease treatment, cation exchange, selective precipitation.
    [0069] Depending on the agent used for eluting the vaccinia virus or recombinant virus, a post-treatment can be performed in order to enhance the purity of the virus preparation. Post-treatment may include ultrafiltration or diafiltration for the additional removal of specific or non-specific impurities and / or agents used for elution.
    [0070] Normally, the pH value increases after the elution of the virus, especially for a pH value of up to 9 or more, especially for pH 7.5, 7.6, 7.8, 8.0, 8 , 2, 8.4, 8.5, 8.6, 8.8, 9.0, 9.2, 9.4, 9.5, 9.6, 9.8, 10.0, 10.2 , 10.4 or 10.5.
    [0071] The practice of the invention employs techniques of molecular biology, protein analysis and microbiology, which are known to those skilled in the art. These techniques are explained in full, for example, in Ausubel et al. 1995, eds, Current Protocols in Molecular Biology, John Wiley & Sons, New York. II. EXAMPLES
    [0072] The following examples are given for the purpose of illustrating various embodiments of the invention and are not intended to limit the present invention in any way whatsoever. Those skilled in the art will readily recognize that the present invention is quite adequate to achieve the objectives and achieve the purposes and advantages mentioned, as well as the objectives, purposes and advantages implied in this document. The present examples, together with the methods described in this document, are currently representative of the preferred embodiments, are exemplary and are not intended as limitations on the scope of the invention. Changes and other uses within the essence of the invention, as defined by the scope of the Claims, will occur to those skilled in the art.
    [0073] Several human cell lines were tested for the production of JX-594. In short, HeLa cells, A549 cells, MCF-7 cells, M14 cells, OS U-2 cells and suspended and adhering HeLa S3 cells were infected with JX-594 at a multiplicity of infection of 0.1 and lysates were collected 72 hours after infection and the virus titer was determined in OS U-2 cells. Figure 1 shows the results. To everyone's surprise, good results were obtained in adherent HeLa cells.
    [0074] Various parameters were examined in an attempt to optimize the production of the virus in adherent HeLa cells. The pH of the infection medium was found to have an unexpectedly significant influence on viral production (see Fig. 2). The production of JX-594 increased from about 12 pfu / cell, in an infection medium with pH 7.1, to about 60 pfu / cell, in an infection medium with pH 7.3 under conditions, outside this, identical. When the pH of the infection medium was below 7.1, viral production practically did not exist. Further improvements in viral production were observed when the multiplicity of infection was within the range of 0.005 and 0.05, especially between 0.01 and 0.03, and when the temperature was maintained between 36 ° C and 37.5 ° C during and after infection. The use of plastic rotating bottles as an adherent surface also contributed to the high production of the virus.
    [0075] It was also determined that the presence and amount of serum in the medium used to grow the adherent HeLa cells influenced the production of the virus. The ideal production was obtained when the medium contained 10% FBS - the reduction in the amount of serum in the medium resulted in a corresponding drop in the production of the virus. However, the optimization of other parameters (for example, pH, multiplicity of infection, adherent surface) neutralizes the drop in production attributable to lower serum concentrations up to a certain level.
    [0076] The presence and quantity of serum in the infection medium, however, did not significantly influence the production of the virus. Therefore, in order to obtain a surprisingly good production of the vaccinia virus in adherent HeLa cells according to the present invention, the infection medium can comprise or be substantially serum-free.
    [0077] Following is a flowchart outlining a non-limiting example of the optimized upstream process:



    [0078] Defrosting of the ampoules and expansion of the inoculum. The upstream process is initiated by defrosting a sufficient number of ampoules from a cell bank. The resulting cell suspension is incubated in a polystyrene cell culture vessel (636 cm) (Nunc Nalgene). The cells are cultured using a cell culture protocol substantially applied to a certain confluence (about 80% to 95%) determined by visual evaluation.
    [0079] In Passage 1, the medium is removed and the cells are rinsed with PBS and detached from the culture vessel. Viable cells are counted using a Vi-CELL XR (Beckman Coulter) or hemocytometer and then seeded at a density of 1 x 104 to 6 x 104 cells / cm2 in four polystyrene culture vessels (total area of 2,544 cm2 ), each containing 200 ml of Medium 100. The culture is incubated at 37 ° C.
    [0080] If the crop is sown at 2 x 104, the expected date for the next pass is 4 days after sowing. If the crop is sown at 4 x 104, the expected date for the next pass is 3 days after sowing. The process is defined to provide about 1 x 105 cells / cm2 in each pass.
    [0081] Expansion of Cell Culture in Rotating Bottles. On the expected date of Passage 2, the medium is removed and the cells are rinsed with PBS and detached from the polystyrene culture vessels.
    [0082] The cells are seeded at a density of 1 x 104 to 6 x 104 cells / cm2 in two polystyrene rotary bottles (GRs, Cellon) with Extended Surface (SE) in an RC-40 rotary bottle apparatus (Synthecon, total surface area of 8,400 cm2). Each bottle is grown containing 900 ml of Medium 100 and the cell culture is incubated at 37 ° C in an RC-40 Incubator (Sanyo).
    [0083] On the scheduled date of Passage 3, the cell culture medium is removed and the cells are rinsed with PBS and detached. The cell suspension is collected from bottles with extended surface, mixed with Medium 100 to 1: 1 and viable cells are counted. The approximate amount of the cell suspension is then sown in 4 SE grs (total surface area of 16,800 cm2) from 1 x 104 to 6 x 104 cells / cm2. The cell culture is incubated at 37 ° C with 900 ml of medium in each bottle.
    [0084] During Passage 4, the cell culture is transferred to 10 GRs (total surface area of 42,000 cm2) and, during Passage 5, the cell culture is expanded to 20 GRs (total surface area of 84,000 cm2). Passage 6 is the first critical step in the JX-594 production process, as the cell culture is expanded to 40 GRs (total surface area of 168,000 cm2). The sowing density of 40 GRs is fundamental for the production of JX-594 and has a defined target of 4 x 104 cells / cm2. Samples of the cell suspension and conditioned medium are collected during Passage 6 for DNA testing and testing for accidental agents. The temperature of the cell culture during the sowing period in production is maintained at 37 ° C.
    [0085] Infection and Production of JX-594. The time interval between sowing the 40 GRs to infection with ampoules of JX-594 from the Master Virus Bank or the Working Virus Bank is expected to last 72 hours. On the day of infection, the medium is removed from the cell culture and replaced with fresh Medium 100 containing 1 x 104 pfu / ml of the JX-594 virus. The title of the infection medium is an extremely important procedural parameter and has a defined target of 1 x 104 pfu / ml. Cell density is not counted at the time of infection, but is expected to be 1 x 105 cells / cm2 based on history of passage. The duration of the infection is controlled within the range of 40 to 80 hours and is preferably about 44 hours. The temperature of the cell culture during infection is an extremely important procedural parameter and is controlled to remain at 37 ° C.
    [0086] Virus titers of up to or even more than 100 pfu / cell are usually obtained according to the process.
    [0087] Process Downstream of JX-594. Classical methods for small-scale preparation of poxvirus from cells include hypotonic lysis, followed by freeze-thaw cycles and sonication. The present invention has found that both freezing / thawing and sonication are largely unnecessary steps, which generally result in lower virus titers. Therefore, downstream processing of JX-594 makes use of nuclease treatment (Benzonase), to digest HeLa DNA, and protease treatment (TrypLE ™ Select), to digest HeLa proteins, followed by tangential flow filtration (TFF ). Treatment of the crude culture (containing virus and cell residue in hypotonic lysis buffer) with benzonase resulted in significantly less contaminating host DNA (HeLa) in the preparation (see Fig. 3). Thus, nuclease treatment combined with TFF provides a significantly purer virus preparation with significantly less contaminating host proteins and DNA in the preparation while maintaining the infectious virus titer. In particular, the reduction in DNA contamination from 40 μg DNA / dose to 4 μg DNA / dose and the reduction in protein contamination from 12 mg protein / dose to 4 mg protein / dose (dose = 1 x 109 pfu of JX-594) were obtained using a combination of nuclease / protease and TFF treatment. Further reductions can be obtained using one or more chromatographic steps. In one aspect, the one or more chromatographic steps comprise ion exchange chromatography. In another aspect, the one or more chromatographic steps comprise pseudo-affinity chromatography, preferably based on heparin (or molecules similar to heparin) or sulfated cellulose, taking advantage of the heparin binding capacity of the vaccinia virus A47L protein. In yet another aspect, the one or more chromatographic steps comprise membrane absorption chromatography, for example, membrane affinity chromatography. Preferred membranes will have a microporous structure with a pore size of at least 3 μM. REFERENCES
    [0088] The following references, insofar as they indicate exemplary procedures and other details complementary to those defined in this document, are specifically incorporated into this document by reference. 1. B. Moss, in Fields' virology B. N. Fields, D. M. Knipe, P. M. Howley, D. E. Griffin, Eds. (Lippincott Williams & Wilkins, Philadelphia, 2001) pp. 2,849 to 2,883. 2. J. B. Johnston, G. McFadden, Cell Microbiol 6, 695 (August 2004). 3. C. J. Breitbach et al., Mol Ther 15, 1686 (September 2007). 4. J. H. Kim et al., Mol Ther 14, 361 (September 2006). 5. R. M. Buller, G. L. Smith, K. Cremer, A. L. Notkins, B. Moss, Nature 317, 813 (October 31 to November 6, 1985). 6. R. M. Buller, G. J. Palumbo, Microbiol Rev 55, 80 (March 1991). 7. M. Hengstschlager et al., J Biol Chem 269, 13836 (May 13, 1994). 8. T. C. Liu, E. Galanis, D. Kirn, Nat Clin Pract Oncol 4, 101 (February 2007). 9. C. Y. Li, Q. Huang, H. F. Kung, Cell Mol Immunol 2, 81 (April 2005). 10. J. W. Hodge et al., Front Biosci 11, 788 (2006). 11. P. D. Ellner, Infection 26, 263 (September to October 1998). 12. F. Fenner, Prog Med Virol 23, 1 (1977). 13. A. W. Artenstein et al., Vaccine 23, 3301 (May 9, 2005). 14. T. P. Monath et al., Int J Infect Dis 8 Suppl 2, S31 (October 2004). 15. Z. S. Guo, D. L. Bartlett, Expert Opin Biol Ther 4, 901 (June 2004). 16. E. De Clercq, Adv Virus Res 42, 1 (1993). 17. L. A. Palomares, M. Gonzalez, O. T. Ramirez, Enzyme Microb Technol 26, 324 (March 1, 2000). 18. B. Horowitz, M. E. Wiebe, A. Lippin, M. H. Stryker, Transfusion 25, 516 (November to December 1985). 19. M. P. Piet et al., Transfusion 30, 591 (September 1990). 20. B.Chun, Y. Kwon Lee, W. G. Bang, N. Chung, Biotechnol Lett 27, 243 (February 2005). 21. B. H. Chun, Y. K. Lee, B. C. Lee, N. Chung, Biotechnol Lett 26, 807 (May 2004). 22. J. A. McCart et al., Cancer Res 61, 8751 (December 15, 2001). 23. S. H. Thorne et al., J Clin Invest 117, 3350 (November 2007). 24. A. M. Naik et al., Hum Gene Ther 17, 31 (January 2006). 25. J. A. McCart et al., Mol Ther 10, 553 (September 2004). 26. A. Piccini, E. Paoletti, Adv Virus Res 34, 43 (1988). 27. C. S. Chung, J. C. Hsiao, Y. S. Chang, W. Chang, J Virol 72, 1577 (February 1998). 28. A. Karger, B. Bettin, H. Granzow, T. C. Mettenleiter, J Virol Methods 70, 219 (February 1998).
    权利要求:
    Claims (16)
    [0001]
    1. Method for Producing Vaccinia Virus, characterized by comprising: (a) infecting HeLa cells adhered to a cell culture bioreactor surface with a vaccinia virus of the Western Reserve strain by placing HeLa cells in contact with the vaccinia virus to a multiplicity of infection (moi) between 0.005 and 1.0 plaque forming units (pfu) / cell; (b) culturing the infected cells in an infection medium having a pH of 7.2 to 7.6 at a temperature of 30 ° C to 40 ° C; and (c) harvesting the vaccinia virus from the culture, through which at least 50 pfu of vaccinia virus is produced per HeLa cell, calculated as
    [0002]
    2. Method for Producing Vaccinia Virus according to Claim 1, characterized in that the surface of the cell culture bioreactor comprises a cumulative culture area of at least 168,000 cm2.
    [0003]
    3. Method for Producing Vaccinia Virus according to Claim 1, characterized by the fact that the pH of the infection medium is 7.2 to 7.4.
    [0004]
    4. Method for Producing Vaccinia Virus according to Claim 3, characterized by the fact that the pH of the infection medium is 7.3.
    [0005]
    5. Method for Producing Vaccinia Virus according to Claim 1, characterized by the fact that m.o.i. from step (a) is 0.01 pfu / cell to 0.05 pfu / cell.
    [0006]
    6. Method for Producing Vaccinia Virus according to Claim 5, characterized by the fact that m.o.i. from step (a) is 0.02 pfu / cell to 0.03 pfu / cell.
    [0007]
    7. Method for Producing Vaccinia Virus according to Claim 1, characterized by the fact that steps (a) and (b) are carried out at a temperature of 32 ° C to 37.5 ° C.
    [0008]
    8. Method for Producing Vaccinia Virus according to Claim 7, characterized by the fact that steps (a) and (b) are performed at a temperature of 32 ° C, 33 ° C, 34 ° C, 35 ° C , 36 ° C or 37 ° C.
    [0009]
    9. Method for Producing Vaccinia Virus according to Claim 1, characterized by the fact that step (b) is carried out for a period of 40-80 hours.
    [0010]
    10. Method for Producing Vaccinia Virus according to Claim 9, characterized by the fact that step (b) is carried out for a period of 44 hours.
    [0011]
    11. Method for Producing Vaccinia Virus according to Claim 1, characterized in that at least 75 pfu of vaccinia virus per HeLa cell is produced calculated as the total virus yield (pfu) / total number of viable HeLa cells in step (a).
    [0012]
    12. Method for Producing Vaccinia Virus according to Claim 1, characterized in that the medium comprises fetal bovine serum in an amount equal to or less than 10% by volume of the medium.
    [0013]
    13. Method for Producing Vaccinia Virus according to Claim 1, characterized in that the medium no longer comprises serum.
    [0014]
    14. Method for Producing Vaccinia Virus according to Claim 1, characterized by the fact that the harvested virus is subjected to one or more purification steps, wherein said one or more purification steps comprise treatment of the harvested virus with a nuclease for removing nucleic acids from HeLa cells and / or a protease to remove proteins from HeLa cells and / or a tangential flow filtration step and / or a heparin affinity chromatography step and / or an absorption chromatography step membrane.
    [0015]
    15. Method for Producing Vaccinia Virus according to Claim 1, characterized in that the vaccinia virus is a recombinant vaccinia virus, which lacks a functional thymidine kinase gene and / or which lacks a growth factor gene of functional vaccine and / or encoding a granulocyte macrophage colony stimulating factor polypeptide.
    [0016]
    16. Method for Producing Vaccinia Virus according to Claim 13, characterized in that the vaccinia virus lacks a functional thymidine kinase gene and lacks a functional vaccinia growth factor gene.
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    同族专利:
    公开号 | 公开日
    KR20140058601A|2014-05-14|
    US20170298324A1|2017-10-19|
    BR112014000787A2|2017-07-11|
    WO2013022764A1|2013-02-14|
    US10767166B2|2020-09-08|
    AU2012294606B2|2016-10-06|
    CN103732236A|2014-04-16|
    JP2014521353A|2014-08-28|
    US10202581B2|2019-02-12|
    CA2841831A1|2013-02-14|
    US9719105B2|2017-08-01|
    CN103732236B|2017-09-15|
    EP2739293B1|2020-06-10|
    KR102022952B1|2019-09-20|
    DK2739293T3|2020-08-24|
    ES2813413T3|2021-03-23|
    PL2739293T3|2020-11-16|
    CA2841831C|2019-12-31|
    EP2739293A1|2014-06-11|
    US20140162342A1|2014-06-12|
    JP6243333B2|2017-12-06|
    US20190169578A1|2019-06-06|
    AU2012294606A1|2014-01-23|
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    法律状态:
    2018-01-09| B25D| Requested change of name of applicant approved|Owner name: SILLAJEN BIOTHERAPEUTICS, INC. (US) |
    2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-12-03| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|
    2019-12-10| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-10-06| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|
    2021-02-09| B09A| Decision: intention to grant|
    2021-04-13| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 03/08/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US201161515724P| true| 2011-08-05|2011-08-05|
    US61/515,724|2011-08-05|
    PCT/US2012/049550|WO2013022764A1|2011-08-05|2012-08-03|Methods and compositions for production of vaccina virus|
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