Patent Grant
10517943
This application is a U.S. National Stage Application pursuant to 35 U.S.C. § 371 of International Patent Application PCT/EP2015/078239, filed on Dec. 1, 2015, and published as WO 2016/087457 on Jun. 9, 2016, which claims priority to European Patent Application 14306930.0, filed on Dec. 1, 2014, all of which are incorporated herein by reference in their entireties for all purposes.
The present invention is in the field of stable liquid formulations intended for liquid storage of poxviruses, in particular vaccinia viruses. It relates to liquid formulations comprising a) a poxvirus, in particular a vaccinia virus, b) a pharmaceutically acceptable buffer, c) a monovalent salt, d) a pharmaceutically acceptable disaccharide or sugar alcohol, and e) a pharmaceutically acceptable chelating agent, wherein the pH of the formulation is comprised between 6.5 and 8.5.
Poxviruses are complex enveloped viruses having a diameter comprised between 200 and 300 nm that distinguish them principally by their unusual morphology, their large DNA genome and their cytoplasmic site of replication. The genome of several members of Orthopoxviruses, including two strains of vaccinia virus (VV): the Copenhagen Vaccinia Virus strain (GOEBEL et al., 1990, Virol. 179, 247-266 and 517-563; JOHNSON et al., 1993, Virol. 196, 381-401), the Wyeth strain (OSBORNE J D et al. Vaccine. 2007 Dec. 17; 25(52):8807-32) and the modified Vaccinia Virus Ankara (MVA) strain (ANTOINE et al., 1998, Virol. 244:365-396), have been mapped and sequenced. VV has a double-stranded DNA genome of about 192 kb coding for about 200 proteins of which approximately 100 are involved in virus assembly. MVA is a highly attenuated Vaccinia Virus strain generated by more than 500 serial passages of the Ankara strain of Vaccinia Virus on chicken embryo fibroblasts (MAYR et al., 1975, Infection 3:6-16). The MVA virus was deposited at Collection Nationale de Cultures de Microorganismes (CNCM) under depositary N° I-721. Determination of the complete sequence of the MVA genome and comparison with the Copenhagen VV genome allow the precise identification of the alterations which occurred in the viral genome and the definition of seven deletions (I to VII) and numerous mutations leading to fragmented ORFs (Open Reading Frame) (ANTOINE et al., 1998, Virology 244:365-396).
MVA is used as a prophylactic vaccine against smallpox and recombinant MVA is currently the subject of many preclinical and clinical studies for prophylactic and therapeutic vaccination against many types of targets, including cancer (melanoma, non-small cell lung carcinoma, renal cell carcinoma, prostate cancer, colorectal cancer, notably), viral (hepatitis B or C, HIV notably), bacterial (tuberculosis notably) and parasitic diseases (malaria notably) (see GOMEZ et al. Current Gene Therapy, 2008, 8:97-120).
Oncolytic vaccinia viruses are also under preclinical and clinical development (see KIRN et al. Nat Rev Cancer, 2009 January, 9(1):64-71).
In both cases, a live vaccinia virus is used.
A live vaccinia virus prophylactic or therapeutic vaccine is generally not administered to the patient just after production and purification, and thus needs to be stored for days, weeks or even months, without losing its potency.
Like all live viruses, live vaccinia viruses have natural instability, which is further increased by the fact that vaccinia virus is an enveloped virus (enveloped viruses are known to be less stable than non-enveloped viruses, see BURKE C J et al. Crit Rev Ther Drug Carrier Syst. 1999, 16(1):1-83; and REXROAD et al. Cell Preservation Technology. June 2002, 1(2):91-104), has a big size (brick shape of 200 to 300 nm), a large genome and is known to be particularly sensitive to UV damage, see LYTLE et al. J. Virol. 2005, 79(22):14244). Moreover, vaccinia virus envelop is even more complex than that of other enveloped viruses. Stabilizing vaccinia virus is thus particularly challenging. Attempts to stabilize vaccinia virus have been made. In most cases, a freeze-dried formulation has been proposed (BURKE C J et al. Crit Rev Ther Drug Carrier Syst. 1999, 16(1):1-83). Indeed, while the performance of freeze-drying may induce some loss of viral titer, once freeze-dried, low temperature and absence of movement and interaction between compounds in freeze-dried state make freeze-dried viruses generally more stable than viruses in liquid state. For instance, EP1418942 discloses vaccinia virus formulations for freeze-drying comprising a substantially pure vaccinia virus, a disaccharide, a pharmaceutically acceptable polymer and a buffer that is not a phosphate buffer. WO2014/053571 discloses other freeze-dried MVA formulations comprising polyvinylpyrrolidone (PVP) or derivatives thereof, at least one sugar (in particular sucrose), at least two different amino acids (in particular sodium glutamate and L-arginine), at least two pharmaceutical acceptable salts (in particular NaCl, Na2HPO4 and KH2PO4), wherein at least one of said salts is a phosphate salt and, optionally a pharmaceutical acceptable buffer (in particular Tris).
However, freeze-drying is expensive, needs specific equipment and freeze-dried formulations need to be reconstituted before administration. Moreover, freeze-drying involves a freezing step that may lead to some virus aggregation, in particular at high virus titers, which is not suitable for injectable administration. It would thus be very useful to have stable liquid vaccinia virus formulations available.
Previous attempts to stabilize vaccinia virus in the liquid state have not been very successful, since log loss superior to 1 log10 after less than 1 hour at 50° C. were observed in most cases (see BURKE C J et al. Crit Rev Ther Drug Carrier Syst. 1999, 16(1):1-83).
Evans et al disclosed stable liquid adenovirus (non-enveloped DNA virus) formulations buffered between pH 6 and pH 8, comprising a salt (generally NaCl), a sugar (sucrose in most cases), an inhibitor of free radical oxidation (notably EDTA, ethanol or an EDTA/ethanol combination), a non-ionic surfactant and divalent salts (see EVANS et al. J Pharm Sci. 2004 October, 93(10):2458-75; and U.S. Pat. No. 7,456,009). Preferred formulations generally also comprise histidine. Parameters identified as essentials for stability include the presence of an inhibitor of free radical oxidation (in particular EDTA, ethanol, an EDTA/ethanol combination, and/or histidine), and presence of a non-ionic surfactant. The presence of divalent salts is also identified as important for increasing adenovirus stability.
The usefulness of non-ionic surfactants for stabilization purpose has also been documented for papilloma virus (see SHI et al. J Pharm Sci. 2005 July, 94(7):1538-51). US2007/0161085 tested various liquid formulations for stabilization of influenza virus (enveloped RNA virus). Most stable formulations included arginine and gelatin. In this study, EDTA was shown to have no effect on influenza virus stability. A low amount of surfactant, in addition to arginine and gelatin, was found to be beneficial.
U.S. Pat. No. 7,914,979 relates to formulation for stabilization of enveloped Newcastle disease virus, comprising a non-reducing saccharide such as sucrose. Preferred compositions also contain an amino acid selected from lysine and arginine. In contrast, EDTA is indicated to have a negative effect on stability and is preferably absent from the formulation.
In WO2014/029702, various types of formulations have been tested for stabilization of four canine viruses: two small and medium non-enveloped viruses (canine parvovirus and canine adenovirus type 2) and two enveloped viruses of the paramyxoviruses family (canine distemper virus and canine parainfluenza virus). Results show that enveloped viruses are more difficult to stabilize than non-enveloped viruses, and that the optimal formulation significantly varies between viruses, even for two enveloped viruses of the same paramyxoviruses family (canine distemper virus and canine parainfluenza virus). In addition, it is indicated in Example 1 that while sucrose—in particular at a concentration of 17-25%—and amino acids (such as arginine and methionine), are efficient stabilizers, free radical scavengers (such as EDTA) do not significantly change the stability profile, although they might somewhat contribute to the stability.
The above description of prior art clearly illustrates that designing a stable liquid formulation for a particular virus is a difficult task, since many stabilizers candidates are known in the art and since their stabilizing effect greatly varies depending on the specific virus to be stabilized. In addition, as explained above, due to its enveloped nature, its large size and its DNA genome, vaccinia virus is particularly difficult to stabilize, notably in the liquid state.
In the context of the present invention, the inventors identified liquid formulations suitable for maintaining stability of vaccinia virus in the liquid state, at about 5° C. (i.e. 5° C.±3° C.) or more. Essential elements of such formulations are the presence of a pharmaceutically acceptable buffer, a monovalent salt, a pharmaceutically acceptable disaccharide or sugar alcohol, a pharmaceutically acceptable chelating agent; and a pH between 6.5 and 8.5. The additional presence of a C2-C3 alcohol further improves stability of the liquid formulations.
In a first aspect, the present invention thus relates to a liquid formulation comprising, consisting essentially of, or consisting of:
The inventors also surprisingly found that the presence of a chelating agent such as EDTA protects vaccinia virus against UV damage. The present invention thus also relates to the use of a chelating agent for stabilizing a poxvirus, in particular a vaccinia virus against UV damage.
Stable Liquid Formulation
As explained above, the inventors identified liquid formulations suitable for maintaining stability of a poxvirus, in particular a vaccinia virus in the liquid state, at about +5° C. or more. Such formulations should comprise a pharmaceutically acceptable buffer, a monovalent salt, a pharmaceutically acceptable disaccharide or sugar alcohol, a pharmaceutically acceptable chelating agent; and a pH between 6.5 and 8.5.
The present invention thus relates to a liquid formulation comprising, consisting essentially of, or consisting of:
The liquid formulation according to the invention is an aqueous formulation.
“Comprising” and “comprise(s)” are intended to mean that the materials, products, formulations, compositions and methods include the referenced components or steps, but not excluding others. “Consisting essentially of” or “consist(s) essentially of”, when used to define products, formulations, compositions and methods, shall mean excluding other components or steps of any essential significance. Thus, for example, a formulation consisting essentially of the recited components would not exclude trace contaminants and pharmaceutically acceptable carriers. “Consisting of” or “consist(s) of” shall mean excluding more than trace elements of other components or steps.
Stability
Formulations according to the invention are liquid, which has the advantage that there is no need for costly and time-consuming freeze-drying processes, and that they may be administered directly, without a need for previous reconstitution.
Stabilization of biological material such as viruses in particular in the liquid state, is not straightforward due to the ability of viruses to interact with all components of the formulation, as well as with the container, which increases risks of viral titer loss. However, the present invention provides virus formulations that are stable in the liquid state and permit to overcome these difficulties. In particular:
Infectious vaccinia virus titers at day 0 and at any following date may be determined either by measuring the number of Infectious Genomes (IG) per mL (IG/mL) or by using a plaque assay on BHK-21 cells (infectious poxvirus, in particular vaccinia virus, titer is then expressed in Plaque Forming Units (PFU) per mL (PFU/mL). Measure of the number of infectious genomes per mL (IG/mL) may be preferred, since this method is more rapid and more precise. Detailed protocols for measuring the number of infectious genomes (IG) per mL (IG/mL) or for plaque assay on BKH-21 cells are disclosed in Examples.
Poxvirus
Formulations according to the invention comprise a poxvirus.
Said poxvirus may be selected from the following families: Orthopoxvirus (e.g. vaccinia virus, Cowpox virus), Parapoxvirus (e.g. Bovine papular stomatitis virus, Orf virus, Pseudocowpox virus), Suipoxvirus (e.g. Swinepox virus), Yatapoxvirus (e.g. Yaba-like disease virus), Avipoxvirus (e.g. Fowlpox virus and Canarypoxvirus) and Leporipoxvirux (Myxoma virus). Said poxvirus may particularly be selected from Orthopoxviruses (e.g. vaccinia virus, Cowpox virus) and Parapoxviruses (e.g. Bovine papular stomatitis virus, Orf virus, Pseudocowpox virus). Particularly preferred poxviruses are vaccinia virus and pseudocowpox virus.
In a preferred embodiment, said poxvirus is an Orthopoxvirus, and more preferably a vaccinia virus (VV).
The inventors observed that vaccinia virus stability in the liquid state increases with the concentration of vaccinia virus particles in the formulation (see Example 4). As a result, the poxvirus (in particular vaccinia virus and notably MVA, Wyeth or Copenhagen vaccinia virus) is preferably present in liquid formulations according to the invention at a titer of at least 107 PFU/mL, preferably at least 2·107 PFU/mL, at least 3·107 PFU/mL, at least 4·107 PFU/mL, more preferably at least 5·107 PFU/mL, or even at least 108 PFU/mL. Since stability of poxvirus (in particular vaccinia virus) increases with the concentration of poxvirus (in particular vaccinia virus) particles in the formulation, there is no particular restriction concerning the maximal concentration of poxvirus (in particular vaccinia virus) particles in the formulation. However, for practical reasons, poxvirus (in particular vaccinia virus) will generally be comprised in the liquid formulations according to the invention at a titer of at most 1012 PFU/mL, at most 1011 PFU/mL, or even at most 1010 PFU/mL. In particular, poxvirus (in particular vaccinia virus) may be comprised in the liquid formulations according to the invention at a titer of 107 PFU/mL to 1012 PFU/mL, 107 PFU/mL to 1011 PFU/mL, 107 PFU/mL to 1010 PFU/mL, 107 PFU/mL to 5·109 PFU/mL, 107 PFU/mL to 109 PFU/mL, 107 PFU/mL to 5·108 PFU/mL, 107 PFU/mL to 108 PFU/mL, 2·107 PFU/mL to 1012 PFU/mL, 2·107 PFU/mL to 1011 PFU/mL, 2·107 PFU/mL to 1010 PFU/mL, 2·107 PFU/mL to 5·109 PFU/mL, 2·107 PFU/mL to 109 PFU/mL, 2·107 PFU/mL to 5·108 PFU/mL, 2·107 PFU/mL to 108 PFU/mL, 3·107 PFU/mL to 1012 PFU/mL, 3·107 PFU/mL to 1011 PFU/mL, 3·107 PFU/mL to 1010 PFU/mL, 3·107 PFU/mL to 5·109 PFU/mL, 3·107 PFU/mL to 109 PFU/mL, 3·107 PFU/mL to 5·108 PFU/mL, 3·107 PFU/mL to 108 PFU/mL, 4·107 PFU/mL to 1012 PFU/mL, 4·107 PFU/mL to 1011 PFU/mL, 4·107 PFU/mL to 1010 PFU/mL, 4·107 PFU/mL to 5·109 PFU/mL, 4·107 PFU/mL to 109 PFU/mL, 4·107 PFU/mL to 5·108 PFU/mL, 4·107 PFU/mL to 108 PFU/mL, 5·107 PFU/mL to 1012 PFU/mL, 5·107 PFU/mL to 1011 PFU/mL, 5·107 PFU/mL to 1010 PFU/mL, in particular 5·107 PFU/mL to 5·109 PFU/mL, 5·107 PFU/mL to 109 PFU/mL, 5·107 PFU/mL to 5·108 PFU/mL, 5·107 PFU/mL to 108 PFU/mL, 108 PFU/mL to 1012 PFU/mL, 108 PFU/mL to 1011 PFU/mL, 108 PFU/mL to 1010 PFU/mL, 108 PFU/mL to 5·109 PFU/mL, 108 PFU/mL to 109 PFU/mL, 108 PFU/mL to 5·108 PFU/mL.
Poxvirus is preferably purified or semi purified to reduce host cell proteins and host cell DNA in order to be well tolerated after human administration. Moreover, a number of impurities could be at the origin of human allergy reaction after injection. Such semi purification or purification processes are conventional in the art and may vary as a function of various parameters such as the virus itself, the producer cell, the culture medium used, the enzymes and other components introduced during production and purification steps (e.g. nucleases, proteases, salts, etc). For example, impurities like egg ovalbumin and antibiotics are typically present when the virus is produced on primary cells like Chicken Embryo Fibrobast (CEF) whereas host cell nucleic acids and proteins are usual contaminants of virus preparation produced on immortalized cell lines such as duck cell lines and human cell lines. For general guidance, it is recommended that host cell DNA be reduced to less than 10 ng/dose (see regulatory specification) and host cell proteins be lower than 150 μg/dose. Representative examples of suitable techniques to achieve semi-purified or purified virus preparation include without limitation tangential flow filtration, enzymatic digestion, chromatography, frontal filtration and the like. Therefore, in a preferred embodiment, the poxvirus (in particular vaccinia virus) is at least semi-purified, comprising 5 to 500 μg/dose of host cell proteins and lower than 10 ng/dose of host cell DNA.
The inventors found that liquid formulations according to the invention are able to stabilize three distinct strains of vaccinia virus: MVA, Wyeth, and Copenhagen (see Example 4). Various strains of vaccinia virus may thus be stabilized using the liquid formulations according to the invention. In particular, said vaccinia virus may be selected from Elstree, Western Reserve, Wyeth, NYVAC, NYCBOH, Paris, Copenhagen, and modified Vaccinia Virus Ankara (MVA) strains. Said vaccinia virus may preferably be selected from modified Vaccinia Virus Ankara (MVA), Wyeth, and Copenhagen strains. In a preferred embodiment, vaccinia virus present in the formulation is a MVA virus, and in particular MVA 575 (ECACC V00120707) or MVA-BN (ECACC V00083008). In another preferred embodiment, vaccinia virus present in the formulation is a Wyeth vaccinia virus. In another preferred embodiment, vaccinia virus present in the formulation is a Copenhagen vaccinia virus.
The poxvirus, in particular vaccinia virus, comprised in the formulations according to the invention may be a wild-type, an attenuated, or a recombinant poxvirus, in particular vaccinia virus. The term “recombinant poxvirus” refers to a poxvirus comprising at least one exogenous sequence inserted in its genome. As used herein, an “exogenous sequence” refers to a nucleic acid which is not naturally present in the parent poxvirus.
When the poxvirus (in particular vaccinia virus) comprised in the formulations according to the invention is recombinant, the exogenous sequence(s) may be any exogenous sequence of interest.
In a first preferred embodiment, the recombinant poxvirus (in particular vaccinia virus) comprises an exogenous sequence encoding a molecule having a directly or indirectly cytotoxic function. By “directly or indirectly” cytotoxic, we mean that the molecule encoded by the exogenous sequence may itself be toxic (for example toxins; cytokines or enzymes such as ribonuclease, deoxyribonuclease) or it may be metabolised to form a toxic product, or it may act on something else to form a toxic product. In a preferred embodiment, the molecule encoded by the exogenous sequence may be a toxin such as ricin or Pseudomonas exotoxin A. The sequence of ricin cDNA is disclosed in LAMB et at (Eur. J. Biochem., 1985, 148:265-270). In another preferred embodiment, the molecule encoded by the exogenous sequence may be a cytokine. Such cytokine may notably be selected from tumor necrosis factor (TNF), interleukin-2 (IL-2), interferon-gamma (IFNγ), or granulocyte-macrophage colony-stimulating factor (GMCSF). In another preferred embodiment, the exogenous sequence encoding a molecule having a directly or indirectly cytotoxic function may be a suicide gene. A suicide gene encodes a protein able to convert a relatively non-toxic prodrug to a toxic drug. For example, the enzyme cytosine deaminase converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) (MULLEN et al., 1922, PNAS 89:33); the herpes simplex enzyme thymidine kinase sensitises cells to treatment with the antiviral agent ganciclovir (GCV) or aciclovir (MOOLTEN, 1986, Cancer Res. 46:5276; EZZEDINE et al., 1991, New Biol 3:608). The cytosine deaminase of any organism, for example E. coli or Saccharomyces cerevisiae, may be used. Thus, in preferred embodiment of the invention, the suicide gene encodes a protein having a cytosine deaminase activity, and more preferably FCU 1 protein or FCU 1-8 protein disclosed in patent applications WO99/54481, WO2005/007857, WO2009/065546 and WO2009/065547, which are incorporated herein by reference. With this regard, preferred recombinant vaccinia viruses comprised in the liquid formulations according to the invention are:
In a second preferred embodiment, the recombinant vaccinia virus comprises an exogenous gene encoding a ribozyme capable of cleaving targeted RNA or DNA. The targeted RNA or DNA to be cleaved may be RNA or DNA which is essential to the function of the cell and cleavage thereof results in cell death or the RNA or DNA to be cleaved may be RNA or DNA which encodes an undesirable protein, for example an oncogene product, and cleavage of this RNA or DNA may prevent the cell from becoming cancerous.
In a third preferred embodiment, the recombinant poxvirus (in particular vaccinia virus) comprises an exogenous sequence encoding an antisense RNA. By “antisense RNA” we mean an RNA molecule which hybridises to, and interferes with the expression from an mRNA molecule encoding a protein or to another RNA molecule within the cell such as pre-mRNA or tRNA or rRNA, or hybridises to, and interferes with the expression from a gene.
In fourth preferred embodiment, the recombinant poxvirus (in particular vaccinia virus) comprises an exogenous sequence replacing the function of a defective gene in the target cell. There are several thousand inherited genetic diseases of mammals, including humans, which are caused by defective genes. Examples of such genetic diseases include cystic fibrosis, where there is known to be a mutation in the CFTR gene; Duchenne muscular dystrophy, where there is known to be a mutation in the dystrophin gene; sickle cell disease, where there is known to be a mutation in the HbA gene. Many types of cancer are caused by defective genes, especially protooncogenes, and tumor-suppressor genes that have undergone mutation. Examples of protooncogenes are ras, src, bcl and so on; examples of tumor-suppressor genes are p53 and Rb.
In a fifth preferred embodiment, the recombinant poxvirus (in particular vaccinia virus) comprises an exogenous sequence encoding a Tumor Associated Antigen (TAA). TAA refers to a molecule that is detected at a higher frequency or density in tumor cells than in non-tumor cells of the same tissue type. Examples of TAA includes but are not limited to CEA, MART1, MAGE1, MAGE3, GP-100, MUC1 (see WO92/07000, WO95/09241 and ROCHLITZ et al. J Gene Med. 2003 August; 5(8):690-9 incorporated herein by reference), MUC2, pointed mutated ras oncogene, normal or point mutated p53, overexpressed p53, CA-125, PSA, C-erb/B2, BRCA I, BRCA II, PSMA, tyrosinase, TRP1, TRP2, NY-ESO-1, TAG72, KSA, HER-2/neu, bcr-abl, pax3-fkhr, ews-fli-1, surviving and LRP. According to a more preferred embodiment the TAA is MUC1.
In a sixth preferred embodiment, the recombinant poxvirus (in particular vaccinia virus) comprises an exogenous gene encoding an antigen. As used herein, “antigen” refers to a ligand that can be bound by an antibody; an antigen need not itself be immunogenic. Preferably the antigen is derived from:
Of course, the recombinant poxvirus (in particular vaccinia virus) present in the liquid formulations according to the invention can comprise more than one exogenous sequence and each exogenous sequence can encode more than one molecule.
For example, it can be useful to associate in a same recombinant poxvirus (in particular vaccinia virus):
In this respect, preferred recombinant vaccinia viruses present in the liquid formulations according to the invention are:
Another example of useful association of two exogenous sequences in the same poxvirus (in particular vaccinia virus) vector is a poxvirus (in particular vaccinia virus) vector comprising:
In this respect, a preferred recombinant vaccinia virus present in the liquid formulations according to the invention is a vaccinia virus (preferably Wyeth strain) defective for J2R gene, and comprising an exogenous sequence encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) (see KIM J H et al., 2006 September, Mol Ther., 14(3):361-70 and BREITBACH C J et al., 2011, Curr Pharm Biotechnol. Vol 12. No 12).
Methods for preparing and purifying poxviruses and in particular vaccinia virus are known to those skilled in the art. For instance, processes for producing and purifying poxviruses and in particular vaccinia viruses are disclosed in WO2007/147528 and WO2010/130753, which are herein incorporated by reference.
Vaccinia virus may notably be firstly amplified by:
In step a), suitable packaging cells depend on the type of vaccinia virus to be amplified.
MVA is strictly host-restricted and may be amplified on avian cells, either primary avian cells (such as chicken embryo fibroblasts or CEF) or an immortalized avian cell line, and in particular:
For other vaccinia virus or other poxvirus strains, in addition to avian primary cells (such as CEF—“chicken embryo fibroblasts”—also called CEC or “chicken embryo cells”) and avian cell lines, many other non-avian cell lines are available for amplification, including Hela, BHK-21, MRC-5, HEK-293, and Vero cells. In a preferred embodiment, vaccinia virus other than MVA is amplified in Hela cells.
Packaging cells are preferably cultivated in a medium free from animal- or human-derived products, using a chemically defined medium with no product of animal or human origin. In particular, while growth factors may be present, they are preferably recombinantly produced and not purified from animal material. An appropriate animal-free medium may be easily selected by those skilled in the art depending on selected packaging cells. Such media are commercially available. In particular, when CEFs are used as packaging cells, they may be cultivated in VP-SFM cell culture medium (Invitrogen). CEFs are also preferably cultivated for between 1 and 5 days, more preferably between 1 and 2 days and even more preferably 2 days before infection. CEFs are further preferably cultivated at a temperature comprised between +30° C. and +37° C. When non-avian immortalized cell tines cells are used, they are preferably cultivated for between 2 and 7 days before infection. If a high number of non-avian immortalized cells is needed, several passages of 2 to 7 days may be made in order to increase the total number of cells. Non-avian immortalized cells are further preferably cultivated at a temperature comprised between +36° C. and +38° C., more preferably at about +37° C.
In step b), packaging cells are infected by poxvirus (in particular vaccinia virus) under appropriate conditions (in particular using an appropriate multiplicity of infection (MOI)) to permit productive infection of packaging cells. In particular, when vaccinia virus is MVA (in particular those disclosed in WO90/10459, WO92/07000, WO95/09241, WO98/04705, WO99/03885, WO2004/111082, WO2007/121894, WO2014/009438 and WO2014/009433) and is amplified using CEF, it may be seeded in the cell culture vessel containing CEFs at a MOI which is preferably comprised between 0.001 and 0.1, more preferably between 0.03 and 0.07 and even more preferably about 0.05. For other vaccinia virus strains, in particular oncolytic vaccinia virus such as Wyeth and Copenhagen strains (notably those disclosed in WO2007/030668, WO2008/113078, WO2009/065546, WO2009/065547), vaccinia virus may be seeded in the cell culture vessel containing packaging cells at a MOI which is preferably comprised between 0.0001 and 0.1, and more preferably about 0.0001. Infection step is also preferably performed in a medium (which may be the same as or different from the medium used for culture of packaging cells) free from animal- or human-derived products, using a chemically defined medium with no product of animal or human origin. For MVA in CEFs, the culture medium used in step b) is preferably a basal medium, notably Basal Medium Eagle cell culture medium (Invitrogen).
In step c), infected packaging cells are then cultured under appropriate conditions well known to those skilled in the art until progeny poxvirus (in particular vaccinia virus) is produced. Culture of infected packaging cells is also preferably performed in a medium (which may be the same as or different from the medium used for culture of packaging cells and/or for infection step) free from animal- or human-derived products, using a chemically defined medium with no product of animal or human origin. For MVA amplified on CEFs, CEFs may notably be cultured in basal medium, notably Basal Medium Eagle cell culture medium (Invitrogen), at a temperature between +33° C. and +37° C., during 1 to 4 days. For other vaccinia virus strains produced in a non-avian immortalized cell line, step c) may notably be performed between +35° C. and +38° C. during 1 to 4 days.
In step d), poxvirus (in particular vaccinia virus) produced in step c) is collected from the culture supernatant and/or the packaging cells. When poxvirus (in particular vaccinia virus) is collected from packaging cells (and optionally also from culture supernatant), step d) may be preceded by a step allowing the disruption of the packaging cell membrane. This step leads to the liberation of poxvirus (in particular vaccinia virus) from packaging cells. The disruption of packaging cells membrane can be induced by various techniques well known to those skilled in the art, including but not limited to: freeze/thaw, hypotonic lysis, sonication, microfluidization, or high speed homogenization.
Poxvirus (in particular vaccinia virus) may then be further purified, using purification steps well known in the art, such as:
In a preferred embodiment of the present invention, vaccinia virus present in the formulation is a MVA virus (in particular those disclosed in WO90/10459, WO92/07000, WO95/09241, WO98/04705, WO99/03885, WO2004/111082, WO2007/121894, WO2014/009438 and WO2014/009433) amplified on CEFs, more preferably a MVA virus (in particular those disclosed in WO90/10459, WO92/07000, WO95/09241, WO98/04705, WO99/03885, WO2004/111082, WO2007/121894, WO2014/009438 and WO2014/009433) amplified on CEFs and which has not been submitted to a step of treatment with at least one protease.
In another preferred embodiment, vaccinia virus present in the formulation is a MVA virus (in particular those disclosed in WO90/10459, WO92/07000, WO95/09241, WO98/04705, WO99/03885, WO2004/111082, WO2007/121894, WO2014/009438 and WO2014/009433) amplified on an immortalized avian cell line (including a Cairina moschata immortalized avian cell line comprising a nucleic acid sequence coding a telomerase reverse transcriptase (TERT), a Cairina moschata immortalized avian cell line comprising an E1A nucleic acid sequence and a nucleic acid sequence coding a telomerase reverse transcriptase (TERT), a DF1 cell line, an Ebx cell line, or a DEC 99 cell line), more preferably a MVA virus (in particular those disclosed in WO90/10459, WO92/07000, WO95/09241, WO98/04705, WO99/03885, WO2007/111082, WO2007/121894, WO2014/009438 and WO2014/009433) amplified on an immortalized avian cell line (including those mentioned above) that has not been subjected to at least one step of treatment with at least one protease.
In another preferred embodiment, vaccinia virus present in the formulation is a Wyeth or Copenhagen vaccinia virus (in particular those disclosed in WO2007/030668, WO2008/113078, WO2009/065546, WO2009/065547) amplified in Hela cells, more preferably a Wyeth or Copenhagen vaccinia virus (in particular those disclosed in WO2007/030668, WO2008/113078, WO2009/065546, WO2009/065547) amplified in Hela cells that has been subjected to at least one step of treatment with at least one protease.
pH and Buffer
Liquid formulations according to the invention have a pH comprised between 6.5 and 8.5. In particular, liquid formulations according to the invention may have a pH comprised between 6.5 and 8.4, between 6.5 and 8.3, between 6.5 and 8.2, between 6.5 and 8.1, between 6.5 and 8.0, between 6.5 and 7.9, between 6.5 and 7.8, between 6.5 and 7.7, between 6.5 and 7.6, between 6.5 and 7.5, between 6.6 and 8.5, between 6.6 and 8.4, between 6.6 and 8.3, between 6.6 and 8.2, between 6.6 and 8.1, between 6.6 and 8.0, between 6.6 and 7.9, between 6.6 and 7.8, between 6.6 and 7.7, between 6.6 and 7.6, between 6.6 and 7.5, between 6.7 and 8.5, between 6.7 and 8.4, between 6.7 and 8.3, between 6.7 and 8.2, between 6.7 and 8.1, between 6.7 and 8.0, between 6.7 and 7.9, between 6.7 and 7.8, between 6.7 and 7.7, between 6.7 and 7.6, between 6.7 and 7.5, between 6.8 and 8.5, between 6.8 and 8.4, between 6.8 and 8.3, between 6.8 and 8.2, between 6.8 and 8.1, between 6.8 and 8.0, between 6.8 and 7.9, between 6.8 and 7.8, between 6.8 and 7.7, between 6.8 and 7.6, between 6.8 and 7.5, between 6.9 and 8.5, between 6.9 and 8.4, between 6.9 and 8.3, between 6.9 and 8.2, between 6.9 and 8.1, between 6.9 and 8.0, between 6.9 and 7.9, between 6.9 and 7.8, between 6.9 and 7.7, between 6.9 and 7.6, between 6.9 and 7.5, between 7 and 8.5, between 7 and 8.4, between 7 and 8.3, between 7 and 8.2, between 7 and 8.1, between 7 and 8, between 7 and 7.9, between 7 and 7.8, between 7 and 7.7, between 7 and 7.6, between 7 and 7.5, between 7.1 and 8.5, between 7.1 and 8.4, between 7.1 and 8.3, between 7.1 and 8.2, between 7.1 and 8.1, between 7.1 and 8, between 7.1 and 7.9, between 7.1 and 7.8, between 7.1 and 7.7, between 7.1 and 7.6, between 7.1 and 7.5, between 7.2 and 8.5, between 7.2 and 8.4, between 7.2 and 8.3, between 7.2 and 8.2, between 7.2 and 8.1, between 7.2 and 8, between 7.2 and 7.9, between 7.2 and 7.8, between 7.2 and 7.7, between 7.2 and 7.6, between 7.2 and 7.5, between 7.3 and 8.5, between 7.3 and 8.4, between 7.3 and 8.3, between 7.3 and 8.2, between 7.3 and 8.1, between 7.3 and 8, between 7.3 and 7.9, between 7.3 and 7.8, between 7.3 and 7.7, between 7.3 and 7.6, between 7.3 and 7.5, between 7.4 and 8.5, between 7.4 and 8.4, between 7.4 and 8.3, between 7.4 and 8.2, between 7.4 and 8.1, between 7.4 and 8, between 7.4 and 7.9, between 7.4 and 7.8, between 7.4 and 7.7, between 7.4 and 7.6, between 7.4 and 7.5, between 7.5 and 8.5, between 7.5 and 8.4, between 7.5 and 8.3, between 7.5 and 8.2, between 7.5 and 8.1, between 7.5 and 8, between 7.5 and 7.9, between 7.5 and 7.8, between 7.5 and 7.7, or between 7.5 and 7.6. Preferably, liquid formulations according to the invention have a pH between 7 and 8, and more particularly close to 7.5, in particular comprised between 7.2 and 7.8, between 7.3 and 7.7, between 7.4 and 7.6, or about 7.5.
In order to maintain this pH, the liquid formulations according to the invention comprise a buffer with buffering capacity at the pH of the formulation. Such buffers are well known to those skilled in the art, and notably include the following buffers:
Preferably, said buffer is selected from TRIS-HCl, TRIS, Tricine, HEPES and phosphate buffer comprising a mixture of Na2HPO4 and KH2PO4 or a mixture of Na2HPO4 and NaH2PO4. More preferably said buffer is selected from TRIS-HCl, TRIS, or Tricine buffer, and more preferably said buffer is TRIS-HCl or TRIS buffer, even more preferably said buffer is TRIS-HCl buffer.
Said buffer (in particular those mentioned above and notably TRIS-HCl) is preferably present in a concentration of 10 to 50 mM. It may notably be present in a concentration of 10 to 45 mM, 10 to 40 mM, 10 to 35 mM, 10 to 30 mM, 10 to 25 mM, 10 to 20 mM, 10 to 15 mM, 15 to 50 mM, 15 to 45 mM, 15 to 40 mM, 15 to 35 mM, 15 to 30 mM, 15 to 25 mM, 15 to 20 mM, 20 to 50 mM, 20 to 45 mM, 20 to 40 mM, 20 to 35 mM, 20 to 30 mM, 20 to 25 mM, 25 to 50 mM, 25 to 45 mM, 25 to 40 mM, 25 to 35 mM, 25 to 30 mM, 30 to 50 mM, 30 to 45 mM, 30 to 40 mM, 30 to 35 mM, 35 to 50 mM, 35 to 45 mM, 35 to 40 mM, 40 to 50 mM, 40 to 45 mM, or 45 to 50 mM. Preferably, said buffer (in particular those mentioned above and notably TRIS-HCl) is preferably present in a concentration of 10 to 40 mM, in particular of 10 to 30 mM.
Monovalent Salt
Liquid formulations according to the invention comprise a monovalent salt. This monovalent salt is believed to ensure an appropriate osmotic pressure. In addition, said monovalent salt is also believed to have inhibition properties of proteases that may be present in the formulation, thus improving stability. Such proteases include cell proteases liberated when disrupting packaging cells and also, when the vaccinia virus present in the formulation has been purified by a method involving the use of protease, remaining traces of said added protease. The inhibiting effect of significant monovalent salts concentration on proteases has been documented in the art (see TÕUGU V et al. Eur J Biochem. 1994 June, 222(2):475-81). For instance, for Pierce Trypsin Protease (Thermo Scientific), the manufacturer indicates in the Instruction notice that high monovalent salt concentrations, such as >100 mM NaCl, may interfere with trypsin activity.
Said monovalent salt may notably be selected from NaCl and KCl, preferably said monovalent salt is NaCl.
Said monovalent salt (in particular NaCl) is preferably present in a concentration of 10 to 1000 mM. It may notably be present in a concentration of 10 to 950 mM, 10 to 900 mM, 10 to 850 mM, 10 to 800 mM, 10 to 750 mM, 10 to 700 mM, 10 to 650 mM, 10 to 600 mM, 10 to 550 mM, 10 to 500 mM, 10 to 450 mM, 10 to 400 mM, 10 to 350 mM, 10 to 300 mM, 10 to 250 mM, 10 to 200 mM, 10 to 150 mM, 10 to 100 mM, 10 to 90 mM, 10 to 80 mM, 10 to 75 mM, 10 to 70 mM, 10 to 60 mM, 10 to 50 mM, 10 to 40 mM, 10 to 30 mM, 10 to 25 mM, 10 to 20 mM, 20 to 1000 mM, 20 to 950 mM, 20 to 900 mM, 20 to 850 mM, 20 to 800 mM, 20 to 750 mM, 20 to 700 mM, 20 to 650 mM, 20 to 600 mM, 20 to 550 mM, 20 to 500 mM, 20 to 450 mM, 20 to 400 mM, 20 to 350 mM, 20 to 300 mM, 20 to 250 mM, 20 to 200 mM, 20 to 150 mM, 20 to 100 mM, 20 to 90 mM, 20 to 80 mM, 20 to 75 mM, 20 to 70 mM, 20 to 60 mM, 20 to 50 mM, 20 to 40 mM, 20 to 30 mM, 20 to 25 mM, 25 to 1000 mM, 25 to 950 mM, 25 to 900 mM, 25 to 850 mM, 25 to 800 mM, 25 to 750 mM, 25 to 700 mM, 25 to 650 mM, 25 to 600 mM, 25 to 550 mM, 25 to 500 mM, 25 to 450 mM, 25 to 400 mM, 25 to 350 mM, 25 to 300 mM, 25 to 250 mM, 25 to 200 mM, 25 to 150 mM, 25 to 100 mM, 25 to 90 mM, 25 to 80 mM, 25 to 75 mM, 25 to 70 mM, 25 to 60 mM, 25 to 50 mM, 25 to 40 mM, 25 to 30 mM, 30 to 1000 mM, 30 to 950 mM, 30 to 900 mM, 30 to 850 mM, 30 to 800 mM, 30 to 750 mM, 30 to 700 mM, 30 to 650 mM, 30 to 600 mM, 30 to 550 mM, 30 to 500 mM, 30 to 450 mM, 30 to 400 mM, 30 to 350 mM, 30 to 300 mM, 30 to 250 mM, 30 to 200 mM, 30 to 150 mM, 30 to 100 mM, 30 to 90 mM, 30 to 80 mM, 30 to 75 mM, 30 to 70 mM, 30 to 60 mM, 30 to 50 mM, 30 to 40 mM, 40 to 1000 mM, 40 to 950 mM, 40 to 900 mM, 40 to 850 mM, 40 to 800 mM, 40 to 750 mM, 40 to 700 mM, 40 to 650 mM, 40 to 600 mM, 40 to 550 mM, 40 to 500 mM, 40 to 450 mM, 40 to 400 mM, 40 to 350 mM, 40 to 300 mM, 40 to 250 mM, 40 to 200 mM, 40 to 150 mM, 40 to 100 mM, 40 to 90 mM, 40 to 80 mM, 40 to 75 mM, 40 to 70 mM, 40 to 60 mM, 40 to 50 mM, 50 to 1000 mM, 50 to 950 mM, 50 to 900 mM, 50 to 850 mM, 50 to 800 mM, 50 to 750 mM, 50 to 700 mM, 50 to 650 mM, 50 to 600 mM, 50 to 550 mM, 50 to 500 mM, 50 to 450 mM, 50 to 400 mM, 50 to 350 mM, 50 to 300 mM, 50 to 250 mM, 50 to 200 mM, 50 to 150 mM, 50 to 100 mM, 50 to 90 mM, 50 to 80 mM, 50 to 75 mM, 50 to 70 mM, 50 to 60 mM, 60 to 1000 mM, 60 to 950 mM, 60 to 900 mM, 60 to 850 mM, 60 to 800 mM, 60 to 750 mM, 60 to 700 mM, 60 to 650 mM, 60 to 600 mM, 60 to 550 mM, 60 to 500 mM, 60 to 450 mM, 60 to 400 mM, 60 to 350 mM, 60 to 300 mM, 60 to 250 mM, 60 to 200 mM, 60 to 150 mM, 60 to 100 mM, 60 to 90 mM, 60 to 80 mM, 60 to 75 mM, 60 to 70 mM, 70 to 1000 mM, 70 to 950 mM, 70 to 900 mM, 70 to 850 mM, 70 to 800 mM, 70 to 750 mM, 70 to 700 mM, 70 to 650 mM, 70 to 600 mM, 70 to 550 mM, 70 to 500 mM, 70 to 450 mM, 70 to 400 mM, 70 to 350 mM, 70 to 300 mM, 70 to 250 mM, 70 to 200 mM, 70 to 150 mM, 70 to 100 mM, 70 to 90 mM, 70 to 80 mM, 70 to 75 mM, 75 to 1000 mM, 75 to 950 mM, 75 to 900 mM, 75 to 850 mM, 75 to 800 mM, 75 to 750 mM, 75 to 700 mM, 75 to 650 mM, 75 to 600 mM, 75 to 550 mM, 75 to 500 mM, 75 to 450 mM, 75 to 400 mM, 75 to 350 mM, 75 to 300 mM, 75 to 250 mM, 75 to 200 mM, 75 to 150 mM, 75 to 100 mM, 75 to 90 mM, 75 to 80 mM, 80 to 1000 mM, 80 to 950 mM, 80 to 900 mM, 80 to 850 mM, 80 to 800 mM, 80 to 750 mM, 80 to 700 mM, 80 to 650 mM, 80 to 600 mM, 80 to 550 mM, 80 to 500 mM, 80 to 450 mM, 80 to 400 mM, 80 to 350 mM, 80 to 300 mM, 80 to 250 mM, 80 to 200 mM, 80 to 150 mM, 80 to 100 mM, 80 to 90 mM, 90 to 1000 mM, 90 to 950 mM, 90 to 900 mM, 90 to 850 mM, 90 to 800 mM, 90 to 750 mM, 90 to 700 mM, 90 to 650 mM, 90 to 600 mM, 90 to 550 mM, 90 to 500 mM, 90 to 450 mM, 90 to 400 mM, 90 to 350 mM, 90 to 300 mM, 90 to 250 mM, 90 to 200 mM, 90 to 150 mM, 90 to 100 mM, 100 to 1000 mM, 100 to 950 mM, 100 to 900 mM, 100 to 850 mM, 100 to 800 mM, 100 to 750 mM, 100 to 700 mM, 100 to 650 mM, 100 to 600 mM, 100 to 550 mM, 100 to 500 mM, 100 to 450 mM, 100 to 400 mM, 100 to 350 mM, 100 to 300 mM, 100 to 250 mM, 100 to 200 mM, 100 to 150 mM, 150 to 1000 mM, 150 to 950 mM, 150 to 900 mM, 150 to 850 mM, 150 to 800 mM, 150 to 750 mM, 150 to 700 mM, 150 to 650 mM, 150 to 600 mM, 150 to 550 mM, 150 to 500 mM, 150 to 450 mM, 150 to 400 mM, 150 to 350 mM, 150 to 300 mM, 150 to 250 mM, 150 to 200, 200 to 1000 mM, 200 to 950 mM, 200 to 900 mM, 200 to 850 mM, 200 to 800 mM, 200 to 750 mM, 200 to 700 mM, 200 to 650 mM, 200 to 600 mM, 200 to 550 mM, 200 to 500 mM, 200 to 450 mM, 200 to 400 mM, 200 to 350 mM, 200 to 300 mM, 200 to 250 mM, 250 to 1000 mM, 250 to 950 mM, 250 to 900 mM, 250 to 850 mM, 250 to 800 mM, 250 to 750 mM, 250 to 700 mM, 250 to 650 mM, 250 to 600 mM, 250 to 550 mM, 250 to 500 mM, 250 to 450 mM, 250 to 400 mM, 250 to 350 mM, 250 to 300 mM, 300 to 1000 mM, 300 to 950 mM, 300 to 900 mM, 300 to 850 mM, 300 to 800 mM, 300 to 750 mM, 300 to 700 mM, 300 to 650 mM, 300 to 600 mM, 300 to 550 mM, 300 to 500 mM, 300 to 450 mM, 300 to 400 mM, 300 to 350 mM, 350 to 1000 mM, 350 to 950 mM, 350 to 900 mM, 350 to 850 mM, 350 to 800 mM, 350 to 750 mM, 350 to 700 mM, 350 to 650 mM, 350 to 600 mM, 350 to 550 mM, 350 to 500 mM, 350 to 450 mM, 350 to 400 mM, 400 to 1000 mM, 400 to 950 mM, 400 to 900 mM, 400 to 850 mM, 400 to 800 mM, 400 to 750 mM, 400 to 700 mM, 400 to 650 mM, 400 to 600 mM, 400 to 550 mM, 400 to 500 mM, 400 to 450 mM, 450 to 1000 mM, 450 to 950 mM, 450 to 900 mM, 450 to 850 mM, 450 to 800 mM, 450 to 750 mM, 450 to 700 mM, 450 to 650 mM, 450 to 600 mM, 450 to 550 mM, or 450 to 500 mM.
MVA (in particular those disclosed in WO90/10459, WO92/07000, WO95/09241, WO98/04705, WO99/03885, WO2004/111082, WO2007/121894, WO2014/009438 and WO2014/009433) is generally amplified in primary avian cells, in which case no protease treatment is necessary for elimination of primary avian cells proteins, since primary cells are not considered as dangerous. Thus, for MVA, and more generally when the poxvirus (preferably vaccinia virus) present in the formulation has been purified by a method that does not involve treatment by at least one protease, said monovalent salt (in particular NaCl) may be present in relatively low concentration, notably in a concentration of 10 to 200 mM, 10 to 150 mM, 10 to 100 mM, 10 to 90 mM, 10 to 80 mM, 10 to 75 mM, 20 to 200 mM, 20 to 150 mM, 20 to 100 mM, 20 to 90 mM, 20 to 80 mM, 20 to 75 mM, 25 to 200 mM, 25 to 150 mM, 25 to 100 mM, 25 to 90 mM, 25 to 80 mM, 25 to 75 mM, 30 to 200 mM, 30 to 150 mM, 30 to 100 mM, 30 to 90 mM, 30 to 80 mM, 30 to 75 mM, 40 to 200 mM, 40 to 150 mM, 40 to 100 mM, 40 to 90 mM, 40 to 80 mM, 40 to 75 mM, 50 to 200 mM, 50 to 150 mM, 50 to 100 mM, 50 to 90 mM, 50 to 80 mM, 50 to 75 mM, 60 to 200 mM, 60 to 150 mM, 60 to 100 mM, 60 to 90 mM, 60 to 80 mM, 60 to 75 mM, 70 to 200 mM, 70 to 150 mM, 70 to 100 mM, 70 to 90 mM, 70 to 80 mM, or 70 to 75 mM, more preferably in a concentration close to 75 mM, such as 50 to 100 mM, 60 to 90 mM, 70 to 80 mM, or about 75 mM.
Other strains of poxviruses, and in particular oncolytic vaccinia viruses, such as Wyeth or Copenhagen vaccinia virus (notably those disclosed in WO2007/030668, WO2008/113078, WO2009/065546, WO2009/065547), are generally amplified on various immortalized cell lines. Some of these cell lines may contain oncogenes and elimination or at least drastic reduction of producing cells DNA and proteins is in this case required by health authorities. For this purpose, the purification process generally includes at least one step of treatment with at least one protease. Remaining traces of protease(s) may, particularly in a liquid formulation, have deleterious effects of vaccinia virus stability. In this respect, the inventors found that increasing the concentration of said monovalent salt (in particular NaCl) results in improved stability of vaccinia virus. Without being bound by theory, it is believed that an increased concentration of said monovalent salt (in particular NaCl) has inhibitory effect on remaining traces of protease(s).
Therefore, when the poxvirus (in particular a vaccinia virus) present in the formulation has been purified by a method that involves at least one step of treatment with at least one protease, said monovalent salt (in particular NaCl) is preferably present in a concentration of 100 to 1000 mM, 100 to 950 mM, 100 to 900 mM, 100 to 850 mM, 100 to 800 mM, 100 to 750 mM, 100 to 700 mM, 100 to 650 mM, 100 to 600 mM, 100 to 550 mM, 100 to 500 mM, 100 to 450 mM, 100 to 400 mM, 100 to 350 mM, 100 to 300 mM, 100 to 250 mM, 100 to 200 mM, 150 to 1000 mM, 150 to 950 mM, 150 to 900 mM, 150 to 850 mM, 150 to 800 mM, 150 to 750 mM, 150 to 700 mM, 150 to 650 mM, 150 to 600 mM, 150 to 550 mM, 150 to 500 mM, 150 to 450 mM, 150 to 400 mM, 150 to 350 mM, 150 to 300 mM, 150 to 250 mM, 150 to 200, 200 to 1000 mM, 200 to 950 mM, 200 to 900 mM, 200 to 850 mM, 200 to 800 mM, 200 to 750 mM, 200 to 700 mM, 200 to 650 mM, 200 to 600 mM, 200 to 550 mM, 200 to 500 mM, 200 to 450 mM, 200 to 400 mM, 200 to 350 mM, 200 to 300 mM, 200 to 250 mM, 250 to 1000 mM, 250 to 950 mM, 250 to 900 mM, 250 to 850 mM, 250 to 800 mM, 250 to 750 mM, 250 to 700 mM, 250 to 650 mM, 250 to 600 mM, 250 to 550 mM, 250 to 500 mM, 250 to 450 mM, 250 to 400 mM, 250 to 350 mM, 250 to 300 mM, 300 to 1000 mM, 300 to 950 mM, 300 to 900 mM, 300 to 850 mM, 300 to 800 mM, 300 to 750 mM, 300 to 700 mM, 300 to 650 mM, 300 to 600 mM, 300 to 550 mM, 300 to 500 mM, 300 to 450 mM, 300 to 400 mM, 300 to 350 mM, 350 to 1000 mM, 350 to 950 mM, 350 to 900 mM, 350 to 850 mM, 350 to 800 mM, 350 to 750 mM, 350 to 700 mM, 350 to 650 mM, 350 to 600 mM, 350 to 550 mM, 350 to 500 mM, 350 to 450 mM, 350 to 400 mM, 400 to 1000 mM, 400 to 950 mM, 400 to 900 mM, 400 to 850 mM, 400 to 800 mM, 400 to 750 mM, 400 to 700 mM, 400 to 650 mM, 400 to 600 mM, 400 to 550 mM, 400 to 500 mM, 400 to 450 mM, 450 to 1000 mM, 450 to 950 mM, 450 to 900 mM, 450 to 850 mM, 450 to 800 mM, 450 to 750 mM, 450 to 700 mM, 450 to 650 mM, 450 to 600 mM, 450 to 550 mM, or 450 to 500 mM. For instance, said monovalent salt may be present in a concentration close to 200 mM, such as 100 to 300 mM, 150 to 250 mM, or about 200 mM. Alternatively, said monovalent salt may be present in a concentration close to 500 mM, such as 250 to 750 mM, 400 to 600 mM, or about 500 mM. In still another embodiment, said monovalent salt may be present in a concentration close to 750 mM, such as 500 to 1000 mM, 700 to 800 mM, or about 750 mM.
Disaccharide or Sugar Alcohol
Liquid formulations according to the invention comprise a pharmaceutically acceptable disaccharide or sugar alcohol.
This pharmaceutically acceptable disaccharide or sugar alcohol is a cryoprotectant and is believed to protect the poxvirus (in particular vaccinia virus) at low storage temperature, such as at about +5° C. In addition, such pharmaceutically acceptable disaccharide or sugar alcohol increases viscosity of the liquid formulation, which might limit interactions between poxvirus (in particular vaccinia virus) and potentially deleterious compounds.
The pharmaceutically acceptable disaccharide or sugar alcohol may notably be selected from sucrose, trehalose, maltose, lactose, mannitol, and sorbitol, preferably said pharmaceutically acceptable disaccharide or sugar alcohol is sucrose.
The pharmaceutically acceptable disaccharide or sugar alcohol (in particular those mentioned above and notably sucrose) is preferably present in a concentration of 5 to 20% (weight in g/volume in L, referred to as w/v). In particular, it may be present in a concentration of 5 to 19% (w/v), 5 to 18% (w/v), 5 to 17% (w/v), 5 to 16% (w/v), 5 to 15% (w/v), 5 to 14% (w/v), 5 to 13% (w/v), 5 to 12% (w/v), 5 to 11% (w/v), 5 to 10% (w/v), 6 to 20% (w/v), 6 to 19% (w/v), 6 to 18% (w/v), 6 to 17% (w/v), 6 to 16% (w/v), 6 to 15% (w/v), 6 to 14% (w/v), 6 to 13% (w/v), 6 to 12% (w/v), 6 to 11% (w/v), 6 to 10% (w/v), 7 to 20% (w/v), 7 to 19% (w/v), 7 to 18% (w/v), 7 to 17% (w/v), 7 to 16% (w/v), 7 to 15% (w/v), 7 to 14% (w/v), 7 to 13% (w/v), 7 to 12% (w/v), 7 to 11% (w/v), 7 to 10% (w/v), 8 to 20% (w/v), 8 to 19% (w/v), 8 to 18% (w/v), 8 to 17% (w/v), 8 to 16% (w/v), 8 to 15% (w/v), 8 to 14% (w/v), 8 to 13% (w/v), 8 to 12% (w/v), 8 to 11% (w/v), 8 to 10% (w/v), 9 to 20% (w/v), 9 to 19% (w/v), 9 to 18% (w/v), 9 to 17% (w/v), 9 to 16% (w/v), 9 to 15% (w/v), 9 to 14% (w/v), 9 to 13% (w/v), 9 to 12% (w/v), 9 to 11% (w/v), or 9 to 10% (w/v). Preferably, said pharmaceutically acceptable disaccharide or sugar alcohol (in particular those mentioned above and notably sucrose) is preferably present in a concentration of close to 10% (w/v), such as 5 to 15% (w/v), 6 to 14% (w/v), 7 to 13% (w/v), 8 to 12% (w/v), 9 to 11% (w/v), or about 10%.
Chelating Agent
Liquid formulations according to the invention comprise a pharmaceutically acceptable chelating agent, and in particular an agent chelating dications.
The reasons why said pharmaceutically acceptable chelating agent improves stability of poxvirus (in particular vaccinia virus) in the liquid state are not really understood.
Indeed, as explained in background art section, the effect of EDTA on virus stability significantly differs between different viruses, and no obvious classification of viruses for which EDTA has beneficial versus viruses for which EDTA has no beneficial or even deleterious effect can be easily made. In particular, while a significant beneficial effect has been found for adenovirus (non-enveloped DNA virus, see EVANS et al. J Pharm Sci. 2004 October, 93(10):2458-75; and U.S. Pat. No. 7,456,009), no significant beneficial effect has been found for influenza virus (enveloped RNA virus, see US2007/0161085), canine parvovirus (non-enveloped DNA virus), canine adenovirus type 2 (non-enveloped DNA virus), canine distemper virus (enveloped RNA paramyxovirus) and canine parainfluenza virus (enveloped RNA paramyxovirus) (see WO2014/029702). Finally, a deleterious effect has been found for Newcastle virus (enveloped RNA paramyxovirus, see U.S. Pat. No. 7,914,979).
However, as demonstrated in the experimental section, said pharmaceutically acceptable chelating agent has an essential role in stabilization of poxvirus (in particular vaccinia virus) in liquid formulations according to the present invention.
The pharmaceutically acceptable chelating agent may notably be selected from ethylenediaminetetraacetic acid (EDTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), ethylene glycol tetraacetic acid (EGTA), dimercaptosuccinic acid (DMSA), diethylene triamine pentaacetic acid (DTPA), and 2,3-Dimercapto-1-propanesulfonic acid (DMPS), preferably said pharmaceutically acceptable chelating agent is EDTA.
The pharmaceutically acceptable chelating agent (in particular those mentioned above and notably EDTA) is preferably present in a concentration of at least 50 μM. In particular, it may be present in a concentration of 50 to 1000 μM, 50 to 750 μM, 50 to 500 μM, 50 to 400 μM, 50 to 300 μM, 50 to 250 μM, 50 to 200 μM, 50 to 150 μM; 50 to 100 μM, 50 to 75 μM, 75 to 1000 μM, 75 to 750 μM, 75 to 500 μM, 75 to 400 μM, 75 to 300 μM, 75 to 250 μM, 75 to 200 μM, 75 to 150 μM; 75 to 100 μM, 100 to 1000 μM, 100 to 750 μM, 100 to 500 μM, 100 to 400 μM, 100 to 300 μM, 100 to 250 μM, 100 to 200 μM, 100 to 150 μM; 150 to 1000 μM, 150 to 750 μM, 150 to 500 μM, 150 to 400 μM, 150 to 300 μM, 150 to 250 μM, 150 to 200 μM. Said pharmaceutically acceptable chelating agent (in particular those mentioned above and notably EDTA) may notably be present in a concentration close to 150 μM, such as 50 to 250 μM, 100 to 200 μM, or about 150 μM. However, higher concentrations may be present, since no deleterious effect on stability has been observed, even at low concentrations.
Optional Additional Components
Liquid formulations according to the invention may further comprise additional compounds with stabilizing effect on vaccinia virus.
C2-C3 Alcohol
While the presence of a pharmaceutically acceptable buffer permitting to have a pH between 6.5 and 8.5, a monovalent salt, a pharmaceutically acceptable disaccharide or sugar-alcohol, and a pharmaceutically acceptable chelating agent has been found to be essential for stabilization of vaccinia virus in the liquid state, the inventors also found that the additional presence of a low concentration of a C2-C3 alcohol, while not necessary for stabilization of vaccinia virus, synergizes with the presence of a chelating agent to further improve stability of vaccinia virus in liquid state. In contrast, a too high concentration of the same C2-C3 alcohol has deleterious effects on vaccinia virus stability in the liquid state. Therefore, the liquid formulations according to the invention preferably further comprise a C2-C3 alcohol in a concentration of 0.05 to 5% (volume/volume or v/v). This finding was quite unexpected, because poxviruses, and in particular vaccinia viruses, are enveloped viruses, for which the addition of a polar solvent might be expected to alter the envelop, contrary to the case of non-enveloped viruses such as adenoviruses.
Said C2-C3 alcohol may notably be selected from ethanol and isopropanol, preferably said C2-C3 alcohol is ethanol.
Said C2-C3 alcohol (in particular those mentioned above and notably ethanol) may notably be present in a concentration of 0.05 to 5% (v/v), 0.05 to 4% (v/v), 0.05 to 3% (v/v), 0.05 to 2% (v/v), 0.05 to 1% (v/v), 0.05 to 0.9% (v/v), 0.05 to 0.8% (v/v), 0.05 to 0.7% (v/v), 0.05 to 0.6% (v/v), 0.05 to 0.5% (v/v), 0.1 to 5% (v/v), 0.1 to 4% (v/v), 0.1 to 3% (v/v), 0.1 to 2% (v/v), 0.1 to 1% (v/v), 0.1 to 0.9% (v/v), 0.1 to 0.8% (v/v), 0.1 to 0.7% (v/v), 0.1 to 0.6% (v/v), 0.1 to 0.5% (v/v), 0.2 to 5% (v/v), 0.2 to 4% (v/v), 0.2 to 3% (v/v), 0.2 to 2% (v/v), 0.2 to 1% (v/v), 0.2 to 0.9% (v/v), 0.2 to 0.8% (v/v), 0.2 to 0.7% (v/v), 0.2 to 0.6% (v/v), 0.2 to 0.5% (v/v), 0.3 to 5% (v/v), 0.3 to 4% (v/v), 0.3 to 3% (v/v), 0.3 to 2% (v/v), 0.3 to 1% (v/v), 0.3 to 0.9% (v/v), 0.3 to 0.8% (v/v), 0.3 to 0.7% (v/v), 0.3 to 0.6% (v/v), 0.3 to 0.5% (v/v), 0.4 to 5% (v/v), 0.4 to 4% (v/v), 0.4 to 3% (v/v), 0.4 to 2% (v/v), 0.4 to 1% (v/v), 0.4 to 0.9% (v/v), 0.4 to 0.8% (v/v), 0.4 to 0.7% (v/v), 0.4 to 0.6% (v/v), 0.4 to 0.5% (v/v), 0.5 to 5% (v/v), 0.5 to 4% (v/v), 0.5 to 3% (v/v), 0.5 to 2% (v/v), 0.5 to 1% (v/v), 0.5 to 0.9% (v/v), 0.5 to 0.8% (v/v), 0.5 to 0.7% (v/v), or 0.5 to 0.6% (v/v). Preferably, said C2-C3 alcohol (in particular those mentioned above and notably ethanol) is present in a concentration not exceeding 2% (v/v) (in particular any range disclosed above with a higher value of at most 2%) and more preferably close to 0.5% (v/v), such as 0.1 to 1% (v/v), 0.1 to 0.9% (v/v), 0.2 to 0.8% (v/v), 0.3 to 0.7% (v/v), 0.4 to 0.6% (v/v), most preferably about 0.5% (v/v).
Sodium Glutamate
White its stabilizing effect is less pronounced, the liquid formulations according to the invention may also comprise sodium glutamate in a concentration tower than 10 mM, such as 0 to 10 mM, 0 to 9 mM, 0 to 8 mM, 0 to 7.5 mM, 0 to 7 mM, 0 to 6.5 mM, 0 to 6 mM, 0 to 5.5 mM, 0 to 5 mM, 1 to 10 mM, 1 to 9 mM, 1 to 8 mM, 1 to 7.5 mM, 1 to 7 mM, 1 to 6.5 mM, 1 to 6 mM, 1 to 5.5 mM, 1 to 5 mM, 2 to 10 mM, 2 to 9 mM, 2 to 8 mM, 2 to 7.5 mM, 2 to 7 mM, 2 to 6.5 mM, 2 to 6 mM, 2 to 5.5 mM, 2 to 5 mM, 2.5 to 10 mM, 2.5 to 9 mM, 2.5 to 8 mM, 2.5 to 7.5 mM, 2.5 to 7 mM, 2.5 to 6.5 mM, 2.5 to 6 mM, 2.5 to 5.5 mM, 2.5 to 5 mM, 3 to 10 mM, 3 to 9 mM, 3 to 8 mM, 3 to 7.5 mM, 3 to 7 mM, 3 to 6.5 mM, 3 to 6 mM, 3 to 5.5 mM, 3 to 5 mM, 3.5 to 10 mM, 3.5 to 9 mM, 3.5 to 8 mM, 3.5 to 7.5 mM, 3.5 to 7 mM, 3.5 to 6.5 mM, 3.5 to 6 mM, 3.5 to 5.5 mM, 3.5 to 5 mM, 4 to 10 mM, 4 to 9 mM, 4 to 8 mM, 4 to 7.5 mM, 4 to 7 mM, 4 to 6.5 mM, 4 to 6 mM, 4 to 5.5 mM, 4 to 5 mM, 4.5 to 10 mM, 4.5 to 9 mM, 4.5 to 8 mM, 4.5 to 7.5 mM, 4.5 to 7 mM, 4.5 to 6.5 mM, 4.5 to 6 mM, 4.5 to 5.5 mM, 4.5 to 5 mM, 5 to 10 mM, 5 to 9 mM, 5 to 8 mM, 5 to 7.5 mM, 5 to 7 mM, 5 to 6.5 mM, 5 to 6 mM, or 5 to 5.5 mM.
In particular, the inventors have found that, notably for MVA, the presence of sodium glutamate in a concentration of about 5 mM is optimal. When sodium glutamate is present in liquid formulations according to the invention, it is thus preferably present in a concentration close to 5 mM, such as 2.5 to 7.5 mM, 3 to 7 mM, 3.5 to 6.5 mM, 4 to 6 mM, 4.5 to 5.5 mM, more preferably about 5 mM.
Potentially Excluded Compounds
Surfactant
Non-ionic surfactants have been shown to induce stabilization of various viruses in the liquid state (see EVANS et al. J Pharm Sci. 2004 October, 93(10):2458-75, U.S. Pat. No. 7,456,009, SHI et al. J Pharm Sci. 2005 July, 94(7):1538-51, US2007/0161085).
However, for vaccinia virus, the inventors found that the presence of a surfactant such as non-ionic surfactant Tween 80 (also known as polysorbate 80) at low concentration has no beneficial effect and that concentrations above 0.02% v/v or even above 0.005% v/v are deleterious to the stability of vaccinia virus (see Example 1).
If polysorbate, or more generally a non-ionic surfactant or even any surfactant is present in a liquid composition according to the invention, it should be present in a concentration lower than 0.1%, preferably lower than 0.05% (v/v), lower than 0.04% (v/v), lower than 0.03% (v/v), lower than 0.02% (v/v), lower than 0.01% (v/v), lower than 0.009% (v/v), lower than 0.008% (v/v), lower, than 0.007% (v/v), tower than 0.006% (v/v), lower than 0.005% (v/v), lower than 0.004% (v/v), lower than 0.003% (v/v), lower than 0.002% (v/v), or even lower than 0.001% (v/v).
In another preferred embodiment of a liquid composition according to the invention, the liquid composition is free of polysorbate, or more generally free of non-ionic surfactants, or even more generally free of any surfactant.
In a preferred embodiment, the liquid formulation according to the invention is free of a surfactant or comprises a surfactant at a concentration lower than 0.1%, preferably lower than 0.05% (v/v), lower than 0.04% (v/v), lower than 0.03% (v/v), tower than 0.02% (v/v), lower than 0.01% (v/v), lower than 0.009% (v/v), lower than 0.008% (v/v), lower than 0.007% (v/v), tower than 0.006% (v/v), lower than 0.005% (v/v), lower than 0.004% (v/v), lower than 0.003% (v/v), lower than 0.002% (v/v), or even lower than 0.001% (v/v).
Divalent Salts
Divalent salts such as MgCl2 or CaCl2 have been found to induce stabilization of various viruses in the liquid state (see EVANS et al. J Pharm Sci. 2004 October, 93(10):2458-75 and U.S. Pat. No. 7,456,009). In such cases, divalent cations were present in the liquid formulation at a concentration of at least 0.5 mM, and preferably at least 1 mM.
The inventors however found that the presence of divalent salts does not have high beneficial effect on vaccinia virus stability (see Example 1), and rather has deleterious effects at higher concentrations (at least 75 mM). Liquid formulations according to the invention may thus be free of MgCl2 and/or CaCl2, or more generally of divalent salts. However, since divalent cations appear to have no deleterious effect on vaccinia virus stability at low concentration, such divalent cations may be present in liquid formulations according to the invention, in particular in low concentration. When such divalent cations (in particular MgCl2 or CaCl2) are present in liquid formulations according to the invention, they are nevertheless preferably present at a concentration lower than 100 mM, preferably lower than 90 mM, lower than 80 mM, lower than 75 mM, lower than 70 mM, lower than 60 mM, tower than 50 mM, lower than 45 mM, lower than 40 mM, lower than 35 mM, lower than 30 mM, lower than 25 mM, lower than 20 mM, lower than 15 mM, lower than 10 mM, lower than 9 mM, lower than 8 mM, lower than 7 mM, lower than 6 mM, lower than 5 mM, lower than 4 mM, lower than 3 mM, lower than 2 mM, more preferably lower than 1 mM, lower than 0.9 mM, lower than 0.8 mM, lower than 0.7 mM, lower than 0.6 mM, lower than 0.5 mM.
In a preferred embodiment, the liquid formulation according to the invention is free of divalent salts or comprises divalent salts at a concentration lower than 100 mM, preferably lower than 90 mM, lower than 80 mM, lower than 75 mM, lower than 70 mM, lower than 60 mM, lower than 50 mM, lower than 45 mM, lower than 40 mM, lower than 35 mM, lower than 30 mM, lower than 25 mM, lower than 20 mM, lower than 15 mM, lower than 10 mM, lower than 9 mM, lower than 8 mM, lower than 7 mM, lower than 6 mM, lower than 5 mM, lower than 4 mM, lower than 3 mM, lower than 2 mM, more preferably lower than 1 mM, lower than 0.9 mM, lower than 0.8 mM, lower than 0.7 mM, lower than 0.6 mM, lower than 0.5 mM.
Amino Acids Other than Glutamic Acid
Amino acids, and in particular histidine, arginine or methionine, have been found to induce stabilization of various viruses in the liquid state (see EVANS et al. J Pharm Sci. 2004 October, 93(10):2458-75, U.S. Pat. No. 7,456,009, US2007/0161085, U.S. Pat. No. 7,914,979, WO2014/029702, WO2014/053571). When histidine is present in a liquid formulation in order to improve stability, histidine is generally present in a concentration of at least 5 mM, and preferably at least 10 mM (see EVANS et al. J Pharm Sci. 2004 October, 93(10):2458-75, U.S. Pat. No. 7,456,009, WO2014/029702). When arginine is present in a liquid formulation in order to improve stability, arginine is generally present in a concentration of at least 50 mM (see US2007/0161085, at least 1% w/v arginine corresponding to at least about 57.4 mM), and sometimes preferably at least 150 mM, and in particular about 300 mM (see WO2014/029702). When methionine is present in a liquid formulation in order to improve stability, methionine is generally present in a concentration of at least 25 mM, and preferably about 67 mM (see WO2014/029702). The inventors however found that the presence of amino acids other than glutamic acid (arginine, histidine or amino acids in general) has no beneficial effect on vaccinia virus stability (see Example 1).
Liquid formulations according to the invention may thus be free of histidine. Alternatively or in addition, liquid formulations according to the invention may be free of arginine. Alternatively or in addition, liquid formulations according to the invention may be free of methionine. In particular, liquid formulations according to the invention may be free of arginine and methionine, or even free of histidine, arginine and methionine. More generally, liquid formulations according to the invention may be free of amino acids other than glutamic acid.
However, while having no beneficial effect, such amino acids other than sodium glutamate were not found to have deleterious effect, and may thus be present in liquid formulations according to the invention, in particular in tow concentration.
When histidine is present in liquid formulations according to the invention, it is nevertheless preferably present at a concentration lower than 10 mM, preferably lower than 9 mM, lower than 8 mM, lower than 7.5 mM, lower than 7 mM, tower than 6 mM, or even lower than 5 mM.
Similarly, when arginine is present in liquid formulations according to the invention, it is nevertheless preferably present at a concentration tower than 300 mM, preferably lower than 150 mM, lower than 100 mM, lower than 75 mM, or even lower than 50 mM. Also, when methionine is present in liquid formulations according to the invention, it is nevertheless preferably present at a concentration lower than 60 mM, preferably lower than 50 mM, tower than 40 mM, lower than 30 mM, or even lower than 25 mM. More generally, when one or more amino acids other than sodium glutamate is/are present in liquid formulations according to the invention, it/they is/are preferably present at a concentration tower than 300 mM, preferably lower than 150 mM, lower than 100 mM, lower than 75 mM, lower than 50 mM, lower than 40 mM, lower than 30 mM, lower than 25 mM, lower than 20 mM, lower than 10 mM, lower than 9 mM, lower than 8 mM, lower than 7.5 mM, lower than 7 mM, lower than 6 mM, or even lower than 5 mM.
In a preferred embodiment, the liquid formulation according to the invention is free of histidine or comprises histidine at a concentration lower than 10 mM, preferably lower than 9 mM, lower than 8 mM, lower than 7.5 mM, lower than 7 mM, lower than 6 mM, or even lower than 5 mM.
In a preferred embodiment, the liquid formulation according to the invention is free of arginine or comprises arginine at a concentration lower than 300 mM, preferably tower than 150 mM, lower than 100 mM, lower than 75 mM, or even lower than 50 mM.
In a preferred embodiment, the liquid formulation according to the invention is free of methionine or comprises methionine at a concentration lower than 60 mM, preferably lower than 50 mM, lower than 40 mM, lower than 30 mM, or even lower than 25 mM.
In a preferred embodiment, the liquid formulation according to the invention is free of amino acids other than sodium glutamate or comprises amino acids other than sodium glutamate at a concentration lower than 300 mM, preferably lower than 150 mM, lower than 100 mM, lower than 75 mM, lower than 50 mM, lower than 40 mM, lower than 30 mM, lower than 25 mM, lower than 20 mM, lower than 10 mM, lower than 9 mM, lower than 8 mM, lower than 7.5 mM, lower than 7 mM, lower than 6 mM, or even lower than 5 mM.
Urea
The inventors also found that urea has no beneficial effect on vaccinia virus stability (see Example 1).
Liquid formulations according to the invention may thus preferably be free of urea.
High Molecular Weight Polymers
Freeze-dried virus formulations generally contain high molecular weight polymers such as dextran or polyvinylpyrrolidone (PVP), which assist in the formation of the cake during freeze-drying (see EP1418942 and WO2014/053571).
However, such high molecular weight polymers are not useful for stabilization of vaccinia virus in the liquid state and liquid formulations according to the invention may thus be free of such high molecular weight polymers. If present in liquid formulations according to the invention, they are preferably present at a concentration lower than 10 g/L, preferably lower than 7.5 g/L, lower than 5 g/L, lower than 2.5 g/L, or even lower than 1 g/L.
In a preferred embodiment, the liquid formulation according to the invention is thus free of dextran, PVP or more generally of high molecular weight polymers or comprises dextran, PVP or more generally of high molecular weight polymers at a concentration lower than 10 g/L, preferably lower than 7.5 g/L, lower than 5 g/L, lower than 2.5 g/L, or even lower than 1 g/L.
Animal- or Human-derived Stabilizers
Animal- or human-derived stabilizers such as serum or gelatin have been used for a long time for stabilization of live viruses (see US2007/0161085). However, such animal- or human-derived stabilizers of animal or human origin potentially involve a health risk, due to potential contamination by viral or non-conventional agents.
Such animal- or human-derived stabilizers are not necessary for stability of vaccinia virus in liquid formulations according to the invention, and liquid formulations according to the invention are thus preferably free of animal- or human-derived stabilizers, such as serum or gelatin.
Preferred Formulations
Various specific compounds belonging to the family of each essential or optional element of the formulations according to the invention have been described above in the section specifically relating to this element. In the context of the invention, each list of appropriate compounds for a particular element and each specific compound disclosed for a particular element may be combined with any generic other element, list of appropriate compounds for said other element or any specific compound disclosed for said other element.
In particular, preferred embodiments of an essential or optional element of the formulations according to the invention may be combined with any generic other element or with preferred embodiments of said other element.
Particularly preferred formulations according to the invention include formulations comprising, consisting essentially of, or consisting of elements mentioned in Table 1 below:
Use of a Pharmaceutically Acceptable Chelating Agent for Stabilizing a Vaccinia Virus Against UV Damage
Vaccinia virus is particularly sensitive to UV damage (see LYTLE et al. J. Virol. 2005, 79 (22): 14244).
Surprisingly, the inventors found that EDTA has a protecting effect on vaccinia virus against UV damage (see Example 6). The present invention thus also relates to the use of a pharmaceutically acceptable chelating agent for stabilizing a poxvirus (in particular a vaccinia virus) against UV damage.
For stabilization of poxvirus (in particular vaccinia virus) against UV damage, the chelating agent is preferably selected from ethylenediaminetetraacetic acid (EDTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), ethylene glycol tetraacetic acid (EGTA), dimercaptosuccinic acid (DMSA), diethylene triamine pentaacetic acid (DTPA), and 2,3-Dimercapto-1-propanesulfonic acid (DMPS), preferably said pharmaceutically acceptable chelating agent is EDTA.
When using a pharmaceutically acceptable chelating agent for stabilizing a poxvirus (in particular a vaccinia virus) against UV damage, said poxvirus (in particular vaccinia virus) is preferably in a liquid composition and said pharmaceutically acceptable chelating agent (in particular those mentioned above and notably EDTA) is preferably present in a concentration of at least 50 μM, preferably 50 to 1000 μM, 50 to 750 μM, 50 to 500 μM, 50 to 400 μM, 50 to 300 μM, 50 to 250 μM, 50 to 200 μM, 50 to 150 μM; 50 to 100 μM, 50 to 75 μM, 75 to 1000 μM, 75 to 750 μM, 75 to 500 μM, 75 to 400 μM, 75 to 300 μM, 75 to 250 μM, 75 to 200 μM, 75 to 150 μM; 75 to 100 μM, 100 to 1000 μM, 100 to 750 μM, 100 to 500 μM, 100 to 400 μM, 100 to 300 μM, 100 to 250 μM, 100 to 200 μM, 100 to 150 μM; 150 to 1000 μM, 150 to 750 μM, 150 to 500 μM, 150 to 400 μM, 150 to 300 μM, 150 to 250 μM, or 150 to 200 μM.
Even more preferably, for stabilizing a poxvirus (in particular a vaccinia virus) against UV damage, a liquid formulation according to the invention (as disclosed above) is used.
The following examples merely intend to illustrate the present invention.
The effect of various candidate compounds for stabilizing MVA virus in a liquid formulation at +5° C. has been tested based on compounds known from prior art to have stabilizing effect of other types of viruses.
Materials and Methods
Viruses
The following MVA viruses were used:
The three MVA viruses were produced in chicken embryo fibroblast, and recovered and purified by a method comprising recovery of infected CEF culture, breakage of cells my mechanical means, and various purification steps that do not involve any step of treatment with a protease.
A recombinant vaccinia virus of strain Wyeth, produced in a human continuous cell line and purified by a method that involves at least one step of treatment with at least one protease (VV Wyeth) was also used, at an initial target titer of 2 108 to 2 109 PFU/mL.
Tested Formulations
Tested formulations are represented in Tables 2 to 11 below:
Analysis of Stability
Stability was analyzed at +37° C. (±2° C.), +25° C. (±2° C.), and/or +5° C. (±3° C.) (see results section).
At +37° C. (±2° C.) (accelerated stability test), samples were kept in relative humidity of 75% and stability was analyzed by measuring infectious losses during at least 28 days (with intermediate measures at days 7 and 14).
At +25° C. (±2° C.) (accelerated stability test), samples were kept in relative humidity of 60% and stability was analyzed by measuring infectious losses during at least 6 months (with intermediate measures at about 1 month, and at 2 and 3 months).
At +5° C. (±3° C.) (target storage temperature test), samples were kept without any control of relative humidity and stability was analyzed by measuring infectious losses during at least 24 months (with intermediate measures at about 1 month, and at 3, 6, 12, 18 and 24 months).
Infectious losses were calculated by subtracting the number of infectious genomes or particle forming units per mL (IG/mL or PFU/mL) at the time measure to the initial number of IG/mL or PFU/mL at day 0, and expressed as decimal logarithm (log10 (IG/mL or PFU/mL)), abbreviated in the present description as log (IG/mL or PFU/mL).
Measure of Infectious Titers
Infectious titers at a given time may be measured either by measuring the number of infectious genomes (IG) per mL (IG/mL) or by using a plaque assay on BHK-21 cells (infectious vaccinia virus titer is then expressed in Plaque forming units (PFU) per mL (PFU/mL)). Measure of the number of infectious genomes per mL (IG/mL) has been preferred here, since this method is more rapid and more precise. However, while no specific data is shown using plaque assay on BHK-21 cells, it should be noted that infectious titers were at some points measured also using plaque assay on BHK-21 cells and that results were always found to be consistent with results obtained using the infectious genomes method.
Measure of the number of infectious genomes per mL (IG/mL) is performed as follows:
This method can measure infectious genomes (IG/mL) for any value of at least 1·105 IG/mL.
Measure of infectious titers using plaque assay on BHK-21 cells is performed as follows:
1. Cells Spreading
Host cells BHK-21 were grown in monolayers in DMEM. At confluency, the cells were washed with 10 mL PBS and then trypsinated. After removing trypsine, cells were then resuspended in 10 mL DMEM with 10% SFV at 37° C.
Then, cells suspension were homogenized and distributed in the multi-well plates (2 mL in each of the 6 wells of the plate). Then, said plates were incubated at 37° C., 5% CO2.
2. Cells Infection
About 1 day after cells spreading, aliquots of virus suspensions were added in each well comprising the BHK-21 cells of step 1. If necessary, said suspensions were firstly diluted serially in PBS, cations 100× and 1% fetal calf serum (FCS), according to method well known by the person skilled in the art. Depending on the case, the virus suspensions which were added to BHK-21 cells of step 1 were either liquid virus-containing compositions before freeze-drying or reconstituted virus-containing composition (i.e after lyophilization, at different time periods and temperatures).
Culture medium was then removed and after stirring during 60 minutes at room temperature, 2 mL of the infection medium (DMEM+5% FCS) were distributed in each well. Plates were then incubated at 37° C., 5% CO2.
3. Cells Fixation
After the medium has been removed, cells were washed with PBS (about 1 mL per well). Then, 1 mL of a solution methanol/acetone (50/50) was added and the resulting mixture was gently stirred at room temperature.
The plates were then let to be dried at room temperature.
4. Detection and Titer Determination
Virus titer determination was performed according to well-known peroxydase reaction using anti-vaccine antibodies and anti-rabbit antibodies combined with peroxydase. More precisely, before reaction anti-vaccine antibodies were diluted 100 times in PBS+2% FCS. Then, 500 μL of said antibodies were added in each well and incubated at 37° C. during about 30 minutes and then washed 3 times with 1 mL PBS+1% Triton X-100.
The reaction with anti-rabbit antibodies combined with peroxydase was carried out in the same manner, except that before reaction, said antibodies are diluted 200 times in PBS+2% FCS.
The DAB solution was prepared by dissolving one commercial DAB tablet in 15 mL of TRIS-HCL 0.05M. Then, the obtained solution was filtrated on a filtration unit NALGENE of 2 μm and the resulting filtrated solution was added to 15 μL of aqueous solution of H2O2 30%. Once prepared, 1 mL of the DAB solution was added to each well and let until a brown coloration has appeared. The coloration solution was subsequently removed and results are visually interpreted.
Then, the infectious titer was calculated in PFU/mL, using the following formula:
[mean of viral plaques numbers×4]×dilution factor=number of PFU/mL
Each of these methods has similar variability, of about ±0.30 log10 for a single determination. However, variability of both methods decreases when increasing the number of determinations (i.e. the number of replicates tested). For a double determination (use of duplicate samples), a variability of ±0.25 log10 is expected. For a triple determination (use of triplicate samples), a variability of ±0.20 log10 is expected. In alt examples, a single determination has been made when measuring the number of infectious genomes (IG) per mL (IG/mL), while determination of Plaque forming units (PFU) per mL (PFU/mL) has been done in triplicates.
Results
Necessity of a Monovalent Salt
The stability of MVA-MUC1 has been tested in formulations containing Tris-HCl, Na glutamate, sucrose, pH 8.0, and containing either 0 mM or 50 mM of NaCl (see Table 2 above).
Infectious losses after 7, 14, or 28 days at 37° C. are presented in
Therefore, addition of NaCl to the formulation significantly improves stability.
Beneficial Effect of EDTA, a Low Concentration of EtOH or EDTA/EtOH
The influence of EDTA on stability of MVA-HCV at +37° C. and +5° C. in a formulation containing Tris-HCl, Na glutamate, sucrose, NaCl, pH 7.5 has been tested using various concentrations of EDTA (see Table 3). Results are presented in
At +37° C. (accelerated stability test), all formulations containing EDTA show less than 1 log infectious loss at 7 and 14 days, and less than 1.5 log infectious loss at 28 days. In marked contrast, formulations without EDTA (control DS and control DS2) showed a very weak stability profile (about 1, 2.5 and 4 log infectious loss at 7, 14 and 28 days, respectively). The concentration of EDTA used (from 50 μM to 1000 μM) does not seem to impact the stabilizing effect. At day 28, formulations containing EtOH in addition to EDTA appear to be further stabilized (see
The influence of ethanol (EtOH) on stability of MVA-HCV at +37° C. and +5° C. in a formulation containing Tris-HCl, Na glutamate, sucrose, NaCl, pH 7.5 has been tested using various concentrations of EtOH (see Table 3). Results are presented in
Therefore, primary tests using EDTA and EtOH show that both compounds independently increase stability of MVA-HCV in liquid formulations, the stabilizing effect of EDTA being higher than the stabilizing effect of EtOH. Moreover, combination of both compounds further increases stability.
Further experiments were performed to confirm stability of MVA-HCV in formulations containing varying EDTA (50 to 250 μM) and EtOH (0.5 to 2.5%) concentrations (see Table 4) at +37° C., 25° C. and 5° C. Results are presented in
Another representation of the results obtained at +37° C. and +5° C. is presented in
Based on this analysis, an optimal formulation using 150 μM EDTA and 0.5% v/v EtOH was defined.
Beneficial Effect of a Low Concentration of Sodium Glutamate
The influence of Na glutamate on stability of MVA-HCV at +37° C. and +25° C. in a formulation containing Tris-HCl, Na glutamate, sucrose, NaCl, pH 7.5 has been tested with or without various concentrations of Na glutamate from 0 to 10 mM (see Table 5). Results are presented in
At +37° C., the three formulations containing at least 5 mM of Na glutamate show infectious losses lower than 1 at day 28, while the two formulations containing 0 mM or 2.5 mM of Na glutamate have infectious losses higher than but close to 1. This shows that Na glutamate does not have high stabilizing effect, but that a low concentration of Na glutamate, between 5 and 10 mM, may have a minor stabilizing effect. In addition,
At +25° C., infectious losses at 12 months confirm that Na glutamate has a small stabilizing effect, and that a concentration of about 5 mM is optimal (
At +5° C., infectious losses from 12 months to 30 months confirm that Na glutamate has a small stabilizing effect, and that a concentration of about 5 mM is optimal (
Beneficial Effect of a Low Concentration of Sucrose
The influence of sucrose on stability of MVA-MUC1 at +37° C. in a formulation containing Tris-HCl, Na glutamate, sucrose, NaCl, pH 8.0 has been tested with various concentrations of sucrose (see Table 6). Results are presented in
No Beneficial Effect and Even Deleterious Effect of Polysorbate 80 or Polysorbate 40
The influence of polysorbate 80 or polysorbate 40 on stability of MVA-HPV at +25° C. and +5° C. in a formulation containing Tris-HCl, Na glutamate, sucrose, NaCl, pH 7.5 (see Table 7) has been tested using various concentrations of polysorbate 80 or polysorbate 40 shown to have stabilizing effect on other viruses (see EVANS et al. J Pharm Sci. 2004 October, 93(10):2458-75, U.S. Pat. No. 7,456,009, SHI et al. J Pharm Sci. 2005 July, 94(7):1538-51, US2007/0161085). Results are presented in
At +25° C. and +5° C., no concentration of polysorbate permits to increase stability compared to control formulation without polysorbate, so that no stabilizing effect is observed.
In contrast, at both temperatures, for concentrations of polysorbate of at least 0.02% v/v, a destabilizing effect can be noted, which increases with the concentration of polysorbate used. At +5° C., even the very low concentration of 0.005% v/v results in some destabilizing effect.
It must therefore be concluded that polysorbate does not have stabilizing effect, and that concentrations of at least 0.02% v/v rather have destabilizing effect. Polysorbate should thus preferably be excluded or present at very low concentrations in liquid formulations of vaccinia virus.
Similarly, stability of a vaccinia virus Wyeth strain produced in a human continuous cell line and purified by a method that involves at least one step of treatment with at least one protease, in a control formulation containing Tris-HCl 30 mM, sucrose 10% (w/v), or in a formulation further containing 150 μg/mL polysorbate 80 was tested after 7, 14, 21, or 28 days at +37° C. Results are presented in
No Beneficial Effect and Even Deleterious Effect at High Concentrations of MgCl2
The influence of MgCl2 on stability of MVA-MUC1 at +37° C. during 14 days in a formulation containing Tris-HCl, Na glutamate, sucrose, NaCl, pH 8.0 (see Table 8) has been tested with or without various concentrations of MgCl2 shown to have stabilizing effect on other viruses (see EVANS et al. J Pharm Sci. 2004 October, 93(10):2458-75 and U.S. Pat. No. 7,456,009). Results are presented in
MgCl2 should thus preferably be excluded or present at low concentrations in liquid formulations of vaccinia virus.
This is all the more true because optimized formulations according to the invention contain a chelating agent (in particular a divalent cations chelating agent and, more preferably EDTA), which may thus interfere with any low beneficial effect of MgCl2 on poxviruses, and more particularly vaccinia viruses, stability.
Further evidence of deleterious effect of MgCl2 in formulations according to the invention is presented in Example 5 below (see also
Similarly, stability of a vaccinia virus Wyeth strain produced in a human continuous cell line and purified by a method that involves at least one step of treatment with at least one protease, in a control formulation containing Tris-HCl 30 mM, sucrose 10% (w/v), or in a formulation further containing 1000 mM (1M) MgCl2 was tested after 7 or 14 days at +37° C. Results are presented in
No Beneficial Effect and Rather Deleterious Effect of Arginine
The influence of arginine on stability of MVA-MUC1 at +37° C. in a formulation containing Tris-HCl, Na glutamate, sucrose, NaCl, pH 8.0 (see Table 9) has been tested with or without various concentrations of arginine shown to have stabilizing effect on other viruses (see US2007/0161085). Results are presented in
Arginine should thus preferably be excluded or present at very low concentrations in liquid formulations of vaccinia virus.
Similarly, stability of a vaccinia virus Wyeth strain produced in a human continuous cell line and purified by a method that involves at least one step of treatment with at least one protease, in a control formulation containing Tris-HCl 30 mM, sucrose 10% (w/v), or in a formulation further containing 50 mM arginine was tested after 7, 14, 21, or 28 days at +37° C. Results are presented in
No Beneficial Effect of a Mixture of Amino Acids
The influence of a mixture of amino acids on stability of MVA-MUC1 at +37° C. and +25° C. in a formulation containing Tris-HCl, Na glutamate, sucrose, NaCl, pH 8.0 (see Table 10) has been tested with or without said mixture of amino acids. Results are presented in
While a mixture of amino acids may be present in liquid formulations of vaccinia virus, it is clearly not essential and does not need to be present.
No Beneficial Effect and Rather Deleterious Effect of Histidine
The influence of histidine on stability of MVA-HPV at +25° C. and +5° C. in a formulation containing Tris-HCl, Na glutamate, sucrose, NaCl, pH 7.5 has been tested with or without 10 mM histidine (see Table 11), a concentration shown to have stabilizing effect on other viruses (see EVANS et al. J Pharm Sci. 2004 October, 93(10):2458-75, U.S. Pat. No. 7,456,009, US2007/0161085, U.S. Pat. No. 7,914,979, WO2014/029702, WO2014/053571).
Results are presented in
No stabilizing effect of histidine has been observed at +25° C. or at 5° C. In contrast, a trend towards a destabilizing effect can be observed at both temperatures.
Histidine should thus preferably be excluded or present at very low concentrations in liquid formulations of vaccinia virus.
Conclusions
The above results clearly show that:
The influence of the pH of the liquid formulation on vaccinia virus stability has been tested, in order to determine a suitable pH range for stable liquid formulations.
Materials and Methods
MVA Viruses
The MVA virus used was MVA-HCV (TG4040), a recombinant MVA virus expressing nonstructural HCV proteins (NS3, NS4 and NS5B) from HCV genotype 1 b strain ja (see WO2004/111082), which was diluted to an initial target concentration of 4-8 107 IG/mL. MVA-HCV was produced in chicken embryo fibroblast (CEF), and recovered and purified by a method comprising recovery of infected CEF culture, breakage of cells by mechanical means, and various purification steps that do not involve any step of treatment with a protease.
Tested Formulations
Tested formulations are represented in Table 12 below:
Analysis of Stability
Analysis of stability was done as described in Example 1.
Measure of Infectious Titers
Measure of infectious titers was done as described in Example 1.
Results
The influence of pH on stability of MVA-HCV at +37° C. and +5° C. in a formulation containing Tris-HCl, Na glutamate, sucrose, NaCl, EDTA and ethanol has been tested at various pH values. Results are presented in
At +37° C. (
At +5° C. also (
Conclusions
The above results show that pH of a liquid formulation of vaccinia virus should preferably be comprised between more than 6 and less than 9. In particular, good results are obtained when the pH is comprised between 7 and 8. A pH comprised between 6.5 and 8.5 might thus be acceptable.
Then influence of vaccinia virus initial titer on subsequent stability in a liquid formulation was also tested.
Materials and Methods
MVA Viruses
The MVA virus used was MVA-HCV (TG4040), a recombinant MVA virus expressing nonstructural HCV proteins (NS3, NS4 and NS5B) from HCV genotype 1 b strain ja (see WO2004/111082), which was diluted to varying initial target concentrations: 1.0-108 PFU/mL, 5.0 107 PFU/mL, 1.0 107 PFU/mL, and 5.0 106 PFU/mL.
MVA-HCV was produced in chicken embryo fibroblast, and recovered and purified by a method comprising recovery of infected CEF culture, breakage of cells by mechanical means, and various purification steps that do not involve any step of treatment with a protease.
Tested Formulations
Tested formulations are represented in Table 13 below:
Analysis of Stability
Analysis of stability was done as described in Example 1.
Measure of Infectious Titers
Measure of infectious titers was done as described in Example 1.
Results
Evolution of infectious losses of MVA-HCV at varying initial titers at +37° C., +25° C. and +5° C. are presented in
At +37° C., infectious titers of all formulations according to the invention were lower than 1 log after 7 days. However, a trend towards higher stability of formulations with higher initial MVA-HCV titer may be observed. At day 14, infectious titers of all formulations according to the invention excepted the formulation containing an initial MVA-HCV titer of 5.0 106 PFU/mL were still lower than 1 log, but a clear trend of higher stability of formulations with higher initial MVA-HCV titer is observed. This observation is confirmed at day 28, only formulations according to the invention containing an initial MVA-HCV titer of 5.0 107 PFU/mL or 1.0 108 PFU/mL showing a infectious loss lower than 1 log (
The same type of observations can be made at +25° C. (
At +5° C., the difference of stability of formulations according to the invention depending on MVA-HCV initial titer distinguishes the same two subfamilies as at 25° C. (
Conclusions
In view of the above results, it appears that an initial titer of at least 1.0 107 PFU/mL is highly preferable for guaranteeing stability of a liquid formulation of MVA.
In order to confirm the stability of optimized formulations defined in previous examples, such optimized formulations were tested on various vaccinia virus strains, in two distinct experiments.
Materials and Methods
Viruses
The following vaccinia viruses were used:
Tested formulations for MVA viruses are represented in Table 14 below:
Experiment 2:
Tested formulations for MVA-HCV/CEC, MVA-HCV/duck cell line, MVA-FCU1/CEC, and Copenhagen-FCU1/CEC viruses are represented in Table 15A below:
Experiment 3:
Tested formulations for VV Wyeth virus are represented in Table 156 below:
Analysis of Stability
Analysis of stability was done as described in Example 1.
Measure of Infectious Titers
Measure of infectious titers was done as described in Example 1.
Results
Experiment 1:
Stability of the three MVA viruses in the optimized formulation of the invention at +37° C., +25° C. and +5° C. is presented in
At +37° C., all three MVA vectors showed less than 1 log loss of infectious titer at day 28, thus showing very good stability at this elevated temperature.
At +25° C., all three MVA vectors also showed less than 1 log loss of infectious titer at 6 months, thus also showing very good stability at this temperature.
Finally, all three MVA vectors also showed less than 0.3 log loss of infectious titer after 30 months at +5° C., thus showing very high stability at this targeted storage temperature.
While some minor variations may be observed depending on the MVA vector used, the above results clearly show that the optimized formulation designed in preceding examples is applicable to any MVA vector, no matter what is/are the heterogeneous sequences inserted into it.
Experiment 2:
In this second experiment, the same formulation previously optimized for MVA was tested on several MVA viruses obtained by various methods, and on another vaccinia virus strain: Copenhagen (vector TG6002).
Results are presented in
The less stabilized virus is MVA-HCV/CEC. This may be explained by the fact that this virus is the virus used at the lowest initial titer.
Experiment 3:
In this third experiment, the same formulation previously optimized for MVA was tested on another strain of vaccinia virus (Wyeth strain), which had been produced in a human continuous cell line and purified by a method that involves at least one step of treatment with at least one protease.
Results are presented in
It should also be noted that, in the context of a virus environment that may contain some residual protease, increasing the monovalent salt (NaCl) from 200 to 500 mM further increases stability of the virus.
Conclusions
The above results clearly demonstrate that the optimized formulations designed by the inventors are applicable to various vaccinia viruses strains, and that the heterogeneous constructions that may be inserted in such viruses do not significantly influence the stabilization provided by the optimized formulations according to the invention.
The above results also show that optimized formulations designed by the inventors are applicable even when the purification process involves treatment with at least one protease and thus when the virus environment may contain residual protease. In this particular case, increasing the monovalent salt concentration of the formulation over 200 mM further improves stability.
In order to confirm whether the individual compounds tested before could be replaced by other compounds of the same family of or other families, an experiment was performed, in which each of the previously tested individual compounds of an optimized formulation was replaced by a compound of the same family of or other families:
The MVA virus used was MVA-MUC1 (TG4010), a recombinant MVA virus expressing MUC1 tumor associated antigen and interleukin 2 (see WO92/07000 and WO95/09241), diluted to an initial target titer between 8.0 107 and 2 108 IG/mL.
MVA-MUC1 was produced in chicken embryo fibroblast, and recovered and purified by a method comprising recovery of infected CEF culture, breakage of cells by mechanical means, and various purification steps that do not involve any step of treatment with a protease.
Tested Formulations
Tested formulations are represented in Table 16 below:
Analysis of Stability
Analysis of stability was done as described in Example 1.
Measure of Infectious Titers
Measure of infectious titers was done as described in Example 1.
Results
Infectious losses of the various tested formulations at +37° C. are represented in
The statistical significance of replacing the initially tested compound by one of the other compounds mentioned above was analyzed using NemrodW® software after 21 (
Infectious losses of the various tested formulations at +5° C. are represented in
Conclusions
The above results clearly demonstrate that, in an optimized formulation according to the invention, initially tested compounds may be replaced by other compounds of the same family (Tris-HCl by another buffer with buffering capacity between pH 7 and 8, NaCl by another monovalent salt, sucrose by another disaccharide or a sugar alcohol, EDTA by another chelating agent, EtOH by another C2-C3 alcohol), without significantly altering the stability of vaccinia virus.
In contrast, NaCl or another monovalent salt should not be replaced by a divalent salt, thus confirming previous results showing deleterious effects of divalent salts when present at significant concentration (here 75 mM).
It was further verified if a stabilized liquid MVA formulation according to the invention had similar immunogenicity in vivo after 12 months storage than a just obtained MVA formulation.
Materials and Methods
MVA Virus
The MVA virus used was MVA-MUC1 (TG4010), a recombinant MVA virus expressing MUC1 tumor associated antigen and interleukin 2 (see WO92/07000 and WO95/09241), diluted to an initial target titer of 1 to 3 108 PFU/mL
MVA-MUC1 was produced in chicken embryo fibroblast, and recovered and purified by a method comprising recovery of infected CEF culture, breakage of cells by mechanical means, and various purification steps that do not involve any step of treatment with a protease.
Tested Formulation and Storage
MVA-MUC1 was formulated in a liquid formulation containing Tris-HCl 20 mM, sucrose 10% w/v, NaCl 75 mM, EDTA 150 μM, EtOH 0.5% v/v, and Na glutamate 5 mM. For the immunogenicity test, were used either a just formulated MVA stored at −80° C. (T=0), or a formulated MVA stored during 12 months at +5° C.±3° C. (T=12 months).
Immunogenicity Test
The model used is a prophylactic model, in which the MVA-MUC1 vector is injected to mice before further administration of tumor cells expressing MUC1 antigen. Further details are given below:
1st Experiment:
The same model has been used to compare immunogenicity of formulation alone or MVA-MUC1 in formulation at T=0 and T=24 months, for a dose of 104 PFU.
Results
1st Experiment:
Table 17A below presents the percentage of mice that were tumor-free at day 86, depending on product (formulation alone or MVA-MUC1 after 0 or 12 months storage) and dose injected.
The above results show that:
Table 17B below presents the percentage of mice that were tumor-free at day 65, depending on product (formulation alone or MVA-MUC1 after 0 or 24 months storage).
The above results show that:
The above data confirm that optimized formulations according to the invention not only maintain infectious virus titers during storage for two years, but also maintain the ability to induce a protective immune response in vivo.
Vaccinia virus is known to be sensitive to UV damage (see LYTLE et al. J. Virol. 2005, 79(22):14244). The ability of various formulations to protect vaccinia virus against UV damage was tested in two independent experiments.
Materials and Methods
MVA Viruses
The MVA virus used was MVA-HCV (TG4040), a recombinant MVA virus expressing nonstructural HCV proteins (NS3, NS4 and NS5B) from HCV genotype 1 b strain ja (see WO2004/111082), which was diluted to an initial target titer of between 5 107 and 7 107 IG/mL for experiment 1 and between 3 108 and 5 108 IG/mL for experiment 2.
MVA-HCV was produced in chicken embryo fibroblast, and recovered and purified by a method comprising recovery of infected CEF culture, breakage of cells by mechanical means, and various purification steps that do not involve any step of treatment with a protease.
Tested Formulations
Tested formulations for MVA-HCV in experiment 1 are represented in Table 18 below:
Tested formulations for MVA-HCV in experiment 2 are represented in Table 19 below:
Light Conditions
Samples were stored in the following light conditions:
Stability was analyzed at +25° C. (±2° C.) during 28 days.
Infectious tosses were calculated by subtracting the number of infectious genomes per mL (IG/mL) at the time measure to the initial number of IG/mL at day 0, and expressed as decimal logarithm (log10 (IG/mL)), abbreviated in the present description as log (IG/mL).
Measure of Infectious Titers
Measure of infectious titers was done as described in Example 1.
Results
Results are presented in
In
In addition, in the case of more reasonable light conditions (PSM conditions), using a liquid formulation according to the invention also results in significantly decreased destabilization of MVA-HCV, the infectious losses after 28 days at +25° C. being largely lower than 0.5 log whereas exceeding 1 log after 21 days at 25° C. with the control formulation.
In
Conclusions
The above results clearly show that formulations according to the invention not only stabilize vaccinia virus in the absence of light, but also further protect vaccinia virus against degradation due to UV damage. This may be very helpful for limiting constraints on vaccinia virus storage in a liquid formulation.
In order to test the robustness of the stabilizing effect of formulations according to the invention, several formulations containing varying concentrations of the distinct ingredients have been tested for their ability to stabilize an MVA vector.
Materials and Methods
Virus
The MVA virus used was MVA-MUC1 (TG4010), a recombinant MVA virus expressing MUC1 tumor associated antigen and interleukin 2 (see WO92/07000 and WO95/09241), diluted to an initial target titer of 1 to 4 108 PFU/mL.
MVA-MUC1 was produced in chicken embryo fibroblast, and recovered and purified by a method comprising recovery of infected CEF culture, breakage of cells by mechanical means, and various purification steps that do not involve any step of treatment with a protease.
Tested Formulations
Tested formulations are represented in Table 20 below:
Analysis of Stability
Analysis of stability was done as described in Example 1.
Measure of Infectious Titers
Measure of infectious titers was done as described in Example 1.
Results
Infectious losses of the various tested formulations at +37° C. are represented in
At day 21 after formulation, all tested formulations had infectious losses inferior to 1 log10. At day 28, most tested formulations also had infectious losses inferior to 1 log10.
Conclusion
The above data confirm that optimized formulations according to the invention have a robust stabilizing effect over a range of concentrations of ingredients contained in the formulation.
Materials and Methods
Virus
A non recombinant pseudocowpox virus (parapoxvirus family) was used at an initial target titer of 1 to 3 108 PFU/mL.
Tested Formulations
Tested formulations are represented in Table 21 below:
Analysis of Stability
Analysis of stability was done as described in Example 1 at 37° C.
Measure of Infectious Titers
Measure of infectious titers was done as described in Example 1.
Results
Results are presented in
Conclusion
The above results clearly show that formulations according to the invention are also suitable to stabilize pseudocowpox virus, a poxvirus of the parapoxvirus family.
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| Number | Date | Country | Kind |
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| 14306930 | Dec 2014 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
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| PCT/EP2015/078239 | 12/1/2015 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
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| WO2016/087457 | 6/9/2016 | WO | A |
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