A compound for stabilizing virus-based therapeutic agents.

Abstract

This invention relates to a viral preparation for administration to a subject.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to pharmaceutical formulations for virus-based therapeutic agents and virus-based cancer vaccines. More specifically, the present invention relates to pharmaceutical formulations for oncolytic viruses as disclosed herein.

[0002] Background of the Invention Oncolytic viruses, such as vesicular stomatitis virus (VSV) (VSV-GP) containing the glycoprotein (GP) of lymphocytic choriomeningitis virus, represent an emerging class of biopharmaceuticals that selectively replicate in cancer cells and selectively kill them. The oncolytic viruses described herein can spread within tumors and efficiently induce lysis of tumor cells, resulting in cell death. Furthermore, additional genes may be cloned into the viral genome, and the expression of such proteins can stimulate the immune system and / or direct the immune system towards tumor cells. Moreover, oncolytic viruses expressing cancer-specific antigens may be co-administered together with the antigens, thereby enhancing and prolonging their immunostimulatory effects. Therefore, oncolytic viruses are beneficial for the treatment and / or prevention of cancer.

[0003] Developing formulations to stabilize oncolytic viruses during storage is challenging but rewarding. Isolated and concentrated live viruses are typically unstable during long-term storage and are sensitive to high temperatures, mechanical stress, and freeze-thaw cycles. To be effective as a therapeutic agent, live viruses need to be formulated so that their activity is preserved and agglutination is prevented, thus preventing the formation of visible and invisible particles (SvPs). Viruses, particularly VSV-GP, tend to self-associate and agglutinate due to their structural complexity. Colloidal stability of the virus particles must be ensured, and agglutination must be reduced to below the limits specified in the official standards through formulation development efforts. For example, the number of freeze-thaw events for filling and labeling vials, as well as the freezing and storage conditions, such as the rate of freezing and thawing and the storage temperature, should be considered so that the virus does not reach the glass transition temperature (T) of the maximum freeze-concentrated solution. g Infectivity and colloidal stability are significantly affected, especially if they are not stored as a frozen liquid at temperatures below ').

[0004] While several formulations for virus-based therapeutics for specific viruses are described in the literature, it is generally understood that each virus species, genus, or family needs to be formulated with its own specific prescription.

[0005] Therefore, there is a need for stable liquid, freeze-liquid, and dried formulations (such as lyophilized formulations) for virus-based therapeutics suitable for parenteral administration, including intravenous or intratumoral injection, to human patients, particularly formulations for oncolytic viruses, such as VSV, especially VSV-GP. There is also a need for formulations that exhibit enhanced colloidal stability and show no or only minimal loss of the biological activity of the therapeutic virus during long-term storage at various temperatures. There is a particular need for lyophilized virus-based therapeutic formulations that explore the possibility of storing drug vials at room temperature or at 5°C for longer periods, while reducing their susceptibility to temperature fluctuations that may occur during cold logistics from the manufacturing site to the treatment site.

[0006] Summary of the Invention The present invention addresses the above-mentioned need by providing a stable formulation for a virus-based therapeutic agent, particularly a stable dried formulation (such as a freeze-dried formulation).

[0007] In a first embodiment, the present invention relates to a pharmaceutical composition comprising an enveloped virus, one or more sugar alcohols, and a protein substance and / or a poly(ethylene oxide) / poly(propylene oxide) block copolymer.

[0008] In one embodiment relating to the first aspect, the poly(ethylene oxide) and poly(propylene oxide) block copolymer is a poloxamer. In one embodiment relating to the first aspect, the poly(ethylene oxide) and poly(propylene oxide) block copolymer is a poloxamer 188.

[0009] In one embodiment relating to the first aspect, the protein substance is albumin or gelatin. In one embodiment relating to the first aspect, the protein substance is human serum albumin or recombinant human albumin.

[0010] In one embodiment relating to the first aspect, the poly(ethylene oxide) and poly(propylene oxide) block copolymer is poloxamer 188, and the protein substance is human serum albumin or recombinant human albumin. In the related embodiments, the pharmaceutical composition is 0.01-50 g / L, 0.1-50 g / L, 0.2-50 g / L, 0.3-50 g / L, 0.4-50 g / L, 0.5-50 g / L, 1-50 g / L, 2-50 g / L, 3-50 g / L, 4-50 g / L, 5-50 g / L, 0.01-40 g / L, 0.01-30 g / L, 0.01-20 g / L L, 0.01~10g / L, 0.01~5g / L, 0.1~40g / L, 0.1~30g / L, 0.1~20g / L, 0.1~10g / L, 0.1~5g / L, 0.2 ~40g / L, 0.2~30g / L, 0.2~20g / L, 0.2~10g / L, 0.2~5g / L, 0.3~40g / L, 0.3~30g / L, 0.3~20g / L, 0.3~10g / L, 0.3~5g / L, 0.4~40g / L, 0.4~30g / L, 0.4~20g / L, 0.4~10g / L, 0.4~5g / L, 0.5~40g / L, 0.5~30g / L, 0.5~20g / L, 0.5~10g / L, 0.5~5g / L, 1~40g / L, 1~30g / L, 1~20g / L, 1~10g / L, 1~ Contains poloxamer 188 at concentrations of 5 g / L, 2-40 g / L, 2-30 g / L, 2-20 g / L, 2-10 g / L, 3-40 g / L, 3-30 g / L, 3-20 g / L, 3-10 g / L, 4-40 g / L, 4-30 g / L, 4-20 g / L, 4-10 g / L, 5-40 g / L, 5-30 g / L, 5-20 g / L, or 5-10 g / L.In further related embodiments, the pharmaceutical composition is expressed in concentrations of 0.05-50 g / L, 0.1-50 g / L, 0.2-50 g / L, 0.3-50 g / L, 0.4-50 g / L, 0.5-50 g / L, 1-50 g / L, 2-50 g / L, 3-50 g / L, 4-50 g / L, 5-50 g / L, 0.05-40 g / L, 0.05-30 g / L, and 0.05-20 g / L. L, 0.05~10g / L, 0.05~5g / L, 0.1~40g / L, 0.1~30g / L, 0.1~20g / L, 0.1~10g / L, 0.1~5g / L, 0.2~4 0g / L, 0.2~30g / L, 0.2~20g / L, 0.2~10g / L, 0.2~5g / L, 0.3~40g / L, 0.3~30g / L, 0.3~20g / L, 0.3~ 10g / L, 0.3~5g / L, 0.4~40g / L, 0.4~30g / L, 0.4~20g / L, 0.4~10g / L, 0.4~5g / L, 0.5~40g / L, 0.5 ~30g / L, 0.5~20g / L, 0.5~10g / L, 0.5~5g / L, 1~40g / L, 1~30g / L, 1~20g / L, 1~10g / L, 1~5g / L, 2~4 Contains human serum albumin or recombinant human albumin at concentrations of 0 g / L, 2-30 g / L, 2-20 g / L, 2-10 g / L, 3-40 g / L, 3-30 g / L, 3-20 g / L, 3-10 g / L, 4-40 g / L, 4-30 g / L, 4-20 g / L, 4-10 g / L, 5-40 g / L, 5-30 g / L, 5-20 g / L, or 5-10 g / L.

[0011] In one embodiment related to the first aspect and in any of the group of embodiments thereof, the pharmaceutical composition further comprises at least one of an amino acid, a buffer, or a sugar. In the related embodiment, the amino acid is selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine. In the related embodiment, the buffer is selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate, and Tris, preferably Tris. In the related embodiment, the buffer has a concentration of 1-100 mM, 1-90 mM, 1-80 mM, 1-70 mM, 1-60 mM, 1-50 mM, 1-40 mM, 1-30 mM, 1-20 mM, or 1-10 mM. In related embodiments, the sugar is selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, and is preferably trehalose. In related embodiments, the sugar is expressed in concentrations of 10-1000 mM, 10-900 mM, 10-800 mM, 10-700 mM, 10-600 mM, 10-500 mM, 10-400 mM, 10-300 mM, 10-200 mM, 20-1000 mM, 20-900 mM, 20-800 mM, 20-700 mM, 20-600 mM, 20-500 mM, 20-400 mM, 20-300 mM, 20-200 mM, 30-1000 mM, 30-900 mM, 30-800 mM, 30-700 mM, and 3 It has concentrations of 0-600 mM, 30-500 mM, 30-400 mM, 30-300 mM, 30-200 mM, 40-1000 mM, 40-900 mM, 40-800 mM, 40-700 mM, 40-600 mM, 40-500 mM, 40-400 mM, 40-300 mM, 40-200 mM, 50-1000 mM, 50-900 mM, 50-800 mM, 50-700 mM, 50-600 mM, 50-500 mM, 50-400 mM, 50-300 mM, or 50-200 mM.

[0012] In one embodiment and any of the group of embodiments relating to the first embodiment, one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch. In further related embodiments, one or more sugar alcohols are mannitol and / or sorbitol, preferably a combination of mannitol and sorbitol.

[0013] In one embodiment relating to the first aspect, the composition is substantially chloride-free, preferably substantially sodium chloride-free.

[0014] In one embodiment relating to the first aspect, the pH of the composition is 5 to 9, or 6 to 9, or 6.5 to 8.5, or 6.5 to 8.0, preferably 7.0 to 8.0. In the related embodiment, the pH of the composition is adjusted using phosphoric acid, lactic acid, citric acid, succinic acid, or sodium phosphate.

[0015] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - A buffer solution, at least one of an amino acid or a sugar, and - Protein materials and / or poly(ethylene oxide) / poly(propylene oxide) block copolymers (The protein substance here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.) Includes.

[0016] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Amino acids and buffer solutions, and - Protein materials and / or poly(ethylene oxide) / poly(propylene oxide) block copolymers (The protein substance here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.) Includes.

[0017] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Amino acids and sugars, and - Protein materials and / or poly(ethylene oxide) / poly(propylene oxide) block copolymers (The protein substance here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.) Includes.

[0018] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Amino acids, buffer solutions, and sugars, - Protein materials and / or poly(ethylene oxide) / poly(propylene oxide) block copolymers (The protein substance here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.) Includes.

[0019] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Buffer and sugar, and - Protein materials and / or poly(ethylene oxide) / poly(propylene oxide) block copolymers (The protein substance here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.) Includes.

[0020] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols (wherein one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch, preferably mannitol and / or sorbitol, and more preferably a combination of mannitol and sorbitol), - A buffer solution, at least one of an amino acid or a sugar, and - Protein materials and / or poly(ethylene oxide) / poly(propylene oxide) block copolymers (The protein substance here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.) Includes.

[0021] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - A buffer, at least one of amino acids or sugars (wherein the buffer is selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate and Tris, preferably Tris), and - Protein materials and / or poly(ethylene oxide) / poly(propylene oxide) block copolymers (The protein substance here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.) Includes.

[0022] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Amino acids and buffer (wherein the buffer is selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate, and Tris, preferably Tris), and - Protein materials and / or poly(ethylene oxide) / poly(propylene oxide) block copolymers (The protein substance here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.) Includes.

[0023] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Amino acids, buffers, and sugars (wherein the buffer is selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate, and Tris, preferably Tris), and - Protein materials and / or poly(ethylene oxide) / poly(propylene oxide) block copolymers (The protein substance here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.) Includes.

[0024] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Buffer and sugar (wherein the buffer is selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, and Tris, preferably Tris), and - Protein materials and / or poly(ethylene oxide) / poly(propylene oxide) block copolymers (The protein substance here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.) Includes.

[0025] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - A buffer solution, at least one of amino acids or sugars (wherein sugar is selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, preferably trehalose), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0026] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Amino acids and buffer solutions (wherein the amino acids are selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0027] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Amino acids and sugars (wherein sugars are selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, preferably trehalose), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0028] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Amino acids and buffers and sugars (wherein sugar is selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, preferably trehalose), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0029] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Buffer solution and sugar (wherein sugar is selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, preferably trehalose), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0030] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - A buffer solution, at least one of an amino acid or a sugar (wherein the amino acid is selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0031] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Amino acids and sugars (wherein amino acids are selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0032] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Amino acids, buffer solutions, and sugars (wherein the amino acids are selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0033] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - A buffer, an amino acid, or a sugar, at least one of these (wherein the buffer is selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate, and Tris, preferably Tris; wherein the amino acid is selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine; wherein the sugar is selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, preferably trehalose), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0034] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Amino acids and buffer (wherein the buffer is selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate and Tris, preferably Tris; wherein the amino acids are selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine and glutamine, preferably glutamic acid and / or arginine), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0035] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Amino acids and sugars (wherein amino acids are selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine; wherein sugars are selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, preferably trehalose), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0036] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Amino acids, buffer solutions, and sugars (wherein the buffer solution is selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate, and Tris, preferably Tris; wherein the amino acid solution is selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine; wherein the sugar solution is selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, preferably trehalose), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0037] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols, - Buffer and sugar (wherein the buffer is selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate and Tris, preferably Tris; wherein the amino acid is selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine and glutamine, preferably glutamic acid and / or arginine), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0038] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols (wherein one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch, preferably mannitol and / or sorbitol, and more preferably a combination of mannitol and sorbitol), - A buffer solution, at least one of amino acids or sugars (wherein sugar is selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, preferably trehalose), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0039] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols (wherein one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch, preferably mannitol and / or sorbitol, and more preferably a combination of mannitol and sorbitol), - Amino acids and buffer solutions (wherein the amino acids are selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0040] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols (wherein one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch, preferably mannitol and / or sorbitol, and more preferably a combination of mannitol and sorbitol), - Amino acids and sugars (wherein sugars are selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, preferably trehalose), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0041] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols (wherein one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch, preferably mannitol and / or sorbitol, and more preferably a combination of mannitol and sorbitol), - Amino acids and buffers and sugars (wherein sugar is selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, preferably trehalose), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0042] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols (wherein one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch, preferably mannitol and / or sorbitol, and more preferably a combination of mannitol and sorbitol), - Buffer solution and sugar (wherein sugar is selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, preferably trehalose), and - Protein material and / or poly(ethylene oxide) / poly(propylene oxide) block copolymer (wherein the protein material is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188) Includes.

[0043] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols (wherein one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch, preferably mannitol and / or sorbitol, and more preferably a combination of mannitol and sorbitol), -At least one of a buffer, an amino acid, or a sugar (wherein the amino acid is selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0044] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols (wherein one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch, preferably mannitol and / or sorbitol, and more preferably a combination of mannitol and sorbitol), - Amino acids and sugars (wherein amino acids are selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0045] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols (wherein one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch, preferably mannitol and / or sorbitol, and more preferably a combination of mannitol and sorbitol), - Amino acids, buffer solutions, and sugars (wherein the amino acids are selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0046] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols (wherein one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch, preferably mannitol and / or sorbitol, and more preferably a combination of mannitol and sorbitol), -At least one of a buffer, an amino acid, or a sugar (wherein the buffer is selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate, and Tris, preferably Tris; wherein the amino acid is selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine; wherein the sugar is selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, preferably trehalose) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0047] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols (wherein one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch, preferably mannitol and / or sorbitol, and more preferably a combination of mannitol and sorbitol), - Amino acids and buffer (wherein the buffer is selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate, and Tris, preferably Tris; wherein the amino acids are selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0048] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols (wherein one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch, preferably mannitol and / or sorbitol, and more preferably a combination of mannitol and sorbitol), - Amino acids and sugars (wherein amino acids are selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine; wherein sugars are selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, preferably trehalose) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0049] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols (wherein one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch, preferably mannitol and / or sorbitol, and more preferably a combination of mannitol and sorbitol), - Amino acids, buffer solutions, and sugars (wherein the buffer solution is selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate, and Tris, preferably Tris; wherein the amino acids are selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine; wherein the sugars are selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, and sucrose, preferably trehalose) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0050] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -One or more sugar alcohols (wherein one or more sugar alcohols are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, and hydrolyzed hydrogenated starch, preferably mannitol and / or sorbitol, and more preferably a combination of mannitol and sorbitol), - Buffer and sugar (wherein the buffer is selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate, and Tris, preferably Tris; wherein the amino acid is selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, and glutamine, preferably glutamic acid and / or arginine) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0051] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, - Mannitol and / or sorbitol, - Buffer solution (the buffer solution here is Tris) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0052] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, - Mannitol and / or sorbitol, - Amino acids (where amino acids refer to arginine or glutamic acid) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0053] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, - Mannitol and / or sorbitol, - Sugar (here, sugar refers to trehalose or sucrose) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0054] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, - Mannitol and / or sorbitol, - Amino acids (where amino acids are arginine or glutamic acid), - Sugar (here, sugar refers to trehalose or sucrose) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0055] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, - Mannitol and / or sorbitol, - Buffer solution (the buffer solution here is Tris), - Sugar (here, sugar refers to trehalose or sucrose) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0056] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, - Mannitol and / or sorbitol, - Buffer solution (the buffer solution here is Tris), - Amino acids (where amino acids refer to arginine or glutamic acid) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0057] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, - Mannitol and / or sorbitol, - Buffer solution (the buffer solution here is Tris), - Amino acids (where amino acids are arginine or glutamic acid), - Sugar (here, sugar refers to trehalose or sucrose) including, and The protein substance used here is selected from human serum albumin or recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0058] In one embodiment and any of the embodiments relating to the first embodiment, which includes a buffer, the buffer has a concentration of 1-100 mM, 1-90 mM, 1-80 mM, 1-70 mM, 1-60 mM, 1-50 mM, 1-40 mM, 1-30 mM, 1-20 mM, or 1-10 mM.

[0059] In one embodiment and any of the embodiments relating to the first embodiment, which contains sugar, the sugar is present in concentrations of 10-1000 mM, 10-900 mM, 10-800 mM, 10-700 mM, 10-600 mM, 10-500 mM, 10-400 mM, 10-300 mM, 10-200 mM, 20-1000 mM, 20-900 mM, 20-800 mM, 20-700 mM, 20-600 mM, 20-500 mM, 20-400 mM, 20-300 mM, 20-200 mM, 30-1000 mM, 30-900 mM, and 30 It has concentrations of ~800mM, 30~700mM, 30~600mM, 30~500mM, 30~400mM, 30~300mM, 30~200mM, 40~1000mM, 40~900mM, 40~800mM, 40~700mM, 40~600mM, 40~500mM, 40~400mM, 40~300mM, 40~200mM, 50~1000mM, 50~900mM, 50~800mM, 50~700mM, 50~600mM, 50~500mM, 50~400mM, 50~300mM, or 50~200mM.

[0060] In one embodiment related to the first aspect and any of the group of embodiments thereof, the pharmaceutical composition is 0.01-50 g / L, 0.1-50 g / L, 0.2-50 g / L, 0.3-50 g / L, 0.4-50 g / L, 0.5-50 g / L, 1-50 g / L, 2-50 g / L, 3-50 g / L, 4-50 g / L, 5-50 g / L, 0.01-40 g / L, 0 .01~30g / L, 0.01~20g / L, 0.01~10g / L, 0.01~5g / L, 0.1~40g / L, 0.1~30g / L, 0.1~20g / L, 0.1~10 g / L, 0.1~5g / L, 0.2~40g / L, 0.2~30g / L, 0.2~20g / L, 0.2~10g / L, 0.2~5g / L, 0.3~40g / L, 0.3~30g / L, 0.3~20g / L, 0.3~10g / L, 0.3~5g / L, 0.4~40g / L, 0.4~30g / L, 0.4~20g / L, 0.4~10g / L, 0.4~5g / L, 0.5~40g / L, 0.5~30g / L, 0.5~20g / L, 0.5~10g / L, 0.5~5g / L, 1~40g / L, 1~30g / L, 1~20g / L, 1~1 Contains poloxamer 188 at concentrations of 0 g / L, 1-5 g / L, 2-40 g / L, 2-30 g / L, 2-20 g / L, 2-10 g / L, 3-40 g / L, 3-30 g / L, 3-20 g / L, 3-10 g / L, 4-40 g / L, 4-30 g / L, 4-20 g / L, 4-10 g / L, 5-40 g / L, 5-30 g / L, 5-20 g / L, or 5-10 g / L.

[0061] In one embodiment related to the first aspect and any of the group of embodiments thereof, the pharmaceutical composition is 0.05-50 g / L, 0.1-50 g / L, 0.2-50 g / L, 0.3-50 g / L, 0.4-50 g / L, 0.5-50 g / L, 1-50 g / L, 2-50 g / L, 3-50 g / L, 4-50 g / L, 5-50 g / L, 0.05-40 g / L, 0.05 ~30g / L, 0.05~20g / L, 0.05~10g / L, 0.05~5g / L, 0.1~40g / L, 0.1~30g / L, 0.1~20g / L, 0.1~10g / L, 0 .1~5g / L, 0.2~40g / L, 0.2~30g / L, 0.2~20g / L, 0.2~10g / L, 0.2~5g / L, 0.3~40g / L, 0.3~30g / L, 0.3 ~20g / L, 0.3~10g / L, 0.3~5g / L, 0.4~40g / L, 0.4~30g / L, 0.4~20g / L, 0.4~10g / L, 0.4~5g / L, 0.5~4 0g / L, 0.5~30g / L, 0.5~20g / L, 0.5~10g / L, 0.5~5g / L, 1~40g / L, 1~30g / L, 1~20g / L, 1~10g / L, 1~5g Contains human serum albumin or recombinant human albumin at concentrations of 1 / L, 2-40 g / L, 2-30 g / L, 2-20 g / L, 2-10 g / L, 3-40 g / L, 3-30 g / L, 3-20 g / L, 3-10 g / L, 4-40 g / L, 4-30 g / L, 4-20 g / L, 4-10 g / L, 5-40 g / L, 5-30 g / L, 5-20 g / L, or 5-10 g / L.

[0062] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, - Tris with a concentration of approximately 1-100 mM, - Approximately 1-100 mM glutamic acid, - Approximately 10-500 mM trehalose, - Approximately 10-200 mM mannitol, - Approximately 10-200 mM sorbitol, - Approximately 0.1-5 mg / ml of poloxamer 188, - Recombinant human albumin at approximately 0.5-10 mg / ml, and -pH about 6~8 Includes.

[0063] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, - Tris with a concentration of approximately 1-100 mM, - Approximately 1-100 mM glutamic acid, - Approximately 10-500 mM arginine, - Approximately 10-500 mM trehalose, - Approximately 10-200 mM mannitol, - Approximately 10-200 mM sorbitol, - Approximately 0.1-5 mg / ml of poloxamer 188, - Recombinant human albumin at approximately 0.5-10 mg / ml, and -pH about 6~8 Includes.

[0064] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, - Tris with a concentration of approximately 1-100 mM, - Approximately 1-100 mM glutamic acid, - Approximately 10-500 mM trehalose, - Approximately 1-100 mM citric acid, - Approximately 10-200 mM mannitol, - Approximately 10-200 mM sorbitol, - Approximately 0.1-5 mg / ml of poloxamer 188, - Recombinant human albumin at approximately 0.5-10 mg / ml, and -pH about 6~8 Includes.

[0065] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, - Tris with a concentration of approximately 1-100 mM, - Approximately 1-100 mM glutamic acid, - Approximately 10-500 mM trehalose, - Approximately 10-200 mM mannitol, - Approximately 10-200 mM sorbitol, - Approximately 0.1-5 mg / ml of poloxamer 188, - Recombinant human albumin at approximately 0.5-10 mg / ml, and -pH about 6~8 Includes.

[0066] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -Approximately 10mM Tris, -Approximately 200 mM trehalose, -Approximately 20 mM glutamic acid, -Approximately 50 mM mannitol, -Approximately 50 mM sorbitol, - Approximately 1.275 mg / ml of poloxamer 188, - Approximately 5 mg / ml of recombinant human albumin, and -pH about 7.4 Includes.

[0067] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -Approximately 10mM Tris, -Approximately 125 mM trehalose, -Approximately 20 mM glutamic acid, -Approximately 100 mM mannitol, -Approximately 40 mM sorbitol, - Approximately 100 mM arginine, - Approximately 2.5 mg / ml of poloxamer 188, - Approximately 5 mg / ml of recombinant human albumin, and -pH about 7.1 Includes.

[0068] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -Approximately 10mM Tris, -Approximately 20 mM glutamic acid, -Approximately 200 mM trehalose, - Approximately 25 mM citric acid, -Approximately 50 mM mannitol, -Approximately 50 mM sorbitol, - Approximately 2.5 mg / ml of poloxamer 188, - Approximately 5 mg / ml of recombinant human albumin, and -pH about 7.4 Includes.

[0069] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -Approximately 10mM Tris, -Approximately 200 mM trehalose, -Approximately 20 mM glutamic acid, -Approximately 50 mM mannitol, -Approximately 50 mM sorbitol, - Approximately 150 mM arginine, - Approximately 1.275 mg / ml of poloxamer 188, - Approximately 5 mg / ml of recombinant human albumin, and -pH about 7.4 Includes.

[0070] In one embodiment relating to the first aspect, the pharmaceutical composition is - Enveloped viruses, -Approximately 10mM Tris, -Approximately 133 mM trehalose, -Approximately 13 mM glutamic acid, -Approximately 33 mM mannitol, - Approximately 33 mM sorbitol, - Approximately 100 mM arginine, - Approximately 1.275 mg / ml of poloxamer 188, - Approximately 5 mg / ml of recombinant human albumin, and -pH about 7.4 Includes.

[0071] In any one of the embodiments and groups of embodiments related to the first aspect, the pH of the composition is adjusted using phosphoric acid, sodium phosphate, or lactic acid.

[0072] In any one of the embodiments and groups of embodiments related to the first aspect, the enveloped virus is from the family Rhabdoviridae, preferably Vesiculovirus or Vesicular stomatitis virus (VSV). In related embodiments, the enveloped virus is a recombinant vesicular stomatitis virus (VSV), where the gene encoding the glycoprotein G of the vesicular stomatitis virus here is replaced by the gene encoding the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), and / or the glycoprotein G is replaced by the glycoprotein GP of LCMV. In related embodiments, the pharmaceutical composition contains at least 1×10 5 of TCID 50 / mL, at least 1×10 6 of TCID 50 / mL, at least 1×10 7 of TCID 50 / mL, at least 1×10 8 of TCID 50 / mL, at least 1×10 9 of TCID 50 / mL, or at least 1×10 9 of TCID 50 / mL concentration of an enveloped virus, preferably Vesiculovirus or Vesicular stomatitis virus (VSV). In further related embodiments, the pharmaceutical composition contains 1×10 5 of TCID 50 / mL to 1×10 12 of TCID 50 / mL, 1×10 6 of TCID 50 / mL to 1×10 12 of TCID 50 / mL, 1×10 7 of TCID 50 / mL to 1×10 12 of TCID 50 / mL, 1×10 8 of TCID50 / mL ~ 1 × 10 12 TCID 50 / mL, 1 × 10 5 TCID 50 / mL ~ 1 × 10 11 TCID 50 / mL, 1 × 10 5 TCID 50 / mL ~ 1 × 10 10 TCID 50 / mL, or 1 × 10 5 TCID 50 / mL ~ 1 × 10 9 TCID 50 It contains an enveloped virus, preferably becyclovirus or vesicular stomatitis virus (VSV), in a concentration range of / mL.

[0073] In one embodiment related to the first aspect and in any of the group of embodiments thereof, the pharmaceutical composition is a liquid or a frozen liquid pharmaceutical composition.

[0074] In one embodiment relating to the first aspect and in any of the group of embodiments thereof, the dry pharmaceutical composition is produced by a method that includes the step of removing water from the pharmaceutical composition.

[0075] In one embodiment related to the first aspect and in any of the group of embodiments thereof, the pharmaceutical composition is a dry pharmaceutical composition. In the relevant embodiment, the dry pharmaceutical composition is obtained by freeze-drying / freeze-drying or spray-drying. In the relevant embodiment, the method includes placing the pharmaceutical composition in a vacuum under controlled temperature and pressure to remove water. In the relevant embodiment, the method is freeze-drying. In the relevant embodiment, the dry pharmaceutical composition contains less than about (0.1% to 10%) w / w of water. In the relevant embodiment, the pharmaceutical composition comprises water and the dry pharmaceutical composition. Further embodiments relate to pharmaceutical compositions comprising water and a product.

[0076] In further embodiments, the freeze-dried preparation is restored with water. - Enveloped viruses, Mannitol at concentrations of -10 mM to 200 mM, and / or sorbitol at concentrations of 10 mM to 200 mM, Poloxamer 188 at concentrations of -0.01 g / L to 50 g / L, and / or recombinant human albumin at concentrations of -0.1 g / L to 50 g / L This results in a restoration solution containing the following: [Brief explanation of the drawing]

[0077] [Figure 1] Geometric mean infectivity of VSV-GP-Cargo 1 after storage of liquid formulations at 5°C. Error bars indicate standard deviation (n=2 for control, n=3 for others, measured with 3 replicates each). Frozen crude samples serve as controls. [Figure 2] Geometric mean infectivity of VSV-GP-Cargo 1 after storage of liquid formulations at 25°C. Error bars indicate standard deviation (n=2 for control, n=3 for others, measured with 3 replicates each). Frozen crude samples serve as controls. [Figure 3] Geometric mean values ​​of SvPs (invisible particles) 10 μm or larger in VSV-GP-Cargo 1 after storing the liquid formulation at 5°C. Error bars indicate the standard deviation (n=2 for the control, n=3 for the others, measured with three replicates each). [Figure 4] Geometric mean of SvPs ≥ 10 μm in VSV-GP-Cargo 1 after storing liquid formulations at 25°C. Error bars indicate standard deviation (n=2 for control, n=3 for others, measured with 3 replicates each). [Figure 5] Geometric mean infectivity of VSV-GP-Cargo 1 after 0, 1, or 3 freeze / thaw cycles and storage of liquid formulations at 25°C. Error bars indicate standard deviation (n=2 for controls, n=3 for others, measured with 3 replicates each). Frozen crude samples serve as controls. [Figure 6]Geometric mean of SvPs ≥ 10 μm in VSV-GP-Cargo 1 after 0, 1, or 3 freeze / thaw cycles of the liquid formulation. Error bars indicate the standard deviation (n=2 for the control, n=3 for the others, measured with 3 replicates each). [Figure 7] Geometric mean infectivity of VSV-GP-Cargo 1 after one, three, or five freeze / thaw cycles at -20°C and +25°C for frozen liquid formulations. Error bars indicate standard deviation (n=2 for controls, n=3 for others, measured with three replicates each). Frozen crude samples serve as controls. [Figure 8] Geometric mean infectivity of VSV-GP-Cargo 1 after one, three, or five freeze / thaw cycles at -80°C and +25°C for frozen liquid formulations. Error bars indicate standard deviation (n=2 for controls, n=3 for others, measured with three replicates each). Frozen crude samples serve as controls. [Figure 9] Geometric mean of SvPs ≥ 10 μm in VSV-GP-Cargo 1 after one, three, or five freeze / thaw cycles at -20°C and +25°C for frozen liquid formulations. Error bars indicate standard deviation (n=2 for control, n=3 for others, measured with three replicates each). [Figure 10] Geometric mean of SvPs ≥ 10 μm in VSV-GP-Cargo 1 after one, three, or five freeze / thaw cycles at -80°C and +25°C for frozen liquid formulations. Error bars indicate standard deviation (n=2 for control, n=3 for others, measured with three replicates each). [Figure 11] Geometric mean of infectivity of VSV-GP-Cargo 1 after storage of freeze-dried preparations at 25°C. Error bars indicate standard deviation (n=2 for controls, n=3 for others, measured with 3 replicates each). Freeze-dried crude samples serve as controls. The first time point shows the measurement before freeze-drying. The second value (week 0) is the value immediately after freeze-drying and reconstitution. [Figure 12]Geometric mean values ​​of SvPs ≥ 10 μm in VSV-GP-Cargo 1 after storage of freeze-dried formulations at 25°C. Error bars indicate the standard deviation (n=2 for the control, n=3 for the others, measured with three replicates each). The first time point shows the measurement before freeze-drying. The second value is the value immediately after freeze-drying and reconstitution. [Figure 13] Tg value of the placebo formulation in work package 7. [Figure 14] Freeze-dried cake of the preparation in work package 7 before and after storage at a temperature of 5°C or 25°C for 14 days. [Figure 15] Figures 15A-B. A) Temperatures of the onset and midpoint of the second melting transition (Tg) peak in the placebo formulation of freeze-dried work package 7. B) Temperatures of the onset and midpoint of the second glass transition (Tg') peak in the placebo formulation of restored work package 7. Formulations 1, 2, and 4 did not contain poloxamer 188. [Figure 16] Geometric mean of infectivity of VSV-GP-Load 1 after storage of the freeze-dried preparation at 25°C. Error bars indicate the standard deviation (each measured with n=2 and 3 replicates). The first time point shows the measurement before freeze-drying. The second value is the value immediately after freeze-drying and reconstitution. Freeze-collected crude samples serve as a control. [Figure 17] Geometric mean of infectivity of VSV-GP-Load 1 after storage of the freeze-dried preparation at 5°C. Error bars indicate the standard deviation (each measured with n=2 and 3 replicates). The first time point shows the measurement before freeze-drying. The second value is the value immediately after freeze-drying and reconstitution. Freeze-collected crude samples serve as a control. [Figure 18]Geometric mean of infectivity of VSV-GP-Load 1 after storage of lyophilized preparations at 25°C. Error bars indicate standard deviation (each measured with 3 replicates, n=2). The first time point shows the measurement before lyophilization. The second value is the value immediately after lyophilization and reconstitution. The values ​​for work packages 09a_03 and 09_04 at 6 and 12 months were below the detection limit. Freeze-crude collected samples serve as controls. [Figure 19] Geometric mean of infectivity of VSV-GP-Load 2 after storage of the freeze-dried preparation at 5°C. Error bars indicate the standard deviation (each measured with n=2 and 3 replicates). The first time point shows the measurement before freeze-drying. The second value is the value immediately after freeze-drying and reconstitution. Freeze-collected crude samples serve as a control. [Figure 20] Geometric mean infectivity of VSV-GP-Load 2 after storage of lyophilized preparations at 25°C. The first time point shows the measurement before lyophilization. The second value is the value immediately after lyophilization and reconstitution. The values ​​for workpackage 09_05 at 6 and 12 months were below the detection limit. Freeze-collected crude samples serve as controls. [Figure 21] Figures 21A-B. Geometric mean infectivity of VSV-GP-Load 1 after storage of lyophilized preparations at 25°C. Error bars indicate standard deviation (each measured with n=2 and 3 replicates). The first time point shows the measurement before lyophilization. The second value is the value immediately after lyophilization and reconstitution. The values ​​for workpackages 010_01, 10_06, and 10_09 at 3 months, 6 months, and 12 months were below the detection limit. Freeze-crude samples serve as controls. [Figure 22]Figures 22A-D. Geometric mean infectivity of VSV-GP-cargo 1 after storage of lyophilized preparations at 25°C using a Design of Experiments (DoE) approach. Error bars indicate the standard deviation (each with n=4 replicates). The first value is the value immediately after lyophilization and reconstitution. The values ​​for work packages 010_06 and 10_09 after 12 months were below the detection limit. Freeze-collected crude samples serve as controls. [Figure 23] Figures 23A-B. (A) Geometric mean of the infectivity of VSV-GP after storing freeze-dried formulations at temperatures of 2-8°C. Error bars indicate the standard deviation (3-9 replicates, each). The first value (t=0) is the value immediately after freeze-drying and reconstitution. (B) Mean of the total particle concentration measured by NTA (nanoparticle tracking analysis) after liquid treatment of five different formulations containing VSV-GP. Each point represents one sample (each acquired 5 times). Error bars indicate the standard deviation (n=2, each acquired 5 times).

[0078] Detailed description of the invention In the following detailed description, specific numerical details are provided to give a full understanding of the invention. However, it will be apparent to those skilled in the art that the subject art can be practiced without using some of these specific details. In other cases, well-known structures and techniques are not described in detail in order not to make the invention difficult to understand. Headings are included simply for convenience to aid in interpretation and should not be understood as limiting the invention to any particular aspect or embodiment.

[0079] In one embodiment, the formulation of the present invention is useful for stabilizing the infectivity titer of enveloped viruses. In another embodiment, the formulation helps maintain the infectivity titer and / or colloidal stability of enveloped viruses after one or more freeze-thaw cycles. In a related embodiment, the formulation is useful for maintaining the infectivity titer and / or colloidal stability of enveloped viruses during storage at room temperature, or even above room temperature, at elevated temperatures, preferably 2 to 8°C. In a related embodiment, the formulation is useful for maintaining the titer and / or activity of enveloped viruses during storage of the virus at various temperatures over a period of time.

[0080] In another embodiment, the preparations of the present invention can reduce and / or slow down the formation of visible and / or invisible particles (SvPs) in preparations containing enveloped viruses. In a related embodiment, the preparations containing enveloped viruses have reduced amounts of visible viruses and / or invisible particles.

[0081] In yet another embodiment, the formulation of the present invention helps maintain the infectivity titer of enveloped viruses. In a related embodiment, the infectivity titer is maintained or the decline in the infectivity titer over time is slowed. In a related embodiment, the decline in the infectivity titer due to long-term storage, repeated freeze-thaw cycles, storage at elevated temperatures, or mechanical stress is slowed.

[0082] In another embodiment, the preparations of the present invention help to inhibit, slow down, or prevent the aggregation of enveloped viruses. In another embodiment, the preparations are useful for inhibiting, reducing, or preventing turbidity in preparations containing enveloped viruses.

[0083] In another embodiment, the preparations of the present invention help to suppress, slow down, or prevent the formation of visible and / or invisible particles in preparations containing enveloped viruses. Preferably, the preparations containing enveloped viruses are substantially free of visible and / or invisible particles.

[0084] In another embodiment, a formulation of the present invention containing an enveloped virus preserves the infectivity of the enveloped virus and / or slows the decline in infectivity when stored at a temperature of 25°C.

[0085] In another embodiment, the preparation is useful for maintaining the infectivity titer and / or colloidal stability of enveloped viruses when stored at a temperature of 2 to 8°C.

[0086] In another embodiment, a preparation of the present invention containing an enveloped virus, after restoration, has visible and / or invisible particles that are below the limits set forth in the official standards.

[0087] In one embodiment relating to any of the above embodiments, the formulation containing the enveloped virus is a liquid formulation, a freeze-liquid formulation, or a dry formulation, such as a freeze-dried formulation.

[0088] In another embodiment, the dry preparation of the present invention containing an enveloped virus is prepared by removing NaCl and adjusting the pH of the preparation using phosphoric acid or sodium phosphate, such as trisodium phosphate (Na3PO4) and / or phosphoric acid (H3PO4) or lactate and / or lactic acid, instead of sodium hydroxide (NaOH) and / or hydrochloric acid (HCl), thereby adjusting the pH of the preparation. g Increasing the value further stabilizes it.

[0089] In another embodiment, a dried preparation of the present invention containing an enveloped virus preserves the infectivity of the enveloped virus and / or slows the decline in infectivity when stored at a temperature of 25°C for at least 1, 2, 3, 6, or at least 12 months.

[0090] In another embodiment, a dried preparation of the present invention containing an enveloped virus can be stored at a temperature of 25°C or 5°C for at least 1, 2, 3, 6, or at least 12 months. In a related embodiment, the overall TCID 50 The decrease is less than 0.25 to 0.3 log units. In a more relevant aspect, the concentration of visible / invisible particles is below the reconstructed official limit.

[0091] In another embodiment, the preparation of the preparation buffer using trisodium phosphate (Na3PO4) and / or phosphoric acid (H3PO4) while omitting sodium chloride reduces the molar osmotic concentration of the restored preparation, and the T of the maximum freeze-concentrated solution. g Increase the value.

[0092] In another embodiment, the preparation of a buffer solution using phosphoric acid (H3PO4) instead of hydrochloric acid reduces the molal osmotic pressure concentration in the preparation without using trehalose, T g Increase the value by 10°C to 20°C.

[0093] In another embodiment, the omission of sodium chloride or chloride ions in general from the preparation eliminates the formation of chloride salts during freezing.

[0094] In relation to any of the above embodiments, the potency of a particular formulation is determined by the method shown in the examples, TCID 50 This can be tested by determining the particle. In relation to any of the above embodiments, the particle may be evaluated according to the method described in the example.

[0095] While we do not wish to dwell on theory, it has been found that poloxamers and specific poloxamer 188 reduce the formation of aggregates and / or particles in liquid and / or frozen liquid oncolytic virus preparations, particularly after freeze / thaw cycles. Even more surprisingly, poloxamer 188 was able to stabilize the infectivity of lyophilized oncolytic virus preparations after storage at various temperatures. This is particularly surprising, given that poloxamers are known to act as decontaminating agents. However, in contrast to other decontaminating agents such as Tween, poloxamers did not destroy the virus or reduce its infectivity. Furthermore, the presence of poloxamers, particularly poloxamer 188, in the preparations has been shown to stabilize the infectivity of lyophilized viruses during storage at various temperatures.

[0096] Furthermore, it was found that recombinant human albumin (rHA) could reduce the formation of aggregates and / or particles in the freeze-liquid and freeze-dried preparations, possibly through interaction with the virus surface. In addition, rHA was found to stabilize the infectivity of freeze-dried viruses in the freeze-liquid and freeze-dried preparations during storage.

[0097] Surprisingly, the combination of recombinant human albumin and poloxamer 188 showed additive / synergistic effects on the infectivity of freeze-dried viruses during storage. This effect was accompanied by a further reduction in particle formation. It was also found that stress-induced particle formation in rHA-containing formulations in liquid and / or freeze-liquid formulations could be effectively prevented by the addition of poloxamer 188.

[0098] The pharmaceutical preparations and compositions described herein are particularly useful for the preparation of enveloped viruses. According to the present invention, the term "enveloped virus" refers to any of the genus enveloped viruses capable of infecting humans, including but not limited to herpesviruses, poxviruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, and filoviruses. The term "rhabdovirus" as used herein refers to any enveloped virus of the genus capable of infecting humans, including but not limited to almendravirus, curiovirus, cytorhabdovirus, dichorhavirus, ephemerovirus, hapavirus, ledantevirus, lyssavirus, novirhabdovirus, nucleorhabdovirus, perhabdovirus, sigmavirus, spribivirus, sripuvirus, tibrovirus, tupavirus, varicosavirus, or becyclovirus.

[0099] Preferably, enveloped viruses are replicable. More preferably, oncolytic viruses and replicable oncolytic viruses. In this regard, the term oncolytic is used in its usual sense, which is well known in the art, and refers to a virus that can infect and lyse (destroy) cancer cells but cannot infect and lyse normal cells (to any significant degree). Preferably, oncolytic viruses can replicate within cancer cells. Oncolytic activity can be tested with various assay systems known to those skilled in the art (an exemplary in vitro assay is described in Muik et al., Cancer Res., 74(13), 3567-78, 2014). It has been found that oncolytic viruses can infect and lyse only certain types of cancer cells. Furthermore, the oncolytic effect may vary depending on the type of cancer cell. It has been found that oncolytic viruses are live viruses that can infect cancer cells and replicate within them.

[0100] In any embodiment, the enveloped virus may be a recombinant or non-recombinant enveloped virus, preferably a recombinant enveloped virus, preferably a recombinant enveloped virus belonging to the Rhabdoviridae family, more preferably a recombinant becyclovirus, and even more preferably a recombinant vesicular stomatitis virus.

[0101] The term “recombinant” refers to a virus, more specifically an enveloped virus, that contains an exogenous nucleic acid sequence inserted into its genome that is not naturally present in the parent virus. Thus, a recombinant virus refers to a nucleic acid or virus created by the artificial combination of two or more nucleic acid sequence segments of synthetic or semi-synthetic origin, linked to another nucleic acid in a permutation that is not naturally present or found in nature. The artificial combination is most commonly achieved by the artificial manipulation of isolated nucleic acid segments using well-established genetic engineering techniques. Generally, “recombinant” enveloped viruses as described herein refer to enveloped viruses created by standard genetic engineering techniques; for example, the enveloped viruses of the present invention are therefore genetically engineered or genetically modified enveloped viruses. Thus, the term “recombinant enveloped virus” includes enveloped viruses in which recombinant nucleic acids are stably incorporated into their genomes.

[0102] Characteristic features of members of the Rhabdoviridae family include mostly unsegmented, 10.8–16.1 kb negative-stranded single-stranded RNA, as well as genomes encoding at least five genes that encode structural proteins: nucleoproteins (N), large proteins (L), phosphorylated proteins (P), matrix proteins (M), and glycoproteins (G).

[0103] Becyclovirus species are primarily defined by serological methods combined with phylogenetic analysis of genomes. Biological characteristics such as host range and transmission mechanism are also used to distinguish virus species within the genus. Thus, the genus Becyclovirus forms a distinct monophyletic group, well supported by maximum likelihood phylogenetic trees inferred from complete L sequences.

[0104] Viruses assigned to different species within the genus Becyclovirus may have one or more of the following characteristics: A) a 20% difference in the minimum amino acid sequence in L; B) a 10% difference in the minimum amino acid sequence in N; C) a 15% difference in the minimum amino acid sequence in G; D) distinguishable by serological testing; and E) occupying different environmental niches, as evidenced by differences in the host and / or vector arthropods.

[0105] In a preferred embodiment, the vesicular stomatitis virus encodes at least the vesicular stomatitis virus nucleoprotein (N), macroprotein (L), phosphorylated protein (P), matrix protein (M), and glycoprotein (G) within its genome.

[0106] In a preferred embodiment, the vesicular stomatitis virus nucleoprotein (N) contains in its genome at least the amino acid sequence shown in SEQ ID NO: 1, or a functional variant that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and contains the amino acid sequence shown in SEQ ID NO: 2, or a functional variant that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. The product encodes a phosphorylated protein (P), a large protein (L) containing a functional variant that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 4, or a functional variant that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 4.

[0107] It is understood by those skilled in the art that modifications to the sequences of the nucleoprotein (N), macroprotein (L), phosphorylated protein (P), matrix protein (M), or glycoprotein (G) of vesicular stomatitis virus can be carried out without impairing the basic function of such proteins. Such functional variants used herein retain all or part of their basic function or activity. For example, protein L is a polymerase and has an essential function in viral transcription and replication. Its functional variant must retain at least part of this ability. A clear indication of retention of basic function or activity is the successful generation of the virus (including these functional variants) that is still able to replicate in tumor cells and infect tumor cells. Tests of viral generation and infection and replication in tumor cells can be performed using various assay systems known to those skilled in the art (exemplary in vitro assays are described by Muik et al., Cancer Res., 74(13), 3567-78, 2014).

[0108] In a preferred embodiment, the vesicular stomatitis virus encodes at least the vesicular stomatitis virus nucleoprotein (N), macroprotein (L), phosphorylated protein (P), matrix protein (M), and glycoprotein (G) within its genome, wherein the macroprotein (L) contains an amino acid sequence having a sequence identity of 80% or more with SEQ ID NO: 3.

[0109] In a preferred embodiment, the vesicular stomatitis virus has a genome that encodes at least a vesicular stomatitis virus nucleoprotein (N), macroprotein (L), phosphorylated protein (P), matrix protein (M), and glycoprotein (G), wherein the nucleoprotein (N) contains an amino acid sequence having a sequence identity of 90% or more with SEQ ID NO: 1.

[0110] In a more preferred embodiment, the vesicular stomatitis virus encodes at least a vesicular stomatitis virus nucleoprotein (N), macroprotein (L), phosphorylated protein (P), matrix protein (M), and glycoprotein (G) within its genome, wherein the macroprotein (L) contains an amino acid sequence having a sequence identity of 80% or more with SEQ ID NO: 3, and the nucleoprotein (N) contains an amino acid sequence having a sequence identity of 90% or more with SEQ ID NO: 1.

[0111] Certain wild-type strains of vesicular stomatitis virus are known to be neurotoxic. Furthermore, infected individuals have been reported to rapidly initiate a potent humoral response with high antibody titers primarily directed towards glycoproteins. Neutralizing antibodies targeting glycoprotein G of vesicular stomatitis virus can limit viral spread and thus mediate protection against reinfection. However, viral neutralization limits the repeated application of the virus to cancer patients.

[0112] To eliminate these drawbacks, the wild-type glycoprotein G may be replaced with a glycoprotein derived from another virus. In this regard, glycoprotein replacement means (i) replacing the gene encoding wild-type glycoprotein G with the gene encoding glycoprotein GP of another virus, and / or (ii) replacing wild-type glycoprotein G with glycoprotein GP of another virus.

[0113] In a preferred embodiment, the enveloped virus is a vesicular stomatitis virus, and the glycoprotein G of the vesicular stomatitis virus is replaced with the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), preferably the glycoprotein of the WE-HPI strain. Such a vesicular stomatitis virus is described, for example, in International Publication No. 2010 / 040526 and named vesicular stomatitis virus-GP.

[0114] Therefore, in the most preferred embodiment, the enveloped virus is recombinant vesicular stomatitis virus, wherein the gene encoding glycoprotein G of vesicular stomatitis virus is replaced by the gene encoding glycoprotein GP of lymphocytic choriomeningitis virus, and / or, the glycoprotein G is replaced by glycoprotein GP of lymphocytic choriomeningitis virus.

[0115] In a preferred embodiment, the gene encoding the glycoprotein GP of lymphocytic choriomeningitis virus encodes a protein having an amino acid sequence as shown in SEQ ID NO: 5, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 5, while maintaining the functional characteristics of an enveloped virus containing glycoprotein GP encoding an amino acid sequence as shown in SEQ ID NO: 5.

[0116] It should be understood that recombinant enveloped viruses may also encode other cargo within their genome, such as tumor antigens, other chemokines, cytokines, or other immunomodulatory elements.

[0117] In a preferred embodiment, the RNA genome of the vesicular stomatitis virus includes or comprises sequences such as those shown in SEQ ID NO: 6, 7, or 8. Furthermore, the RNA genome of the vesicular stomatitis virus may also consist of or include such sequences, where the nucleic acids of the RNA genome are exchanged according to the degeneracy of the gene code without altering the respective amino acid sequences. In a further preferred embodiment, the RNA genome of the vesicular stomatitis virus includes or comprises coding sequences that are identical to or at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6, 7, or 8.

[0118] definition In this specification, the terms “identical” or “identical ratio” in the context of two or more nucleic acid sequences or polypeptide sequences refer to two or more sequences or subsequences that, when compared and aligned to the greatest extent possible, have the same nucleotide residues or amino acid residues, or identical in a specific ratio. To determine the degree of identity, the sequences are aligned for optimal comparison (for example, gaps may be introduced within the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The degree of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., degree of identity % = number of identical positions / total number of positions (e.g., overlapping positions) × 100). In some embodiments, the two sequences being compared are of the same length after introducing gaps within the sequences as appropriate (e.g., extra sequences extending beyond the sequences being compared are excluded).

[0119] The determination of the degree of identity or similarity between two sequences can be achieved using mathematical algorithms. A preferred and non-restrictive example of a mathematical algorithm used for comparing two sequences is the algorithm from Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. By performing a BLAST nucleotide search using the NBLAST program, score=100, and word length=12, homologous nucleotide sequences to the nucleic acid encoding the protein of interest can be obtained. By performing a BLAST protein search using the XBLAST program, score=50, and word length=3, homologous amino acid sequences for the protein of interest can be obtained. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, iterative searches can be performed using PSI-Blast to detect distant relationships between molecules (ibid.). When using the BLAST, gapped BLAST, and PSI-Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. Another preferred non-restrictive example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, gap length penalty 12, and gap penalty 4 may be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM, as described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA, as described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. When ktup=2, similar regions in the two sequences being compared are found by searching for aligned residue pairs; when ktup=1, single aligned amino acids are examined. ktup can be set to 2 or 1 for protein sequences, or to 1-6 for DNA sequences. If ktup is not specified, the default is 2 for proteins and 6 for DNA. Alternatively, the ordering of protein sequences can be performed using the CLUSTAL W algorithm, as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0120] The term "approximately" generally refers to the degree of acceptable error or deviation of a measured quantity, taking into account the nature or accuracy of the measurement. Typical exemplary degrees of error or deviation are within 5%, 3%, or 1% of a given number or range of numbers. For example, the expression "approximately 100" includes 105 and 95, or 103 and 97, or 101 and 99, and all numbers in between (e.g., 95.1, 95.2, etc. for the range of 95-105; or 97.1 or 97.2, etc. for the range of 97-103; or 99.1, 99.2, etc. for the range of 99-101). Unless otherwise specified, quantities shown herein are approximate, meaning that the term "approximately" can be inferred unless explicitly stated.

[0121] As used herein, the general embodiments of “contains” or “includes” encompass the more specific embodiment of “consistes of.” Furthermore, singular and plural forms are not used in a restrictive sense. As used herein, the singular forms “a,” “an,” and “the” refer to both singular and plural forms unless otherwise specified.

[0122] "Pharmaceutical preparation" or "compounding" refers to the process of producing a final pharmaceutical or drug by combining an active drug or active substance with a chemical substance, but also refers to the product of the process. Therefore, a final preparation refers to a pharmaceutical product such as a liquid, frozen liquid, dried preparation, or composition. Thus, in one embodiment, a pharmaceutical preparation is a pharmaceutical composition.

[0123] In this context, "pharmaceutical composition" refers to a dosage form that allows the biological activity of an active ingredient(s) to be clearly effective, and which contains no additional ingredients that are significantly toxic to the subject to whom the composition is to be administered, whether liquid, frozen, or dried. Such a composition is sterile.

[0124] It will be understood that the various formulations described herein may be provided as liquids, frozen liquids, and / or dried formulations. Therefore, unless otherwise specified, the general terms formulation or pharmaceutical formulation or composition or pharmaceutical composition encompass all liquids, frozen liquids, and / or dried compositions / formulations.

[0125] A "dried preparation" or "dried pharmaceutical composition" is prepared by removing the liquid from a preparation containing an enveloped virus, which has been prepared in a liquid solution. Removal of the liquid can be achieved, for example, by evaporation, such as by applying the liquid solution to a solid substrate and allowing the liquid to evaporate, and / or by sublimation, such as by freeze-drying. The dried preparations / dried pharmaceutical compositions of the present invention are generally stored as dried preparations having a residual moisture content (RMC) of 0.5% to 10.0% (w / w). The dried preparations / dried pharmaceutical compositions can be restored before administration in an aqueous solution (such as, but not limited to, sterile water, saline solution, buffer solution, aqueous dextrose, or glycerol). In certain embodiments, the dried preparations / dried pharmaceutical compositions of the present invention are stored as dried preparations having a residual moisture content of 0.1% to 5% (w / w). In more specific embodiments, the dried preparation / dried pharmaceutical composition of the present invention is stored as a dried preparation containing a residual moisture content of 0.25% to 2.5% (w / w).

[0126] It will be understood that the dry pharmaceutical composition described in the present invention will first be prepared as a liquid pharmaceutical composition. Therefore, when a concentration range and / or pH range for a dry pharmaceutical composition is indicated, it will be further understood that the concentration range and / or pH range refers to the liquid pharmaceutical composition initially prepared before it is dried, and / or the liquid pharmaceutical composition obtained after the restoration of the dry pharmaceutical composition using an aqueous solution such as water.

[0127] In one embodiment, the pharmaceutical composition described in the present invention is a liquid pharmaceutical composition comprising an aqueous solution, preferably water, and any (dry) pharmaceutical composition as described herein.

[0128] Furthermore, in another embodiment, it can be seen that the pharmaceutical composition described in the present invention is a liquid pharmaceutical composition obtained by restoring a dry pharmaceutical composition in an aqueous solution, preferably in water.

[0129] As used herein, the term "water" refers to water for injection.

[0130] "Pharmacologically acceptable" excipients (vehicles, additives) are suitable for parenteral administration to subjects.

[0131] In one embodiment, the pharmaceutical formulation of the present invention is stable.

[0132] "Stability" refers to chemical and physical stability, and can be qualitatively and / or quantitatively evaluated using various analytical techniques described in this technical field, for example, in Moving oncolytic viruses into the clinic: clinical-grade production, purification, and characterization of diverse oncolytic viruses. Mol Ther Methods Clin Dev. 2016 Apr 6;3:16018. doi: 10.1038 / mtm.2016.18. PMID: 27088104; PMCID: PMC4822647. Such methods include evaluating the formation of aggregates and particles (for example, by measuring turbidity, particles invisible to the naked eye, by light-blocking (LO) or microflow imaging (MFI = flow imaging microscopy (FIM), dynamic image analysis (DIA), and / or visual inspection of color and clarity) using high-performance size exclusion chromatography (HP-SEC); by evaluating charge heterogeneity using cation exchange chromatography (CEX) or capillary isoelectric focusing electrophoresis; by mass spectrometry; by capillary gel electrophoresis (CGE) analysis; by peptide mapping (e.g., digestion with trypsin or digestion with lysyl endopeptidase (Lys-C)) analysis; and by evaluating biological activity (infectivity). To measure stability, samples of the preparations of the present invention may be tested in a stability test, where the sample is exposed to stress conditions for a selected period and subsequently subjected to quantitative and qualitative analysis of chemical stability, physical stability, and infectivity using appropriate analytical techniques.

[0133] Therefore, stability can be measured by storing the sample at various temperatures such as -80°C, -20°C, 2 - 8°C, room temperature (RT), 25°C or 30°C, for a selected period at the selected temperature, for example, up to a maximum of 12 months, and by using, for example, high performance size exclusion chromatography, cation exchange chromatography, flow imaging microscopy, light blocking method, capillary isoelectric focusing electrophoresis, or infectivity can be measured by qualitative and quantitative analysis.

[0134] According to the above, a "stable formulation" is a formulation containing an enveloped virus that is physically and chemically stable and / or retains its biological activity upon storage.

[0135] "Physical stability" substantially refers to an enveloped virus that has little or no signs of aggregation, sedimentation, and / or decrease in infectivity in the context of the present invention. Methods for obtaining physical stability include, for example, size exclusion chromatography, light blocking method or microflow imaging, or dynamic image analysis (DIA), and visual inspection. In size exclusion chromatography, broadening or tailing of a broad peak may be determined to be significant under the test conditions and in the context of the present invention, depending on the column used, the operating pressure, and the flow rate of the buffer. Using microflow imaging, a significant increase in the number of particles, particularly particles larger than 2 μm and / or 10 μm, may be determined to be significant, especially if the number of particles exceeds the limit value in the official document. The method for obtaining infectivity is the 50% tissue culture infective dose, that is, the amount of a cytopathic agent that will cause cytopathic effects in 50% of the seeded cells.

[0136] In the context of this invention, the terms “stress” or “stress conditions” refer to, for example, mechanical stress, thermal stress, photostress, or freeze-thaw and the resulting stress, as shown in particular in the Examples section. A variety of methods and conditions for stimulating mechanical stress, thermal stress, photostress, or stress resulting from freeze-thaw are known to those skilled in the art. Mechanical stress may be, for example, shaking at 300 rpm for up to 48 hours at room temperature, or carefully shaking a virus-containing vial manually. Thermal stress may refer to, for example, storage at a reduced or increased temperature over a period of time; in one example, the sample may be stored at temperatures of 5°C, 25°C, or 30°C, where 25°C and 30°C refer to accelerated stress conditions. Photostress may be, for example, storage of the sample at a light intensity of about 1100 lux over 5 days at various temperatures. The sample can be subjected to stress from freezing and thawing by repeatedly subjecting it to a cycle of freezing at a temperature of, for example, -80°C and thawing at room temperature for 2 hours, with this cycle being repeated 3 to 5 times.

[0137] The term "substantially chloride-free" means that no chloride ion source is added to the pharmaceutical composition, preferably no chloride ion source is added to the pharmaceutical composition from an external source. More preferably, the pharmaceutical composition is chloride ion-free.

[0138] buffer solution As used herein, “buffer solution” refers to a buffered solution that can withstand changes in pH due to the action of its acid-base conjugated components. As used herein, “pH” refers to the acidity or alkalinity of the composition at room temperature. Standard methods for measuring the pH of a composition are known to those skilled in the art. Typically, pH measurement involves calibrating the instrument, placing electrodes in a well-mixed sample, and then directly decoding the pH from a pH meter.

[0139] In various embodiments, the pharmaceutical composition may include a buffer. Exemplary buffers of the present invention include acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate, and Tris.

[0140] sugar In various embodiments, the pharmaceutical composition may contain a sugar or a combination of several sugars. Exemplary sugars of the present invention include dextrose, fructose, galactose, glucose, raffinose, trehalose, or sucrose.

[0141] sugar alcohol In various embodiments, the pharmaceutical composition may contain a sugar alcohol or a combination of several sugar alcohols. Exemplary sugar alcohols of the present invention include mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, or hydrolyzed hydrogenated starch.

[0142] Poloxamer As used herein, "EO-PO block copolymer" means a copolymer consisting of blocks of poly(ethylene oxide) and poly(propylene oxide).

[0143] As used herein, "Pluronic®" is an EO (Registered Trademark) x -PO y -EO z This refers to the EO-PO block copolymer within the structure. This structure is also called a "poloxamer."

[0144] Regarding the general term poloxamer, these copolymers are typically named using a three-digit number following the letter P (for poloxamer): multiplying the first two digits by 100 gives the approximate molecular weight of the polyoxypropylene core, and multiplying the last digit by 10 gives the polyoxyethylene content (for example, P188 = a poloxamer with a polyoxypropylene molecular weight of 7680-9510 g / mol and a polyoxyethylene content of 20%). For poloxamer 188, the poly(propylene oxide) chain contains a number of units ranging from 25 to 30, and each poly(ethylene oxide) block consists of an average of 75-85 ethylene oxide units (L. Bollenbach, J. Buske, K. Mader, P. Garidel, International Journal of Pharmaceutics, Volume 620, 2022).

[0145] The "protein substance" used herein refers to albumin, gelatin, serum albumin, recombinant albumin, bovine serum albumin, porcine serum albumin, human serum albumin, recombinant human albumin, preferably human serum albumin (HSA) or recombinant human albumin (rHA).

[0146] In some embodiments, the pharmaceutical composition comprises human serum albumin (HSA), preferably recombinant human albumin (rHA). HSA is the most abundant protein found in human plasma. As used herein, the term “recombinant” means, in the context of “HA,” that rHA is either a genetically engineered product or produced by a recombinant production method. rHA is not derived from (isolated from or purified from) a natural product (e.g., human plasma), but can be produced, for example, through genetically engineered cells; however, other methods for obtaining rHA may be equally used by those skilled in the art.

[0147] All of the following tables should be read such that the formulation contains or consists of the components within the specified concentration ranges. Optional components may or may not be part of the formulation. When the term "at least one" or "one or more" is used with a concentration range, the concentration range is understood to apply individually to each component. For example, if there are two amino acids and only one concentration range of 1 - 300 mM is indicated, each amino acid individually has a concentration range of 1 - 300 mM. When the term "one or more" is used in conjunction with a total concentration range, the said total concentration range is the total concentration of the component in the formulation.

[0148]

Table 1

[0149]

Table 2

[0150] The formulations described in the present invention are shown below. Preferably, the formulation is provided as a dry formulation. The dry formulation can be made from a liquid formulation, where the liquid formulation contains or consists of the described components and has the described concentration ranges before being dried. By reconstituting the dry formulation with an appropriate volume of an aqueous solution such as water, a liquid formulation having the following components and concentration ranges will then be obtained again.

[0151]

Table 3

[0152]

Table 4

[0153]

Table 5

[0154] Table 6

[0155] Table 7

[0156] Table 8

[0157] Table 9

[0158] Table 10

[0159] Table 11

[0160] Table 12

[0161] Table 13

[0162] Table 14

[0163] Table 15

[0164] [Table 16]

[0165] [Table 17]

[0166] [Table 18]

[0167] [Table 19]

[0168] [Table 20]

[0169] [Table 21]

[0170] [Table 22]

[0171] [Table 23]

[0172] In another embodiment, the preparation, preferably a dry preparation, comprises an enveloped virus, about 10-50 mM Tris, about 100-500 mM Trehalose, about 0-50 mM Mannitol, about 50-100 mM Sorbitol, about 5-25 mM Glutamic Acid, about 3-10 g / L rHA, and about 1.5-5 g / L Poloxamer 188, with a pH of 7.0-8.0, preferably about 7.4.

[0173] In another embodiment, the preparation, preferably a dry preparation, contains an enveloped virus, about 10 mM Tris, about 200 mM Trehalose, about 50 mM Sorbitol, about 20 mM Glutamic Acid, about 5 g / L rHA, and about 1.5 to 5 g / L Poloxamer 188, with a pH of 7.0 to 8.0, preferably about 7.4.

[0174] In another embodiment, the preparation, preferably a dry preparation, comprises an enveloped virus, about 8–12 mM Tris, about 180–220 mM Trehalose, about 40–60 mM Sorbitol, about 15–25 mM Glutamic Acid, about 4–6 g / L rHA, and about 1.5–5 g / L Poloxamer 188, with a pH of 7.0–8.0, preferably about 7.4.

[0175] In another embodiment, the preparation, preferably a dry preparation, comprises an enveloped virus, about 10 mM Tris, about 200 mM trehalose, about 50 mM sorbitol, about 50 mM mannitol, about 20 mM glutamic acid, about 5 g / L rHA, and about 2 g / L poloxamer 188, with a pH of 7.0 to 8.0, preferably about 7.4.

[0176] In another embodiment, the preparation, preferably a dry preparation, comprises an enveloped virus, about 8–12 mM Tris, about 180–220 mM Trehalose, about 40–60 mM Sorbitol, about 40–60 mM Mannitol, about 15–25 mM Glutamic Acid, about 4–6 g / L rHA, and about 1.5–2.5 g / L Poloxamer 188, with a pH of 7.0–8.0, preferably about 7.4.

[0177] In another embodiment, the preparation, preferably a dry preparation, contains an enveloped virus, about 20 mM Tris, about 150 mM sucrose, about 50 mM mannitol, about 50 mM sorbitol, about 20 mM glutamic acid, about 5 g / L rHA, and a pH of 7.0 to 8.0, preferably about 7.4.

[0178] In another embodiment, the preparation, preferably a dry preparation, contains an enveloped virus, about 15-25 mM Tris, about 140-160 mM sucrose, about 40-60 mM mannitol, about 40-60 mM sorbitol, about 15-25 mM glutamic acid, about 4-6 g / L rHA, and a pH of 7.0-8.0, preferably about 7.4.

[0179] In another embodiment, the preparation, preferably a dry preparation, contains an enveloped virus, about 10 mM Tris, about 200 mM Trehalose, about 50 mM Mannitol, about 50 mM Sorbitol, about 20 mM Glutamic Acid, about 10 g / L rHA, and about 0.05 g / L Poloxamer 188, with a pH of 7.0 to 8.0, preferably about 7.4.

[0180] In another embodiment, the preparation, preferably a dry preparation, comprises an enveloped virus, about 8–12 mM Tris, about 180–220 mM Trehalose, about 40–60 mM Mannitol, about 40–60 mM Sorbitol, about 15–25 mM Glutamic Acid, about 8–12 g / L rHA, and about 0.05–1.5 g / L Poloxamer 188, with a pH of 7.0–8.0, preferably about 7.4.

[0181] In another embodiment, the preparation, preferably a dry preparation, comprises an enveloped virus, about 10 mM Tris, about 200 mM trehalose, about 50 mM mannitol, about 50 mM sorbitol, about 20 mM glutamic acid, about 10 g / L rHA, and about 2.5 g / L poloxamer 188, with a pH of 7.0 to 8.0, preferably about 7.4.

[0182] In another embodiment, the preparation, preferably a dry preparation, comprises an enveloped virus, about 8–12 mM Tris, about 180–220 mM Trehalose, about 40–60 mM Mannitol, about 40–60 mM Sorbitol, about 15–25 mM Glutamic Acid, about 8–12 g / L rHA, and about 1.5–3.5 g / L Poloxamer 188, with a pH of 7.0–8.0, preferably about 7.4.

[0183] In another embodiment, the preparation, preferably a dry preparation, comprises an enveloped virus, about 10 mM Tris, about 200 mM Trehalose, about 50 mM Mannitol, about 50 mM Sorbitol, about 20 mM Glutamic Acid, about 10 g / L rHA, and about 5 g / L Poloxamer 188, with a pH of 7.0 to 8.0, preferably about 7.4.

[0184] In another embodiment, the preparation, preferably a dry preparation, comprises an enveloped virus, about 8–12 mM Tris, about 180–220 mM Trehalose, about 40–60 mM Mannitol, about 40–60 mM Sorbitol, about 15–25 mM Glutamic Acid, about 8–12 g / L rHA, and about 2.5–6 g / L Poloxamer 188, with a pH of 7.0–8.0, preferably about 7.4.

[0185] In another embodiment, the preparation, preferably a dry preparation, comprises an enveloped virus, about 10 mM Tris, about 200 mM Trehalose, about 20.7–34.6 mM Mannitol, about 49–49.6 mM Sorbitol, about 20 mM Glutamic Acid, about 3.4–4.7 g / L rHA, and about 1.4 g / L Poloxamer 188, with a pH of 7.0–8.0, preferably about 7.4.

[0186] In any of the above embodiments of the preparations, preferably the dry preparations, the concentration of the buffer may be within any concentration range of 1 to 100 mM, for example, 1 to 90 mM, 1 to 80 mM, 1 to 70 mM, 1 to 60 mM, or 1 to 50 mM. Preferably, the concentration of the buffer is 5 mM to 50 mM, 5 mM to 40 mM, 5 mM to 30 mM, or 5 mM to 20 mM. Preferably, the dry preparation contains Tris buffer, more preferably Tris buffer at a concentration of 5 to 50 mM.

[0187] In any of the above formulations, preferably dry formulations, the concentration of the sugar is within any concentration range of 1 to 500 mM, for example, 1 to 490 mM, 1 to 480 mM, 1 to 470 mM, 1 to 460 mM, 1 to 450 mM, 1 to 440 mM, 1 to 430 mM, 1 to 420 mM, 1 to 410 mM, 1 to 400 mM, 1 to 390 mM, 1 to 380 mM The concentration may be mM, 1-370 mM, 1-360 mM, 1-350 mM, 1-340 mM, 1-330 mM, 1-320 mM, 1-310 mM, 1-300 mM, 1-290 mM, 1-280 mM, 1-270 mM, 1-260 mM, 1-250 mM, 1-240 mM, 1-230 mM, 1-220 mM, 1-210 mM, or 1-200 mM. Preferably, the concentration of the sugar is 50 mM-300 mM, 50 mM-250 mM, or 50 mM-200 mM. Preferably, the preparation contains a sugar, such as dextrose, fructose, galactose, glucose, raffinose, trehalose, or sucrose. More preferably, the sugar is trehalose, at a concentration of 50 to 250 mM.

[0188] In any of the above formulations, preferably dry formulations, the amino acid concentration is within any concentration range of 1 to 300 mM, for example, 1 to 290 mM, 1 to 280 mM, 1 to 270 mM, 1 to 260 mM, 1 to 250 mM, 1 to 240 mM, 1 to 230 mM, 1 to 220 mM, 1 to 210 mM, 1 to 200 mM, 1 to 19 The concentration may be 0 mM, 1-180 mM, 1-170 mM, 1-160 mM, 1-150 mM, 1-140 mM, 1-130 mM, 1-120 mM, 1-110 mM, 1-100 mM, 1-90 mM, 1-80 mM, 1-70 mM, 1-60 mM, 1-50 mM, 1-40 mM, 1-30 mM, 1-20 mM, or 1-10 mM. Preferably, the amino acid concentration is 5 mM-100 mM, 5 mM-90 mM, 5 mM-80 mM, 5 mM-70 mM, 5 mM-60 mM, or 5 mM-50 mM. Preferably, the dry preparation contains an amino acid, such as arginine, alanine, phenylalanine, glycine, glutamine, glutamic acid, methionine, or lysine. More preferably, the amino acid is glutamic acid. Most preferably, the concentration of glutamic acid is 50-100 mM, or 5-50 mM, or 5-20 mM.

[0189] In any of the above embodiments of the preparation, preferably the dry preparation, the concentration of the sugar alcohol may be within any concentration range of 1 to 200 mM, for example, 1 to 190 mM, 1 to 180 mM, 1 to 170 mM, 1 to 160 mM, 1 to 150 mM, 1 to 140 mM, 1 to 130 mM, 1 to 120 mM, 1 to 110 mM, or 1 to 100 mM. Preferably, the concentration of the sugar alcohol is 5 to 500 mM, 10 to 100 mM, 10 to 80 mM, 10 to 70 mM, 10 to 60 mM, or 10 to 50 mM. Preferably, the dry preparation contains one or more sugar alcohols, for example, mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, or hydrolyzed hydrogenated starch. More preferably, the sugar alcohol is mannitol and / or sorbitol, more preferably a combination of mannitol and sorbitol. Most preferably, the combination of mannitol and sorbitol is one in which each is independently concentrated at a concentration of 5 to 100 mM.In any of the above formulations, preferably in the dry formulation embodiment, the concentration of poloxamer is within any concentration range of 0.01 g / L to 50 g / L, for example, 0.02 g / L to 50 g / L, 0.03 g / L to 50 g / L, 0.04 g / L to 50 g / L, 0.05 g / L to 50 g / L, 0.05 g / L to 49 g / L, 0.05 g / L to 48 g / L, 0.05 g / L to 47 g / L, 0. 05g / L~46g / L, 0.05g / L~45g / L, 0.05g / L~44g / L, 0.05g / L~43g / L, 0.05g / L~42g / L, 0.05g / L~41g / L, 0.0 5g / L~40g / L, 0.05g / L~39g / L, 0.05g / L~38g / L, 0.05g / L~37g / L, 0.05g / L~36g / L, 0.05g / L~35g / L, 0.05 g / L~34g / L, 0.05g / L~33g / L, 0.05g / L~32g / L, 0.05g / L~31g / L, 0.05g / L~30g / L, 0.05g / L~29g / L, 0.05 g / L~28g / L, 0.05g / L~27g / L, 0.05g / L~26g / L, 0.05g / L~25g / L, 0.05g / L~24g / L, 0.05g / L~23g / L, 0.05g It may be 0.05g / L~22g / L, 0.05g / L~21g / L, 0.05g / L~19g / L, 0.05g / L~18g / L, 0.05g / L~17g / L, 0.05g / L~16g / L, 0.05g / L~15g / L, 0.05g / L~14g / L, 0.05g / L~13g / L, 0.05g / L~12g / L, 0.05g / L~11g / L, or 0.05g / L~40g / L. More preferred ranges include 0.05 g / L to 15 g / L, 0.05 g / L to 14 g / L, 0.05 g / L to 13 g / L, 0.05 g / L to 12 g / L, 0.05 g / L to 11 g / L, or 0.05 g / L to 10 g / L. Preferably, the formulation contains pharmaceutically acceptable poloxamer. More preferably, the poloxamer is poloxamer 188. Most preferred concentrations of poloxamer 188 are 0.05 g / L to 20 g / L, 0.05 g / L to 15 g / L, 0.05 g / L to 10 g / L, 0.5 g / L to 10 g / L, or 1 g / L to 10 g / L.

[0190] In any of the above formulations, preferably in the dry formulation embodiment, the concentration of the protein substance may be within any concentration range of 0.1 g / L to 50 g / L, for example, 1 g / L to 50 g / L, 1.1 g / L to 50 g / L, 1.2 g / L to 50 g / L, 1.3 g / L to 50 g / L, 1.4 g / L to 50 g / L, 1.5 g / L to 50 g / L, 1.6 g / L to 50 g / L, 1.7 g / L to 50 g / L, 1.8 g / L to 50 g / L, 1.9 g / L to 50 g / L, 2.0 g / L to 50 g / L, 2.1 g / L to 50 g / L, 2.2 g / L to 50 g / L, 2.3 g / L to 50 g / L, 2.4 g / L to 50 g / L, or 2.5 g / L to 50 g / L. More preferred ranges include 1 g / L to 45 g / L, 1 g / L to 40 g / L, 1 g / L to 35 g / L, 1 g / L to 30 g / L, 1 g / L to 25 g / L, 1 g / L to 20 g / L, 1 g / L to 15 g / L, 1.5 g / L to 10 g / L, 2.0 g / L to 10 g / L, or 2.5 g / L to 10 g / L. Preferably, the formulation contains recombinant human albumin. Most preferred is recombinant human albumin at concentrations of 1 g / L to 20 g / L, 1 g / L to 15 g / L, 1.5 g / L to 10 g / L, 2.0 g / L to 10 g / L, or 2.5 g / L to 10 g / L.

[0191] The pH of the above-described formulations, preferably the dry formulations, is typically maintained within the range of 5 to 9, or 6 to 9, or 6.5 to 8.5, or 6.5 to 8.0, preferably 7.0 to 8.0. Those skilled in the art will understand that the pH of the formulation refers to the pH of the formulation when it is in aqueous solution form.

[0192] In any of the above formulations, preferably in the dry formulation embodiment, the enveloped virus is preferably becyclovirus, more preferably vesicular stomatitis virus, and most preferably vesicular stomatitis virus having glycoprotein G substituted with glycoprotein GP of lymphocytic choriomeningitis virus (LCMV). In any of the above formulation embodiments, the viral concentration is 1 × 10⁻⁶ 5 TCID 50 / mL ~ 1 × 10 12 TCID 50Any concentration range within / mL, or at least 1 × 10 5 TCID 50 / mL, at least 1 × 10⁻⁶ 6 TCID 50 / mL, at least 1 × 10⁻⁶ 7 TCID 50 / mL, at least 1 × 10⁻⁶ 8 TCID 50 / mL, or at least 1 × 10 9 TCID 50 The concentration can be as low as / mL. Other ranges are 1 × 10 6 TCID 50 / mL ~ 1 × 10 12 TCID 50 / mL, 1 × 10 6 TCID 50 / mL ~ 1 × 10 11 TCID 50 Includes / mL, etc.

[0193] Further formulations described in the present invention are shown below.

[0194] Hereinafter, all tables should be read as indicating that the preparation contains or consists of components within the specified concentration range. Optional components may or may not be part of the preparation. Where the terms “at least one” or “one or more” are used with a concentration range, the concentration range is understood to apply individually to each component; for example, if there are two amino acids and only one concentration range of 1–300 mM is indicated, each amino acid individually has a concentration range of 1–300 mM. Furthermore, where the terms “one or more” are used with a total concentration range, the total concentration range is the total concentration of one or more of the components in the preparation.

[0195] [Table 24]

[0196] [Table 25]

[0197] Table 26

[0198] Table 27

[0199] Table 28

[0200] Table 29

[0201] Table 30

[0202] Table 31

[0203] Table 32

[0204] Table 33

[0205] Table 34

[0206] Table 35

[0207] [Table 36]

[0208] [Table 37]

[0209] [Table 38]

[0210] In its broadest form, the present invention relates to a pharmaceutical composition comprising an enveloped virus, one or more sugar alcohols, and a protein substance and / or a poly(ethylene oxide) / poly(propylene oxide) block copolymer. The phrase "at least one of buffers, sugars, or amino acids" means that the formulation must contain at least one of such components, but may contain two or all three of such components. For example, a formulation containing at least one of buffers, sugars, or amino acids may contain buffer; sugar; amino acid; buffer and sugar; buffer and amino acid; buffer, sugar and amino acid; or sugar and amino acid. It will also be understood that the formulation may contain, for example, two or more amino acids, two or more sugars, etc. Preferably, the formulation is provided as a dry formulation, for example, a lyophilized formulation.

[0211] In any of the above formulations, preferably dry formulations, such as freeze-dried formulations, the concentration of poloxamer is 0.01-50 g / L, 0.1-50 g / L, 0.2-50 g / L, 0.3-50 g / L, 0.4-50 g / L, 0.5-50 g / L, 1-50 g / L, 2-50 g / L, 3-50 g / L, 4-50 g / L, 5-50 g / L, 0.01-40 g / L, 0.01~30g / L, 0.01~20g / L, 0.01~10g / L, 0.01~5g / L, 0.1~40g / L, 0.1~30g / L, 0.1~20g / L, 0. 1~10g / L, 0.1~5g / L, 0.2~40g / L, 0.2~30g / L, 0.2~20g / L, 0.2~10g / L, 0.2~5g / L, 0.3~40g / L, 0.3 ~30g / L, 0.3~20g / L, 0.3~10g / L, 0.3~5g / L, 0.4~40g / L, 0.4~30g / L, 0.4~20g / L, 0.4~10g / L, 0.4 ~5g / L, 0.5~40g / L, 0.5~30g / L, 0.5~20g / L, 0.5~10g / L, 0.5~5g / L, 1~40g / L, 1~30g / L, 1~20g / L, The concentration can be in any of the following ranges: 1-10 g / L, 1-5 g / L, 2-40 g / L, 2-30 g / L, 2-20 g / L, 2-10 g / L, 3-40 g / L, 3-30 g / L, 3-20 g / L, 3-10 g / L, 4-40 g / L, 4-30 g / L, 4-20 g / L, 4-10 g / L, 5-40 g / L, 5-30 g / L, 5-20 g / L, or 5-10 g / L. Preferably, the poloxamer is poloxamer 188, and the concentration is in the range of 0.1-10 g / L.

[0212] In any of the above formulations, preferably dry formulations, such as freeze-dried formulations, the concentration of the protein substance is 0.05-50 g / L, 0.1-50 g / L, 0.2-50 g / L, 0.3-50 g / L, 0.4-50 g / L, 0.5-50 g / L, 1-50 g / L, 2-50 g / L, 3-50 g / L, 4-50 g / L, 5-50 g / L, 0.05- 40g / L, 0.05~30g / L, 0.05~20g / L, 0.05~10g / L, 0.05~5g / L, 0.1~40g / L, 0.1~30g / L, 0.1~20g / L, 0.1~10g / L, 0.1~5g / L, 0.2~40g / L, 0.2~30g / L, 0.2~20g / L, 0.2~10g / L, 0.2~5g / L, 0.3~40g / L, 0. 3~30g / L, 0.3~20g / L, 0.3~10g / L, 0.3~5g / L, 0.4~40g / L, 0.4~30g / L, 0.4~20g / L, 0.4~10g / L, 0. 4~5g / L, 0.5~40g / L, 0.5~30g / L, 0.5~20g / L, 0.5~10g / L, 0.5~5g / L, 1~40g / L, 1~30g / L, 1~20g / L, The concentration can be within any of the following ranges: 1-10 g / L, 1-5 g / L, 2-40 g / L, 2-30 g / L, 2-20 g / L, 2-10 g / L, 3-40 g / L, 3-30 g / L, 3-20 g / L, 3-10 g / L, 4-40 g / L, 4-30 g / L, 4-20 g / L, 4-10 g / L, 5-40 g / L, 5-30 g / L, 5-20 g / L, or 5-10 g / L. Preferably, the protein substance is recombinant human albumin or human serum albumin, and its concentration is within the range of 0.5-10 g / L.

[0213] In any of the above formulations, preferably dry formulations, such as freeze-dried formulations, the concentration of the buffer may be within any of the following concentration ranges: 1 to 100 mM, 1 to 90 mM, 1 to 80 mM, 1 to 70 mM, 1 to 60 mM, 1 to 50 mM, 1 to 40 mM, 1 to 30 mM, 1 to 20 mM, or 1 to 10 mM. Preferably, the formulation, preferably a liquid or freeze-liquid formulation, contains Tris buffer, more preferably Tris buffer at a concentration of 5 to 50 mM.

[0214] In any of the above formulations, preferably dry formulations, such as freeze-dried formulations, the total concentration of one or more sugar alcohols is 1-1000 mM, 1-900 mM, 1-800 mM, 1-700 mM, 1-600 mM, 1-550 mM, 1-500 mM, 1-450 mM, 1-400 mM, 1-350 mM, 1-300 mM, 1-250 mM, 1-200 mM, 1-150 mM, 1-100 mM, 1-50 mM, 1-20 mM, 10-1000 mM, 10-900 mM, 10-800 mM, 10-700 mM, 10-600 mM, or 10-550 mM. The total concentration may fall within the following ranges: 10-500 mM, 10-450 mM, 10-400 mM, 10-350 mM, 10-300 mM, 10-250 mM, 10-200 mM, 10-150 mM, 10-100 mM, 10-50 mM, 20-1000 mM, 20-900 mM, 20-800 mM, 20-700 mM, 20-600 mM, 20-550 mM, 20-500 mM, 20-450 mM, 20-400 mM, 20-350 mM, 20-300 mM, 20-250 mM, 20-200 mM, 20-150 mM, 20-100 mM, or 20-50 mM. In another embodiment, the total concentration of one or more sugar alcohols may be at least 10 mM, 20 mM, 30 mM, 40 mM, or at least 50 mM. Total concentration or having a total concentration refers to the total amount of one or more sugar alcohols in the preparation; for example, if 200 mM mannitol is present in the preparation, the total concentration will be 200 mM. However, if, for example, 200 mM mannitol and 300 mM sorbitol are present in the preparation, the total concentration of sugar alcohols will be 500 mM. Preferably, the preparation, preferably a dry preparation, for example a freeze-dried preparation, contains mannitol and / or sorbitol, more preferably a combination of mannitol and sorbitol, at a total concentration of at least 10 mM, 20 mM, 30 mM, 40 mM, or at least 50 mM.

[0215] In any of the above formulations, preferably dry formulations, such as freeze-dried formulations, the sugar concentration is 10-1000 mM, 10-900 mM, 10-800 mM, 10-700 mM, 10-600 mM, 10-500 mM, 10-400 mM, 10-300 mM, 10-200 mM, 20-1000 mM, 20-900 mM, 20-800 mM, 20-700 mM, 20-600 mM, 20-500 mM, 20-400 mM, 20-300 mM, 20-200 mM, 30-1000 mM, 30-900 mM, 30-800 mM The concentration can be any of the following ranges: mM, 30-700 mM, 30-600 mM, 30-500 mM, 30-400 mM, 30-300 mM, 30-200 mM, 40-1000 mM, 40-900 mM, 40-800 mM, 40-700 mM, 40-600 mM, 40-500 mM, 40-400 mM, 40-300 mM, 40-200 mM, 50-1000 mM, 50-900 mM, 50-800 mM, 50-700 mM, 50-600 mM, 50-500 mM, 50-400 mM, 50-300 mM, or 50-200 mM. Preferably, the concentration of the sugar is within any range of 50 mM to 300 mM, 50 mM to 250 mM, or 50 mM to 200 mM. Preferably, the dried preparation, such as a freeze-dried preparation, contains a sugar, such as dextrose, fructose, galactose, glucose, raffinose, trehalose, or sucrose. More preferably, the sugar is trehalose and is at a concentration of 50 to 250 mM.

[0216] The pH of the above-mentioned formulations, preferably dry formulations, such as freeze-dried formulations, is typically maintained within the range of 5 to 9, or 6 to 9, or 6 to 8, or 6.5 to 8.5, or 6.5 to 8.0, preferably 7.0 to 8.0.

[0217] In any of the above-mentioned preparations, preferably in a dry preparation, such as a lyophilized preparation, the envelope virus is preferably a vesicular stomatitis virus in which the glycoprotein G has been replaced with the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), more preferably vesicular stomatitis virus, and most preferably lymphocytic choriomeningitis virus (LCMV). In any of the above-mentioned preparations, preferably in a dry preparation, such as a lyophilized preparation, the virus concentration is within any concentration range of 1×10 5 TCID 50 / mL to 1×10 12 TCID 50 / mL, at least 1×10 5 TCID 50 / mL, at least 1×10 6 TCID 50 / mL, at least 1×10 7 TCID 50 / mL, at least 1×10 8 TCID 50 / mL, or at least 1×10 9 TCID 50 / mL. Other ranges include 1×10 6 TCID 50 / mL to 1×10 12 TCID 50 / mL, 1×10 6 TCID 50 / mL to 1×10 11 TCID 50 / mL, etc.

[0218] In a preferred embodiment, the dry preparation, such as a lyophilized preparation, contains an envelope virus, about 10 mM Tris, about 20 mM glutamic acid, about 50 mM mannitol, about 50 mM sorbitol, about 200 mM trehalose, about 1.275 mg / mL poloxamer 188, about 5 mg / ml recombinant human albumin, pH 7.0 to 8.0, preferably about pH 7.4. Preferably, the pH of the composition is adjusted using phosphoric acid or sodium phosphate.

[0219] In a preferred embodiment, the dry preparation, for example, the freeze-dried preparation, contains an enveloped virus, about 8–12 mM Tris, about 15–25 mM glutamic acid, about 40–60 mM mannitol, about 40–60 mM sorbitol, about 180–220 mM trehalose, about 1–2 mg / mL poloxamer 188, about 4–6 mg / mL recombinant human albumin, and a pH of 7.0–8.0, preferably about 7.4. Preferably, the pH of the composition is adjusted using phosphoric acid or sodium phosphate.

[0220] In a preferred embodiment, the dry preparation, for example, the freeze-dried preparation, contains an enveloped virus, about 10 mM Tris, about 20 mM glutamic acid, about 100 mM arginine, about 100 mM mannitol, about 40 mM sorbitol, about 125 mM trehalose, about 2.5 mg / ml poloxamer 188, about 5 mg / ml recombinant human albumin, and a pH of 7.0 to 8.0, preferably about 7.1. Preferably, the pH of the composition is adjusted using phosphoric acid or sodium phosphate.

[0221] In a preferred embodiment, the dry preparation, for example, the freeze-dried preparation, contains an enveloped virus, about 8–12 mM Tris, about 15–25 mM glutamic acid, about 80–120 mM arginine, about 80–120 mM mannitol, about 30–50 mM sorbitol, about 100–150 mM trehalose, about 1.5–3.5 mg / ml poloxamer 188, about 4–6 mg / ml recombinant human albumin, and a pH of 7.0–8.0, preferably about 7.1. Preferably, the pH of the composition is adjusted using phosphoric acid or sodium phosphate.

[0222] In a preferred embodiment, the dry preparation, for example, the freeze-dried preparation, contains an enveloped virus, about 10 mM Tris, about 20 mM glutamic acid, about 25 mM citrate, about 50 mM mannitol, about 50 mM sorbitol, about 200 mM trehalose, about 2.5 mg / ml poloxamer 188, about 5 mg / ml recombinant human albumin, and a pH of 7.0 to 8.0, preferably about 7.4. Preferably, the pH of the composition is adjusted using phosphoric acid or sodium phosphate.

[0223] In a preferred embodiment, the dry preparation, for example, the freeze-dried preparation, contains an enveloped virus, about 8–12 mM Tris, about 15–25 mM glutamic acid, about 20–30 mM citrate, about 40–60 mM mannitol, about 40–60 mM sorbitol, about 180–220 mM trehalose, about 2–3 mg / ml poloxamer 188, about 4–6 mg / ml recombinant human albumin, and a pH of 7.0–8.0, preferably about 7.4. Preferably, the pH of the composition is adjusted using phosphoric acid or sodium phosphate.

[0224] In a preferred embodiment, the dry preparation, such as a freeze-dried preparation, contains an enveloped virus, about 10 mM Tris, about 20 mM glutamic acid, about 150 mM arginine, about 50 mM mannitol, about 50 mM sorbitol, about 200 mM trehalose, about 1.275 mg / ml poloxamer 188, about 5 mg / ml recombinant human albumin, and a pH of 7.0 to 8.0, preferably about 7.4. Preferably, the pH of the composition is adjusted using lactic acid.

[0225] In a preferred embodiment, the dry preparation, such as a freeze-dried preparation, contains an enveloped virus, about 8–12 mM Tris, about 15–25 mM glutamic acid, about 120–180 mM arginine, about 40–60 mM mannitol, about 40–60 mM sorbitol, about 180–220 mM trehalose, about 1–2 mg / mL poloxamer 188, about 4–6 mg / mL recombinant human albumin, and a pH of 7.0–8.0, preferably about 7.4. Preferably, the pH of the composition is adjusted using lactic acid.

[0226] In a preferred embodiment, the dry preparation, for example, the freeze-dried preparation, contains an enveloped virus, about 10 mM Tris, about 13 mM glutamic acid, about 100 mM arginine, about 33 mM mannitol, about 33 mM sorbitol, about 133 mM trehalose, about 1.275 mg / mL poloxamer 188, about 5 mg / mL recombinant human albumin, and a pH of 7.0 to 8.0, preferably about 7.4. Preferably, the pH of the composition is adjusted using lactic acid.

[0227] In a preferred embodiment, the dry preparation, for example, the freeze-dried preparation, contains an enveloped virus, about 8–12 mM Tris, about 10–15 mM glutamic acid, about 80–120 mM arginine, about 25–40 mM mannitol, about 25–40 mM sorbitol, about 100–150 mM trehalose, about 1–1.5 mg / mL poloxamer 188, about 4–6 mg / mL recombinant human albumin, and a pH of 7.0–8.0, preferably about 7.4. Preferably, the pH of the composition is adjusted using lactic acid.

[0228] In a preferred embodiment, the dry preparation, for example, the freeze-dried preparation, contains an enveloped virus, about 1 to 50 mM Tris, about 10 to 30 mM glutamic acid, about 100 to 200 mM trehalose, about 20 to 100 mM mannitol, about 20 to 100 mM sorbitol, about 0.5 to 5 mg / mL poloxamer 188, about 2 to 10 mg / mL recombinant human albumin, and a pH of 7 to 8.

[0229] In a preferred embodiment, the dry preparation, for example, the freeze-dried preparation, contains an enveloped virus, about 1–50 mM Tris, about 20–50 mM citrate, about 10–30 mM glutamic acid, about 100–200 mM trehalose, about 20–100 mM mannitol, about 20–100 mM sorbitol, about 0.5–5 mg / mL poloxamer 188, about 2–10 mg / mL recombinant human albumin, and a pH of 7–8.

[0230] In a preferred embodiment, the dry preparation, for example, the freeze-dried preparation, contains an enveloped virus, about 1–50 mM Tris, about 10–30 mM glutamic acid, about 50–200 mM arginine, about 100–200 mM trehalose, about 20–100 mM mannitol, about 20–100 mM sorbitol, about 0.5–5 mg / mL poloxamer 188, about 2–10 mg / mL recombinant human albumin, and pH 7–8.

[0231] In a preferred embodiment, the dry preparation, for example, the freeze-dried preparation, contains an enveloped virus, about 20–50 mM citric acid, about 10–30 mM glutamic acid, about 100–200 mM trehalose, about 20–100 mM mannitol, about 20–100 mM sorbitol, about 0.5–5 mg / mL poloxamer 188, about 2–10 mg / mL recombinant human albumin, and a pH of 7–8.

[0232] In a preferred embodiment, the dry preparation, for example, the freeze-dried preparation, contains an enveloped virus, about 10–30 mM glutamic acid, about 50–200 mM arginine, about 100–200 mM trehalose, about 20–100 mM mannitol, about 20–100 mM sorbitol, about 0.5–5 mg / mL poloxamer 188, about 2–10 mg / mL recombinant human albumin, and a pH of 7–8.

[0233] In a preferred embodiment, the dry preparation, for example, the freeze-dried preparation, contains an enveloped virus, about 10–30 mM glutamic acid, about 100–200 mM trehalose, about 20–100 mM mannitol, about 20–100 mM sorbitol, about 0.5–5 mg / ml poloxamer 188, about 2–10 mg / ml recombinant human albumin, and a pH of 7–8.

[0234] [Table 39] TIFF2026519453000040.tif18170

[0235] [Table 40]

[0236] [Table 41]

[0237] [Table 42]

[0238] [Table 43] TIFF2026519453000045.tif30170

[0239] Further aspects: In a second aspect, the present invention relates to a pharmaceutical composition comprising an enveloped virus, (optionally) a buffer, a sugar, at least one amino acid, one or more sugar alcohols, and a protein substance and / or a block copolymer of poly(ethylene oxide) and poly(propylene oxide).

[0240] In one embodiment relating to the second aspect, the amino acid is selected from the group consisting of alanine, arginine, phenylalanine, glutamic acid, glycine, methionine, lysine, or glutamine, and is preferably glutamic acid and / or arginine.

[0241] In one embodiment relating to the second aspect, the composition is substantially chloride-free, preferably substantially sodium chloride-free.

[0242] In one embodiment relating to the second aspect, the pH of the composition is 5 to 9, or 6 to 9, or 6.5 to 8.5, or 6.5 to 8.0, preferably 7.0 to 8.0.

[0243] In one embodiment relating to the second aspect, the pH of the composition is adjusted using phosphoric acid, lactic acid, citric acid, succinic acid, or sodium phosphate.

[0244] In one embodiment relating to the second aspect, the buffer is selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate, and Tris, and is preferably Tris.

[0245] In one embodiment relating to the second aspect, the sugar is sucrose or trehalose, preferably trehalose.

[0246] In one embodiment relating to the second aspect, one or more sugar alcohols(s) are selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, inositol, glycerol, erythritol, isomalt, or hydrolyzed hydrogenated starch.

[0247] In one embodiment relating to the second aspect, one or more sugar alcohols are mannitol and / or sorbitol, preferably a combination of mannitol and sorbitol.

[0248] In one embodiment relating to the second aspect, the poly(ethylene oxide) and poly(propylene oxide) block copolymer is a poloxamer, preferably a pharmaceutically acceptable poloxamer, more preferably a poloxamer 188.

[0249] In one embodiment relating to the second aspect, the protein substance is albumin, gelatin, preferably human serum albumin, or recombinant human albumin.

[0250] In one embodiment relating to the first aspect, the composition comprises both a protein substance and a poly(ethylene oxide) / poly(propylene oxide) block copolymer.

[0251] In one embodiment relating to the second aspect, the protein substance is recombinant human albumin, and the poly(ethylene oxide) / poly(propylene oxide) block copolymer is poloxamer 188.

[0252] In one embodiment relating to the second aspect, the composition comprises: an enveloped virus; a buffer at a concentration of 1 mM to 100 mM, selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate, and Tris, preferably Tris buffer; glutamic acid at a concentration of 10 mM to 500 mM; a sugar at a concentration of 10 mM to 1000 mM, selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, or sucrose, preferably trehalose; mannitol at a concentration of 1 to 100 mM and / or sorbitol at a concentration of 1 to 100 mM; and poloxamer 188 at a concentration of 0.01 g / L to 50 g / L and / or recombinant human albumin at a concentration of 0.1 g / L to 50 g / L.

[0253] In a third aspect, the present invention relates to a pharmaceutical composition comprising an enveloped virus, Tris buffer, glutamic acid and / or arginine, trehalose, mannitol and / or sorbitol, and poloxamer 188 and / or recombinant human albumin.

[0254] In one embodiment relating to a third aspect, the pharmaceutical composition comprises an enveloped virus, Tris buffer, glutamic acid and / or arginine, trehalose, mannitol and / or sorbitol, poloxamer 188, and recombinant human albumin.

[0255] In one embodiment relating to the third aspect, the poloxamer 188 is present in a concentration of 0.01 g / L to 50 g / L.

[0256] In one embodiment related to the third aspect, the recombinant human albumin is present in a concentration of 0.1 g / L to 50 g / L.

[0257] In a fourth aspect, the present invention relates to a pharmaceutical composition comprising: an enveloped virus; a buffer solution in a concentration of 1 mM to 100 mM, selected from the group consisting of acetate, citrate, histidine, succinate, HEPES, tartrate, phosphate, citrate / phosphate, lactate, and Tris, preferably Tris buffer; glutamic acid in a concentration of 10 mM to 500 mM; a sugar in a concentration of 10 mM to 1000 mM, selected from the group consisting of dextrose, fructose, galactose, glucose, raffinose, trehalose, or sucrose, preferably trehalose; mannitol in a concentration of 1 to 100 mM and / or sorbitol in a concentration of 1 to 100 mM; and poloxamer 188 in a concentration of 0.01 g / L to 50 g / L and / or recombinant human albumin in a concentration of 0.1 g / L to 50 g / L.

[0258] In one embodiment relating to the second aspect, the pharmaceutical composition comprises an enveloped virus, Tris buffer in a concentration of 1 mM to 100 mM, glutamic acid in a concentration of 10 mM to 500 mM, trehalose in a concentration of 10 mM to 1000 mM, mannitol in a concentration of 1 mM to 100 mM and / or sorbitol in a concentration of 1 mM to 100 mM, and poloxamer 188 in a concentration of 0.01 g / L to 50 g / L and / or recombinant human albumin in a concentration of 0.1 g / L to 50 g / L.

[0259] In one embodiment and a group of embodiments relating to any of the above embodiments, the enveloped virus is a Rhabdoviridae, preferably a becyclovirus or a vesicular stomatitis virus (VSV). In the relevant embodiments, the enveloped virus is recombinant vesicular stomatitis virus (VSV), wherein the gene encoding glycoprotein G of vesicular stomatitis virus is replaced by the gene encoding glycoprotein GP of lymphocytic choriomeningitis virus, and / or glycoprotein G is replaced by glycoprotein GP of lymphocytic choriomeningitis virus.

[0260] In one embodiment related to any of the above embodiments and in a group of such embodiments, the pharmaceutical composition is a liquid or frozen liquid pharmaceutical composition.

[0261] In a fifth embodiment, the present invention relates to a dried pharmaceutical composition produced by a method comprising the step of removing water from a pharmaceutical composition, the composition comprising an enveloped virus, Tris buffer at a concentration of 1 mM to 100 mM, glutamic acid at a concentration of 10 mM to 500 mM, trehalose at a concentration of 10 mM to 1000 mM, mannitol at a concentration of 1 mM to 100 mM and / or sorbitol at a concentration of 1 mM to 100 mM, and poloxamer 188 at a concentration of 0.01 g / L to 50 g / L and / or recombinant human albumin at a concentration of 0.1 g / L to 50 g / L. In related embodiments, the dried pharmaceutical composition is obtained by freeze-drying / freeze-drying or spray-drying. In further related embodiments, the method comprises the step of removing water from the pharmaceutical composition by placing it under a vacuum at a controlled temperature and pressure. In related embodiments, the method is freeze-drying. In another embodiment, the dry pharmaceutical composition contains less than about (0.1% to 10%) w / w of water.

[0262] In the sixth aspect, the present invention relates to a pharmaceutical composition containing water, and to a dry pharmaceutical composition according to the third aspect and any of its embodiments.

[0263] In one embodiment relating to any of the above embodiments and their sets of embodiments, the pharmaceutical composition is a dry pharmaceutical composition.

[0264] In another embodiment relating to any of the above embodiments and their sets of embodiments, the pharmaceutical composition thus further comprises arginine, preferably at a concentration of 10 mM to 500 mM.

[0265] In a seventh aspect, the present invention results in a reconstitution solution that, when reconstituted with water, contains an enveloped virus, Tris buffer at a concentration of 1 mM to 100 mM, glutamic acid at a concentration of 10 mM to 500 mM, trehalose at a concentration of 10 mM to 1000 mM, mannitol at a concentration of 1 mM to 100 mM, and / or sorbitol at a concentration of 1 mM to 100 mM, and poloxamer 188 at a concentration of 0.01 g / L to 50 g / L, and / or recombinant human albumin at a concentration of 0.1 g / L to 50 g / L.

[0266] Examples method: Preparations and test viruses Preparations and viruses to be tested The virus being tested is a vesicular stomatitis virus in which the wild-type glycoprotein G is replaced by the glycoprotein GP of lymphocytic choriomeningitis virus; such viruses are subsequently named VSV-GP (vesicular stomatitis virus-glycoprotein). In some cases, VSV-GP further codes for a cargo, and such viruses are subsequently named VSV-GP-cargo 1 / 2 / or 3. The viral material VSV-GP or VSV-GP-cargo 1 / 2 / or 3 is approximately 5 × 10⁻⁶. 9 TCID 50 It is used undiluted at a concentration of / ml. Dialysis cassettes (Slide-a-Lyzer dialysis cassettes, Thermo, MWCO (molecular weight cutoff) 10kDa or 20kDa, 12-30ml) are used to introduce the virus into specific formulations. The sample is dialyzed three times over 2 hours each time, with slow agitation at 2-8°C or room temperature. The final step is performed overnight. Sterile filtration is performed using a 0.22 μm polyethersulfone (PES) filter. For macromolecules that do not pass through the dialysis membrane, namely recombinant human albumin (rHA), dextran, and poloxamer 188, an appropriate volume of stock solution is added after dialysis. For placebo samples, a certain amount of recombinant human albumin and poloxamer 188 is added to 15 ml of each sterile-filtered formulation.

[0267] Infectivity (TCID) 50 ) Method for determining Cells and viruses: BHK (Baby Hamster Kidney)-21 cells (No. 603126 (C13), CLS) are cultured at a temperature of 37°C in 5% CO2. The medium (GMEM No. 21710082, Thermo) is supplemented with 8.7% fetal bovine serum and 4.3% tryptose phosphate broth. The BHK-21 cells are washed with phosphate-buffered saline and detached from the cell culture flask by incubation with TrypLE (trademark) Select enzyme at a temperature of 37°C for 6 - 8 minutes. The cells in the medium are picked up, counted using Flex2 (Novo Biomedical), and seeded into 96-well plates.

[0268] TCID 50 Assay: Seed 10 4 BHK-21 cells per well into 100 μl of supplemented Glasgow minimum essential medium (GMEM) per well of a 96-well plate. After 24 hours of incubation, the adherent cells are infected with a 11×0.5 log 10 serial dilution of the virus or only the diluent (negative control), and then incubated for 3 days at a temperature of 37°C and 5% carbon dioxide. The bright-field images of the wells of the cell culture fluid are taken with a Cytation5 multimode imaging reader (BioTek) using a 4× objective lens. Whether the cytopathic effect of the imaged wells is positive or negative is evaluated visually (by the naked eye). The final titer [TCID 50 / ml] is calculated by the Spearman - Karber formula. For each virus sample, infection with serial dilutions is performed on a total of 8 plates on the same day. Based on such 8 replicates, TCID 50 / ml is calculated. As an assay control, a crude collection of vesicular stomatitis virus stored at a temperature of -80°C is used and performed in separate 96-well plates at each time point.

[0269] Method for determining visible and invisible particles Detection of visible particles by visual inspection (VI) Visual inspections are conducted using a stepwise procedure in accordance with company standards. The inspections are performed by two trained inspectors.

[0270] Step 1: Before testing, allow the sample to equilibrate to room temperature in the dark.

[0271] Step 2: Place the vial outside the inspection zone on the workbench without rotating or reversing it. Inspect the bottom of the vial and the curved section of the vial for particles.

[0272] Step 3: Inspect the vial for sedimentation at the bottom of the vial, directly in front of the light source and outside the inspection zone. Inspect the vial in an upright position, handling it carefully to avoid causing any sediment to swirl up.

[0273] Step 4: Inspect the vial outside the inspection zone, directly in front of the light source. Keep the vial upright and rotating. If there is any sediment at the bottom of the vial, it will rise and form a sediment of particles invisible to the naked eye that may swirl around.

[0274] Step 5: Examine the vial for the presence of visible particles at 2,000–3,750 lux in accordance with the European Pharmacopoeia (9th edition; monograph 2.9.20). Homogenize the solution by gently rotating it to avoid the formation of bubbles, and examine the liquid for 5 seconds in front of a white background and 5 seconds in front of a black background within the examination area.

[0275] Step 6: As the final step, the vial is inspected outside the inspection zone, directly in front of the light source. The vial is carefully rotated. The sample is inspected for very small particles, which are invisible to the naked eye only if present in large numbers.

[0276] Detection of particles invisible to the naked eye using microfluidic imaging (MFI) Microfluid imaging measurements are performed using the MFI-5200 particle analyzer system equipped with a silane-coated, high-resolution 100 μm flow cell. Briefly, the sample is diluted 5-fold with ADB. A 0.25 ml pre-lane volume is followed by 0.6 ml of the sample. Between measurements, the flow cell is rinsed with water. Background irradiation is optimized by using water. Measurements are performed using MFI's MVSS software version 2-R5.0.0.43, and samples are analyzed using MFI MVAS software version 1.3.0.1007.

[0277] Freeze drying Samples in 2R vials (0.4 ml) with Flurotec® stoppers are freeze-dried using a pilot-scale LCD-2-6D or LCD-2-10D freeze-dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany). Vacuum during the freeze-drying process is controlled by a diaphragm vacuum gauge. A conservative freeze-drying process is applied based on the calculated solid content and the estimated glass transition temperature (Tg') of the maximum freeze-concentrated solution. During the freeze-drying process, the product temperature, shelf temperature, condenser temperature, and chamber pressure (diaphragm vacuum gauge and Pirani vacuum gauge) are monitored. The product temperature is controlled by eight Pt 100 The samples are monitored by sensors (one sensor per preparation), and these sensors are located in different vials in the center of the samples on the third shelf. At the end of the process, the stoppers of the vials are closed in a freeze-drying oven at a pressure of 600 millibars under a nitrogen atmosphere. After stoppering the vials, the chamber is aerated to atmospheric pressure using nitrogen, and the samples are removed. After removing the samples from the freeze-drying oven, they are crimped, labeled, and stored under their respective storage conditions for further analysis. The freeze-dried preparations are stored at a temperature of 25°C or 30°C in an ICH110 cabinet (Memmert GmbH & Co. KG, Schwabach, Germany).

[0278] Determination of glass transition temperature Glass transition temperature T g and T g The result is determined using a DSC (Differential Scanning Calorimeter) 214 polymer oven (Erich Netzsch GmbH & Co Holding KG, Selb, Germany). 2–10 mg of the freeze-dried product was weighed in an aluminum pot in a humidity-controlled glove box (approximately 8% relative humidity), and the aluminum pot was subsequently sealed. g The values ​​are analyzed using Netzsch Proteus analysis software. All measurements are performed twice, and the results are calculated as the mean ± standard deviation.

[0279] Preparation of frozen liquid samples The sample is frozen by placing it in a freezer at -70°C using a CoolCell LX (BioCision LLC) (Larkspur, California). The temperature is recorded by monitoring it with a temperature logger. For the freeze / thaw cycle, the sample is removed from the freezer and placed in an ICH110 cabinet (Memmert GmbH & Co. KG, Schwabach, Germany) at a temperature of 25°C ± 2°C / 60% ± 5% relative humidity, or 30°C ± 2°C / 65% ± 5% relative humidity, respectively. The temperature cycle is repeated one, three, or five times.

[0280] Determination of particle number by nanoparticle tracking analysis (NTA) The particle count is determined by nanoparticle tracking analysis (NTA) using NanoSight NS300 (Malvern, Worcestershire, UK). The sample is diluted with an appropriate particle-free dilution buffer to contain approximately 30–120 particles per frame. The particles are tracked five times for 60 seconds each in dynamic mode. Two independent dilutions are prepared for each sample. The particle count is analyzed using NanoSight NTA3.4 software (Malvern, Worcestershire, UK) in raw data mode with a detection threshold of approximately 6.

[0281] Example 1: Initial screening formulation Summary: The objective of the initial formulation development activities is to increase infectivity (TCID). 50 The objective was to characterize various virus formulations (Tables 5a-c) by the effects of storage and freeze / thaw (F / T) cycles on the formulations, and by influencing particle formation in formulations designed to be stored as liquid, freeze-liquid formulations, or dried, i.e., freeze-dried formulations. In this example, VSV-GP-Load 1 was tested. Hereinafter, workpackage 3a refers to a liquid formulation, workpackage 3b refers to a freeze-liquid formulation, and workpackage 3d refers to a freeze-dried formulation. The general formulations were the same as the initial liquid, freeze-liquid, or freeze-dried formulations.

[0282] [Table 44]

[0283] [Table 45]

[0284] [Table 46]

[0285] Infectivity titer and the effect of preserving the preparation at 5°C or 25°C on particles invisible to the naked eye in the liquid preparation (work package 3a). Figures 1-4 All liquid formulations (with the exception of workpackage 3a_03, which contains polysorbate at 0.02 g / L) showed reasonable stability during storage at 5°C for 28 days (Figure 1), with an average titer decrease of 0.2 log units during storage. Some formulations also showed a titer decrease of 0.2 to 2.1 log units at 25°C (Figure 2). The most stable formulation was workpackage 3a_General, which contains recombinant human albumin (at 5 g / L). All formulations contained very low levels of microscopic particles (SvPs). After storage at 5°C (Figure 3) or 25°C (Figure 4), the formulation with the lowest amount of microscopic particles larger than 10 μm was workpackage 3a_03, which contains polysorbate.

[0286] Infectivity titer and the effect of freeze / thaw cycles on invisible particles in liquid formulations (work package 3a). Figures 5-6 When the liquid formulation of work package 3a was subjected to stress from one or three freeze / thaw cycles at temperatures between -80°C and +25°C (Table 6), three freeze / thaw cycles resulted in a greater decrease in infectivity titer than one freeze / thaw cycle. The most stable formulation was work package 3a_General, which contained recombinant human albumin. The formulation containing polysorbate (work package 3a_03) showed the greatest decrease in titer (Figure 5), and therefore, despite the positive effect of polysorbate on particle formation, it was no longer considered a suitable excipient for formulation development. The negative effect of polysorbate on the stability of vesicular stomatitis virus likely stems from its effect on its phospholipid envelope, which can be easily damaged by detergents. The freeze / thaw cycle increased the amount of particles invisible to the naked eye in all formulations, except for work package 3a_General, which contains recombinant human albumin for particles larger than 10 μM, and work package 3a_03, which contains polysorbate for particles larger than 10 μM (Figure 6).

[0287] [Table 47]

[0288] Effect of the freeze / thaw cycle on infectivity titer and the formation of microscopic particles in the frozen liquid preparation (work package 3b). Figures 7-10

[0289] When VSV-GP was circulated from -20°C or -80°C to +25°C one, three, or five times (Table 7), the most stable formulation was again Workpackage 3a_General containing recombinant human albumin, followed by the formulation containing poloxamer 188, regardless of whether the formulation was circulated from -20°C to +25°C (Figure 7) or from -80°C to +25°C (Figure 8). Only in these formulations did five freeze / thaw cycles result in no further decrease in infectivity compared to three freeze / thaw cycles. Furthermore, in these formulations, the number of microscopic particles larger than 10 μM did not increase with freeze / thaw cycles, whether the formulation was circulated from -20°C to +25°C (Figure 9) or from -80°C to +25°C (Figure 10). When circulated at -20°C compared to -80°C, the number of particles more than doubled. Given previous observations that cleaning agents such as polysorbate damaged the envelope of VSV-GP, causing an unacceptable decrease in infectivity titer, it was highly surprising that the cleaning agent poloxamer 188 did not have such an effect on the envelope and titer. In contrast, the addition of poloxamer 188 to the formulation was beneficial for both infectivity titer and suppression of the formation of particles invisible to the naked eye.

[0290] [Table 48]

[0291] The effect of storing the preparation at 25°C on infectivity titer and the formation of microscopic particles in the freeze-dried preparation (work package 3d). Figures 11-12 The infectivity of all formulations was stable immediately after freeze-drying, but decreased almost linearly when stored at 25°C (Figure 11). All formulations contained very low levels of particles invisible to the naked eye. The formulations with the fewest amounts of particles larger than 10 μm invisible to the naked eye (Figure 12) were, as expected, work package 3d_General containing rHA and work package 3d_03 containing polysorbate. Stable freeze-dried cakes were obtained from all freeze-dried formulations except work package 3d_General. This was because, in this formulation, sucrose was the sugar component instead of trehalose, which was present in all the other freeze-dried formulations. The glass transition temperature of work package 3d_General (T g Since the value was approximately 25°C, which is close to the storage temperature, sucrose was replaced with trehalose in the following experiment.

[0292] Overall, WP3a_General=WP3b_General=WP3d_General, which contain rHA, performed well under all stress conditions and served as a foundation for further development efforts of freeze-dried formulations. The composition was 20 mM Tris (titrated with NaOH), 150 mM sucrose, 50 mM mannitol, 50 mM sorbitol, 20 mM glutamic acid, and 5 g / L rHA (pH 7.4).

[0293] Example 2: Initial optimization for freeze-dried formulations Figures 15A-B The objective of Work Package 7 was to identify key parameters of the formulation, as well as the freeze-thaw process that stabilizes the oncolytic virus after lyophilization and storage, based on previous learning from Work Package 3. Therefore, the selected formulations were stored at 5°C or 25°C for two weeks to determine their infectivity titers. To optimize the formulations, the amount of Tris buffer was reduced, sucrose was replaced with trehalose, and its concentration was increased to 200 mM. Furthermore, poloxamer 188 was added to the formulation. However, in this particular experiment, poloxamer 188 and dextran were not added to the formulation after dialysis, but were mistakenly added to the dialysis buffer. In this setting, only the monomers of poloxamer 188 with an average molecular weight of 8.6 kDa permeated the dialysis membrane with a MWCO (molecule cutoff) of 10 kDa, while the micelles of poloxamer did not. As a result, the actual poloxamer concentration in the formulation could only be determined in the placebo sample, and even then, it was below the assay's limit of quantification. Numerical poloxamer concentrations were subsequently determined externally at concentrations of 0.03–0.06 g / L, approximately 100 times lower than the target concentration. Nevertheless, the T values ​​of the lyophilized formulation and the reconstituted placebo formulations were determined. g and T g The curve represents the second melting transition (T) in all poloxamer-containing formulations. g The presence of poloxamer 188 in the formulation was confirmed by the ) peak and the second thermal event (although the TG curve is not shown, the temperature start and midpoints of the second melting transition and second glass transition peaks are shown in Figures 15A and 15B). Formulations 1, 2, and 4, which did not contain poloxamer 188, lacked these peaks. Therefore, the concentration of poloxamer 188 was reported as approximately 0.05 g / L in the formulation table. The concentration of dextran could not be determined and was indicated by a question mark. All formulations with inaccurate amounts of excipients were retested with the correct amounts in subsequent work packages.

[0294] [Table 49]

[0295] [Table 50]

[0296] The effect of sugar seeds on the stability of freeze-dried cakes, and the effect of storing the freeze-dried preparation at 5°C or 25°C on the infectivity of the freeze-dried preparation of work package 7. Figures 13, 14, and 16 Freeze-dried cakes containing sucrose, which have a Tg value close to 25°C, melted when stored at 25°C. However, this melting could be avoided by using trehalose, which has a higher Tg value, instead of sucrose in the preparation. Trehalose is particularly beneficial in freeze-dried preparations because it has an unusually high Tg value, approximately 60°C higher than sucrose (Zhang, M., Oldenhof, H., Sydykov, B. et al. Freeze-drying of mammalian cells using trehalose: preservation of DNA integrity. Sci Rep 7, 6198 (2017)). The previous results for work package 3 were confirmed even when the preparation was slightly adapted. The starting Tg value of the sucrose-containing preparation, titrated with HCl / NaOH, was approximately 25°C. When the preparation was titrated with Na3PO4 / H3PO4, the starting Tg value rose to approximately 25°C, a temperature slightly above room temperature. When the preparation was titrated with Na3PO4 / H3PO4 and sucrose was replaced with trehalose, the starting Tg value rose to approximately 90°C (Figure 13). Therefore, the freeze-dried cake remained stable when stored at 25°C (Figure 14).

[0297] Infectivity titers after lyophilization and 14 days at room temperature of 25°C remained within assay variability (approximately 0.5 log units) for the four lead formulations. Despite the presence of a small amount of poloxamer 188 (approximately 0.05 g / L instead of 5 g / L) in the formulations, its presence was not compared to the lyophilized placebo formulation. g Curves and T g 'We were able to detect it on the curve. T g The peak starts at approximately 52°C, which is the melting point of poloxamer 188 (Figure 15A). g The peak initiation point was approximately -17°C (Figure 15B). Neither peak was present in formulations that did not contain poloxamer 188 (1, 2, and 4).

[0298] The infectivity results after storing the lyophilized preparations at 25°C for up to two weeks (Figure 16) showed that preparations lacking poloxamer 188 exhibited the greatest decrease in infectivity. However, even a small amount of poloxamer 188, approximately 0.05 g / l, in the preparation was beneficial in stabilizing the lyophilized preparation during storage, especially when combined with rHA. These effects were further investigated in the next steps of this study (workpackages 9-11). The excipients alanine, arginine, and lysine offered no further advantages in the lyophilized preparations and were therefore not included in further testing. By titrating the pH with trisodium Na3PO4 / H3PO4 instead of NaOH / HCl, T g Since the values ​​increased slightly, the pH of all subsequent preparations was titrated using Na3PO4 / H3PO4. The best-performing composition in this experiment was 10 mM Tris (titrated with Na3PO4), 200 mM trehalose, 50 mM mannitol, 50 mM sorbitol, 20 mM glutamic acid, 10 g / L rHA, and approximately 0.05 g / L poloxamer 188, with a pH of 7.4.

[0299] [Table 51]

[0300] Example 3: Third Optimization Recipe The purpose of this optimization was to confirm previous results using a representative VSV-GP-cargo 1 (work package 9a) batch and a novel viral variant, namely VSV-GP-cargo 2. VSV-GP-cargo 2 (work package 9b) is intended for a different therapeutic indication and is therefore manufactured and applied at a concentration approximately 100 times lower than that of VSV-GP-cargo 1. Both viruses were compounded in four selected formulations. In this optimization, poloxamer 188 was added at the intended concentration of 5 g / L, not the incorrectly lower concentration of 0.05 g / L. VSV-GP-cargo 2 was also stored in two additional formulations containing poloxamer at 2.5 g / L or 0 g / L, and all formulations were stored at temperatures of 5°C or 25°C for up to 12 months. The infectivity titer and the concentration of microscopic particles after reconstitution were determined at regular time intervals. Furthermore, the advantages of a mixture of sugar alcohols and rHA at an optimal concentration were evaluated.

[0301] [Table 52]

[0302] [Table 53]

[0303] The effect of storing a freeze-dried preparation at 5°C or 25°C on its infectious titer, for a typical batch containing VSV-GP-Load 1 (work package 9a) or VSV-GP-Load 2 (work package 9b). Figures 17-20 The rHA concentration of 5 g / L in the VSV-GP-Load 1 formulation (lower concentrations were not tested) was sufficient to maintain infectivity after initial decline after freeze-drying and storage at 5°C for up to 12 months for VSV-GP-Load 1 (Figure 17) and VSV-GP-Load 2 (Figure 19), or at 25°C for up to 12 months for VSV-GP-Load 1 (Figure 18) and VSV-GP-Load 2 (Figure 20). Note that due to the limited number of materials for VSV-GP-Load 1, VSV-GP-Load 1 was tested in four formulations (work packages 09_01 to 09_04), and VSV-GP-Load 2 (work packages 9_01 to 09_06) was tested in six formulations. More rHA (10 g / L instead of 5 g / L) or more glutamic acid (30 mM instead of 20 mM) offered no further benefit. The formulation without mannitol melted when stored at 25°C compared to the VSV-GP-Load 1 formulation, showing the greatest decrease in infectivity (Figure 18), demonstrating the cake-stabilizing effect of mannitol. The beneficial effect of poloxamer 188 in the formulation was also clearly confirmed, as the VSV-GP-Load 2 formulation without poloxamer 188 showed the greatest decrease in infectivity when stored at 5°C (Figure 19) or 25°C (Figure 20), and TCID 50 This was because the levels continued to decrease over time to below the detection limit of the assay. Furthermore, a poloxamer 188 concentration of 2.5 g / L in the VSV-GP-cargo 2 formulation was sufficient to maintain the infectivity titer after lyophilization (lower concentrations were not tested). These experiments revealed that the infectivity titer of VSV-GP variants could be maintained after lyophilization and storage at 25°C for up to 12 months, following an initial decrease of approximately 1 log unit. As expected, no differences in stabilization effects were observed for VSV-GP and VSV-GP expressing other cargoes. Among the best-performing compositions was 10 mM Tris (titrated with Na3PO4), 200 mM trehalose, 50 mM mannitol, 50 mM sorbitol, 20 mM glutamic acid, 10 g / L rHA, and 2.5 g / L poloxamer 188, pH 7.4.

[0304] Example 4: Fourth optimization and verification The effects of poloxamer 188 and the alternative dextran 70 as a second polymer on the stability of VSV-GP were determined, and several formulations were also subjected to long-term stability tests. Furthermore, the stability of VSV-GP formulations containing rHA was investigated in two formulations titrated with NaOH instead of phosphoric acid at a reduced concentration of 5 g / L.

[0305] [Table 54]

[0306] [Table 55]

[0307] The effect of storing the freeze-dried preparation (work package 10) at a temperature of 5°C or 25°C on its infectivity titer. Figures 21A-B A poloxamer 188 concentration of 5 g / L in the VSV-GP formulation was slightly more advantageous than a concentration of 2.5 g / L in maintaining the infectivity titer after lyophilization compared to a concentration of 2.5 g / L, when stored at 25°C for up to 12 months after lyophilization (Figure 21A-B). The infectivity titer after lyophilization could be maintained for up to 12 months at 25°C after an initial decrease of approximately 1 log unit. All formulations without poloxamer 188 failed to maintain the infectivity titer after the initial decrease after lyophilization, and the infectivity titer continued to decrease with storage time. The presence of dextran or titration with NaOH offered no advantage whatsoever in stabilizing VSV-GP. The optimal composition was 10 mM Tris(phosphate), 200 mM Trehalose, 50 mM Mannitol, 50 mM Sorbitol, 20 mM Glutamic Acid, 10 g / L rHA, and 5 g / L Poloxamer 188, at pH 7.4.

[0308] Example 5: Fifth optimization Further formulation development efforts using an experimental design approach led to the identification of optimal combinations and concentrations of excipients for further optimizing the current lead formulation for lyophilized oncolytic virus using VSV-GP-Cargo 1, based on 3 months of data from work package 10.

[0309] [Table 56]

[0310] [Table 57]

[0311] [Table 58]

[0312] [Table 59]

[0313] The effect of storing freeze-dried preparations at 25°C on the infectivity titer of freeze-dried preparations in an experimental design approach (work package 11). Figures 22A-D In this section, we evaluated whether the formulation could be further optimized using a Design of Experiments (DoE) approach. The variable concentrations were those of mannitol, sorbitol, poloxamer 188, and rHA. The concentrations of tris, trehalose, and glutamic acid were fixed. Essentially, the results of the traditional one-factor-at-a-time formulation development approach were confirmed by the Design of Experiments approach (Figures 22A-D). Using various optimization methods with response surface models, Design of Experiments predicted the following optimal formulation within the workpackage 11 design space: 10 mM tris (phosphate), 200 mM trehalose, 20–46 mM mannitol, 20–50 mM sorbitol, 20 mM glutamic acid, 3–5 g / L rHA, and 1.4–1.5 g / L poloxamer 188, pH 7.4.

[0314] [Table 60]

[0315] The experimental design method, based on various optimization techniques, predicted the following optimal formulations outside the design space of work package 11: 10 mM Tris(phosphate), 200 mM Trehalose, 20–69 mM Mannitol, 19–76 mM Sorbitol, 20 mM Glutamic Acid, 3.4–5.6 g / L rHA, and 1.4–1.5 g / L Poloxamer 188, pH 7.4.

[0316] [Table 61]

[0317] [Table 62]

[0318] Example 6: Enhanced colloidal stability Based on the results of the experimental design, additional tests were conducted by adding various excipients that could act as colloid stabilizers to the further optimized formulation, which allowed us to increase the overall number and stability of particles during liquid processing.

[0319] [Table 63]

[0320] An additional excipient that enhances the recovery of viruses during handling in liquids and improves processing in liquids, without negatively affecting the stability of long-term storage in dry conditions. Figures 23A-B In the presence of additional excipients known to act as colloidal stabilizers (e.g., arginine or citrate), a greater overall particle count was achieved during handling and processing in liquid, starting from the same VSV-GP starting material. Stability was confirmed after dry storage at temperatures of 2–8°C for up to 6 months.

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