Process for producing purified rhabdovirus from cell culture

Abstract

To provide a method for producing a purified oncolytic rhabdovirus and in particular a vesicular stomatitis virus.SOLUTION: There is provided a method of producing a rhabdovirus in a cell culture comprising the step of: (i) obtaining a rhabdovirus harvest from the cell culture by: a. adding directly to the cell culture a viral release agent, followed by clarifying the cell culture, preferably via depth filtration, tangential flow filtration or centrifugation, and recovery of the rhabdovirus harvest in the supernatant, or b. subjecting the cell culture to a filtration step, preferably depth filtration followed by rinsing of the filter, preferably the depth filter with a viral release agent, and recovery of the rhabdovirus harvest in the supernatant.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the field of upstream and downstream processing, and provides methods and processes for preparing, producing and / or purifying rhabdovirus, preferably an oncolytic rhabdovirus, in particular a vesicular stomatitis virus, from cell culture, including pharmaceutical compositions comprising rhabdovirus.

[0002] 2. Background of the Invention Manufacturing process design for oncolytic viruses must consider several important quality attributes of the final drug substance and drug product. Among these are high infectious virus content per volume and low tolerance levels of product and process related impurities. The virus content per volume in the drug product should be 10 9 ~10 12 The concentration of the active virus can be in the range of infectious units or genome units, and during downstream purification, at least one order of magnitude is required for the concentration of the active virus. The acceptable impurity threshold of a typical biological product may still apply, for example, 10 ng / dose of host cell DNA (WHO limit), which, when combined with the required high product content, poses another significant challenge. Both limitations are typically several orders of magnitude more severe than those found in other active virus products for pharmaceutical use, such as viral vaccines. Therefore, commercial-scale manufacturing techniques need to be optimized and newly developed for this intended purpose. In particular, there is a need for improved methods for preparing, producing or purifying rhabdovirus from cell culture.

[0003] For example, WO 2007 / 123961 discloses a purification process for isolating purified vesicular stomatitis virus from a cell culture in which the cell culture is loaded onto an anion exchange membrane adsorber after clarification and filtration, and the virus is then eluted, followed by further purification and filtration steps.

[0004] Summary of the Invention The present invention addresses the above needs by providing methods and processes for obtaining purified rhabdoviruses, such as vesicular stomatitis viruses, from cell culture.

[0005] It should also be understood that any embodiment relating to a particular aspect can be combined with another embodiment relating to that particular aspect, and that in multiple layers, a combination can include several embodiments for that particular aspect.

[0006] In a first aspect, the present invention provides a method for producing a rhabdovirus in cell culture, comprising: (i) a. by adding a virus releasing agent directly to the cell culture followed by clarification of the cell culture, preferably by depth filtration, tangential flow filtration or centrifugation, and recovering the rhabdovirus harvest in the supernatant; or b. The method comprises obtaining a rhabdovirus harvest from a cell culture by subjecting the cell culture to a filtration step, preferably depth filtration, followed by rinsing the filter, preferably the depth filter, with a virus releasing agent and recovering the rhabdovirus harvest in the supernatant.

[0007] In one embodiment of the first aspect, the rhabdovirus is a vesiculovirus, preferably a vesicular stomatitis virus. In a further related embodiment, the glycoprotein G of the vesicular stomatitis virus is replaced by the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV).

[0008] In one embodiment of the first aspect or any of its embodiments, the virus releasing agent in step (ia) is a solid salt or an aqueous salt solution and the virus releasing agent in step (ib) is an aqueous salt solution. In a further related embodiment, in step (ia), the salt concentration in the cell culture is increased to at least about 0.01M, 0.05M, 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M or 0.5M and the aqueous salt solution in step (ib) has a concentration of at least about 0.01M, 0.05M, 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M or 0.5M. In further related embodiments, the increase in salt concentration in the cell culture in step (ia) and the concentration of the aqueous salt solution in step (ib) are from about 0.01 M to about 5 M, from about 0.05 M to about 5 M, from about 0.1 M to about 5 M, from about 0.15 M to about 5 M, from about 0.2 M to about 5 M, from about 0.25 M to about 5 M, from about 0.3 M to about 5 M, from about 0.35 M to about 5 M, from about 0.4 M to about 5 M, from about 0.45 M to about 5 M, or from about 0.5 M to about 5 M.

[0009] In one embodiment of the first aspect or any of its embodiments, the salt is NaCl, KCl, MgCl 2 , CaCl 2 , N.H. 4 Cl, sulfate NH 4 , NH acetate 4 or NH bicarbonate 4 It is.

[0010] In one embodiment of the first aspect or any of the embodiments thereof, the virus release agent is an amino acid, preferably a polar, acidic or basic amino acid, more preferably arginine.

[0011] In one embodiment of the first aspect or any of the embodiments thereof, the virus release agent is a sulfated polysaccharide, preferably dextran sulfate.

[0012] In one embodiment of the first aspect or any of its embodiments, in step (ia), the virus releasing agent is added to the cell culture to increase the ionic strength of the cell culture to preferably at least about 0.01M, or at least about 0.05M, or at least about 0.1M, or at least about 0.15M, or at least about 0.2M, or at least about 0.25M, or at least about 0.3M, or at least about 0.35M, or at least about 0.4M, or at least about 0.45M, or at least about 0.5M, or from about 0.01M to about 5M, or from about 0.05M to about 5M, or from about 0.1M to about 5M, or from about 0.15M to about 5M or about 0.2M to about 5M, and in step (ib), the filter is rinsed with an aqueous solution containing a virus releasing agent having an ionic strength of at least about 0.01M, or at least about 0.05M, or at least about 0.1M, or at least about 0.15M, or at least about 0.2M, or at least about 0.25M, or at least about 0.3M, or at least about 0.35M, or at least about 0.4M, or at least about 0.45M, or at least about 0.5M, or about 0.01M to about 5M, or about 0.05M to about 5M, or about 0.1M to about 5M, or about 0.15M to about 5M, or about 0.2M to about 5M.

[0013] In one embodiment of the first aspect or any of its embodiments, the rhabdovirus is produced in a mammalian host cell, preferably a HEK293 cell. In a further related embodiment, the mammalian host cell is cultured in suspension.

[0014] In one embodiment of the first aspect or any of its embodiments, a method for producing a rhabdovirus in cell culture comprises: (ii) (optionally) reducing the salt concentration of the harvest obtained after step (ia) or (ib), preferably by dilution, diafiltration or dialysis; (iii) (optionally) treating the rhabdovirus collection with a DNA degrading nuclease, preferably benzonase or salt active nuclease; (iv) capturing the rhabdovirus by loading the solution obtained after any of steps (i) to (iii) onto a cation exchanger; (v) eluting the rhabdovirus and recovering the eluate; (vi) (optionally) refining the rhabdovirus eluate of step (v), preferably by size exclusion, multimodal size exclusion, ion exchange and / or tangential flow filtration; (vii) (optionally) exchanging the buffer of the refined rhabdovirus eluate, preferably by ultrafiltration and diafiltration or dialysis; (viii) (optionally) sterile filtering the rhabdovirus.

[0015] In a related embodiment, the cation exchanger is a monolith, a resin or a membrane. In a further related embodiment, the cation exchanger is a monolith adsorbent.

[0016] In one embodiment of the first aspect or any of its embodiments, the rhabdovirus is formulated in a pharmaceutical composition.

[0017] In a second aspect, the present invention provides a method for purifying a rhabdovirus from a cell culture infected with the rhabdovirus, comprising: (i) a. by adding a virus releasing agent directly to the cell culture followed by clarification of the cell culture, preferably by depth filtration, tangential flow filtration or centrifugation, and recovering the rhabdovirus harvest in the supernatant; or b. The method comprises obtaining a rhabdovirus harvest from a cell culture by subjecting the cell culture to a filtration step, preferably depth filtration, followed by rinsing the filter, preferably the depth filter, with a virus releasing agent and recovering the rhabdovirus harvest in the supernatant.

[0018] In one embodiment of the second aspect, the rhabdovirus is a vesiculovirus, preferably a vesicular stomatitis virus. In a further related embodiment, the glycoprotein G of the vesicular stomatitis virus is replaced by the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV).

[0019] In one embodiment of the second aspect or any of its embodiments, the virus releasing agent in step (ia) is a solid salt or an aqueous salt solution and the virus releasing agent in step (ib) is an aqueous salt solution. In a further related embodiment, in step (ia), the salt concentration in the cell culture is increased to at least about 0.01M, 0.05M, 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M or 0.5M and the aqueous salt solution in step (ib) has a concentration of at least about 0.01M, 0.05M, 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M or 0.5M. In further related embodiments, the increase in salt concentration in the cell culture in step (ia) and the concentration of the aqueous salt solution in step (ib) are from about 0.01 M to about 5 M, from about 0.05 M to about 5 M, from about 0.1 M to about 5 M, from about 0.15 M to about 5 M, from about 0.2 M to about 5 M, from about 0.25 M to about 5 M, from about 0.3 M to about 5 M, from about 0.35 M to about 5 M, from about 0.4 M to about 5 M, from about 0.45 M to about 5 M, or from about 0.5 M to about 5 M.

[0020] In one embodiment of the second aspect or any of its embodiments, the salt is NaCl, KCl, MgCl 2 , CaCl 2 , N.H. 4 Cl, sulfate NH 4 , NH acetate 4 or NH bicarbonate 4 It is.

[0021] In one embodiment of the second aspect or any of the embodiments thereof, the virus release agent is an amino acid, preferably a polar, acidic or basic amino acid, more preferably arginine.

[0022] In one embodiment of the second aspect or any of the embodiments thereof, the virus release agent is a sulfated polysaccharide, preferably dextran sulfate.

[0023] In one embodiment of the second aspect or any of its embodiments, in step (ia), the virus releasing agent is added to the cell culture to increase the ionic strength of the cell culture to preferably at least about 0.01M, or at least about 0.05M, or at least about 0.1M, or at least about 0.15M, or at least about 0.2M, or at least about 0.25M, or at least about 0.3M, or at least about 0.35M, or at least about 0.4M, or at least about 0.45M, or at least about 0.5M, or from about 0.01M to about 5M, or from about 0.05M to about 5M, or from about 0.1M to about 5M, or from about 0.15M to about 5M or about 0.2M to about 5M, and in step (ib), the filter is rinsed with an aqueous solution containing a virus releasing agent having an ionic strength of at least about 0.01M, or at least about 0.05M, or at least about 0.1M, or at least about 0.15M, or at least about 0.2M, or at least about 0.25M, or at least about 0.3M, or at least about 0.35M, or at least about 0.4M, or at least about 0.45M, or at least about 0.5M, or about 0.01M to about 5M, or about 0.05M to about 5M, or about 0.1M to about 5M, or about 0.15M to about 5M, or about 0.2M to about 5M.

[0024] In one embodiment of the second aspect or any of its embodiments, the rhabdovirus is produced in a mammalian host cell, preferably a HEK293 cell. In a further related embodiment, the mammalian host cell is cultured in suspension.

[0025] In one embodiment of the second aspect or any of its embodiments, a method for purifying a rhabdovirus from a cell culture infected with the rhabdovirus comprises: (ii) (optionally) reducing the salt concentration of the harvest obtained after step (ia) or (ib), preferably by dilution, diafiltration or dialysis; (iii) (optionally) treating the rhabdovirus collection with a DNA degrading nuclease, preferably benzonase or salt active nuclease; (iv) capturing the rhabdovirus by loading the solution obtained after any of steps (i) to (iii) onto a cation exchanger; (v) eluting the rhabdovirus and recovering the eluate; (vi) (optionally) refining the rhabdovirus eluate of step (vii), preferably by size exclusion, multimodal size exclusion, ion exchange and / or tangential flow filtration; (vii) (optionally) exchanging the buffer of the refined rhabdovirus eluate, preferably by ultrafiltration and diafiltration or dialysis; (viii) (optionally) sterile filtering the rhabdovirus.

[0026] In a related embodiment, the cation exchanger is a monolith, a resin or a membrane. In a further related embodiment, the cation exchanger is a monolith adsorbent.

[0027] In one embodiment of the second aspect or any of its embodiments, the rhabdovirus is formulated in a pharmaceutical composition.

[0028] In a third aspect, the invention relates to a vesicular stomatitis virus, wherein the glycoprotein G of the vesicular stomatitis virus is replaced by the glycoprotein GP of Lymphocytic Choriomeningitis Virus (LCMV), produced according to the method of any of the preceding aspects or embodiments or purified according to the method of any of the preceding aspects or embodiments. In an embodiment of the third aspect, the RNA genome of the vesicular stomatitis virus produced according to the method of any of the preceding aspects or embodiments or purified according to the method of any of the preceding aspects or embodiments consists of a coding sequence at least 98%, at least 99% or 100% identical to SEQ ID NO: 12. In a further related embodiment of the third aspect or any of its embodiments, the amount of infectious particles is determined by measuring the TCID 50 At least about 1 × 10 / mL 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 or at least about 1 × 10 10 It is. [Brief description of the drawings]

[0029] [Figure 1] FIG. 1: Exemplary flow chart showing the upstream and downstream process steps performed for the purification / production of vesicular stomatitis virus pseudotyped with the GP of LCMV (VSV-GP). [Diagram 2]Figure 2: Bar chart of host cell DNA purity sorted over time for process intermediates per dose of 1x1011 TCID50 VSV-GP (-cargo) in large scale manufacturing. The upstream clarified sample (USP) was obtained by taking a sample of the crude harvest of the cell culture, adding NaCl to this crude harvest sample, and then clarifying the crude harvest sample by centrifugation to yield the USP. The host cell DNA purity was then measured by USP and the host cell DNA purity was then calculated for the entire cell culture harvest. The sterile filtered material obtained at the end of the process represents the host cell DNA purity at the drug substance level. [Diagram 3] Figure 3: Bar chart of host cell protein purity sorted over time for process intermediates per dose of 1x1011 TCID50 VSV-GP (-cargo) in large scale manufacturing. The upstream clarified sample (USP) was obtained by taking a sample of the crude harvest of the cell culture, adding NaCl to this crude harvest sample, and then clarifying the crude harvest sample by centrifugation to yield the USP. The host cell protein purity was then measured by USP and the host cell protein purity was then calculated for the entire cell culture harvest. The sterile filtered material obtained at the end of the process represents the host cell protein purity at drug substance level. [Figure 4] Figure 4: Bar chart showing TCID50 / mL levels of VSV-GP at the indicated time points post-infection (pi) of HEK293F cells. TCID50 / mL levels were determined either in untreated crude harvest (black bars) or in the supernatant of centrifuged samples of treated crude harvest (grey bars). For this purpose, samples of crude harvest were treated with a range of different additives and then clarified by centrifugation. TCID50 / mL levels were then determined in the supernatant of treated crude harvest samples after centrifugation. TCID50 levels of crude harvest were measured for the control 30 and 48 h pi and only for dextran sulfate treatment. [Diagram 5]Figure 5: Bar chart showing genome titer / mL levels (black bars) and TCID50 / mL (grey bars) of VSV-GP after infection of HEK293F cells. Genome titer / mL or TCID50 / mL levels were determined in the supernatants of untreated crude harvest (no additives, no centrifugation) and centrifuged samples of treated crude harvest samples. For this purpose, samples of crude harvest were treated with a range of additives and then clarified by centrifugation. Genome titer / mL or TCID50 / mL levels were then determined in the supernatants of treated crude harvest samples after centrifugation. [Figure 6] Figure 6: Bar chart showing the VSV-GP genome titer / mL level (black bars) and the recovery of VSV-GP as measured by TCID50 / mL (gray bars) after infection of HEK293F cells. The genome titer / mL or TCID50 / mL levels were determined in untreated crude harvest (without additives) and set to 100%. The % recovery of VSV-GP after treatment with different concentrations of salt was measured in the supernatant of centrifuged samples of treated crude harvest samples. For this purpose, samples of crude harvest were treated with different ranges of salt and then clarified by centrifugation. The genome titer / mL or TCID50 / mL levels were then determined in the supernatant of treated crude harvest samples after centrifugation. [Figure 7] Figures 7A-7B: Representative performance data for (A) VSV-GP scalable process control data at 50 L scale and (B) VSV-GP scalable process control data at 4 L. TCID50 / mL and genomic titer / mL levels were measured at various unit operations starting from saline harvest and ending with drug substance (DS). [Figure 8]Figure 8: Bar chart showing genome titer / mL levels (black bars) and TCID50 / mL (gray bars) of VSV-GP after infection of HEK293F cells. Genome titer / mL or TCID50 / mL levels were determined in the supernatant of centrifuged samples of processed crude harvest samples. For this purpose, samples of crude harvest were treated with different ranges of MgCl2 or NaCl2 and then clarified by centrifugation. Genome titer / mL or TCID50 / mL levels were then determined in the supernatant of processed crude harvest samples after centrifugation. [Figure 9] Figure 9: Bar chart showing total TCID50 of VSV-GP in harvests of HEK293F infected cells. Total TCID50 levels were determined in the same run for (i) crude harvest, (ii) nuclease-treated crude harvest, (iii) nuclease-treated crude harvest after depth filtration (clarified harvest), (iv) first filter flush with Tris buffer containing 0.25 M sodium chloride (eluate), and (v) second filter flush with Tris buffer containing 0.5 M sodium chloride (eluate).

[0030] Detailed Description of the Invention In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the subject technology can be practiced without some of these specific details. In other instances, well-known structures and techniques are not shown in detail so as not to obscure the present invention. Headings are included merely for convenience to aid in reading comprehension and should not be understood to limit the present invention to any particular aspect or embodiment.

[0031] The inventors have surprisingly found that by following the method or process of the present invention, the recovery and / or purification of rhabdovirus from cell culture is significantly improved compared to the method and process known to those skilled in the art.The inventors have found that by directly adding a virus release agent, in particular salt or dextran sulfate, to the cell culture infected with rhabdovirus before the harvesting procedure is started, the amount of rhabdovirus that can be recovered from cell culture in the subsequent step is significantly improved.Furthermore, the recovery and / or purification of rhabdovirus is further improved by capturing rhabdovirus on a cation exchanger.The recovered rhabdovirus can be, for example, 100% or more of the total TCID 50 Hit or TCID 50 Quantification can be based on genome copy number / mL in crude supernatants, by determining infectious virus concentration in / mL, by using the Spearman-Karber method or by qPCR.

[0032] In the broadest sense, the present invention provides a method for producing rhabdovirus, in particular vesicular stomatitis virus.In one aspect, the implementation of the method or process of the present invention allows the preparation, production and / or purification of rhabdovirus, preferably under cGMP conditions, and results in a high total infectious virus or infectious virus content per volume.In a related aspect, the implementation of the method or process of the present invention allows the preparation, production and / or purification of rhabdovirus sufficient for commercial purposes, i.e., to meet the demands of commercial scale.In relation to this aspect, the method / process of the present invention is preferably carried out in cell culture with cell culture volume of at least 2L, 5L, 10L, 20L, 30L, 40L, 50L, 60L, 70L, 80L, 90L, 100L, 110L, 120L, 130L, 140L, 150L, ​​160L, 170L, 180L, 190L or at least 200L. Further, in relation to this embodiment, the total amount of rhabdovirus recovered is 50 At least about 10 8 ~10 14 In particular, the infectious particles may be in the range of at least about 108 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 TCID 50 Preferably, the amount of infectious particles is measured in TCID 50 At least about 10 13 Further, in accordance with this embodiment, the amount of rhabdovirus recovered is 50 At least about 10 / mL 8 ~10 11 In particular, the infectious particles may be in the range of at least about 10 8 , 10 9 , 10 10 or 10 11 TCID 50 Infectious particles measured in TCID 50 At least about 1 × 10 / mL 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 or at least about 9×10 9 More preferably, the amount of infectious particles is TCID 50 At least about 10 / mL 10 It is.

[0033] In another embodiment, practice of the methods or processes of the invention will result in host cell DNA levels of less than 10 ng per dose, less than 9 ng per dose, less than 8 ng per dose, less than 7 ng per dose, less than 6 ng per dose, less than 5 ng per dose, less than 4 ng per dose, less than 3 ng per dose, less than 2 ng per dose, or less than 1 ng per dose, where the dose is less than 1×10 11 Total TCID 50Preferably, the host cell DNA level per dose is less than 1 ng per dose, where the dose is less than 1×10 11 Total TCID 50 It's a virus.

[0034] In yet another aspect, practice of the methods or processes of the invention will result in host cell protein levels of less than 10 μg per dose, less than 9 μg per dose, less than 8 μg per dose, less than 7 μg per dose, less than 6 μg per dose, less than 5 μg per dose, less than 4 μg per dose, less than 3 μg per dose, less than 2 μg per dose, or less than 1 μg of host cell protein per dose, where the dose is less than 1×10 11 Total TCID 50 Preferably, the host cell protein level per dose is less than 1 μg per dose, where the dose is less than 1×10 11 Total TCID 50 It's a virus.

[0035] In another aspect, the total TCID 50 At least 1 x 10 measured per 12 TCID 50 , 2×10 12 TCID 50 , 3×10 12 TCID 50 , 4×10 12 TCID 50 , 5×10 12 TCID 50 , 6×10 12 TCID 50 , 7×10 12 TCID 50 , 8×10 12 TCID 50 , 9×10 12 TCID 50 , 1×10 13 TCID 50 , 1.5×10 13 TCID 50 , 2×10 13 TCID 50 , 2.5×1013 TCID 50 , 3×10 13 TCID 50 Or 3.5 x 10 13 TCID 50 Preferably, implementation of the method or process of the present invention at a 200 L upstream scale will result in a drug substance yield of infectious titer of TCID 50 At least 1 x 10 measured per 13 TCID 50 This will result in a drug substance yield of infectious titer of

[0036] In the first step, the host cells are infected with a rhabdovirus, preferably a vesicular stomatitis virus. Infection of the host cells is carried out by techniques routinely available to those skilled in the art and usually involves inoculating a cell culture with a rhabdovirus seed at a specific multiplicity of infection. Preferably, the infection is carried out at a concentration of 1.0-2.0×10 6 The cell culture is then cultured for a period of time that allows sufficient rhabdovirus replication, i.e. under conditions that allow rhabdovirus replication.The exact conditions that allow rhabdovirus replication are selected by those skilled in the art according to specific host cell lineage.Preferably, cells are infected with master seed virus at a low multiplicity of infection, for example 0.0005 (or 5 infectious particles per 10,000 cells).Therefore, those skilled in the art will understand that in the first step, the method of the present invention will include the infection of host cells suitable for rhabdovirus and the culture of the infected host cells under conditions that allow rhabdovirus replication.

[0037] The host cell can be of any origin and can be present as an isolated cell or as a cell contained in a cell population.The host cell producing rhabdovirus is preferably a mammalian cell.Alternatively, the host cell can be a human cell, a monkey cell, a mouse cell or a hamster cell.Those skilled in the art will know the suitable method to use when testing whether a given cell produces virus and therefore whether a particular host cell can be used within the scope of the present invention.In this respect, the amount of virus produced by the host cell is not particularly limited.Preferred virus titer is 1x10 in the crude supernatant of a given cultured cell product after infection without further downstream processing. 7 More than TCID 50 / mL or 1 × 10 8 or more genome copies / mL.

[0038] In one embodiment, the mammalian cells are multipotent adult progenitor cells (MAPCs), neural stem cells (NSCs), mesenchymal stem cells (MSCs), HeLa cells, HEK cells, any HEK293 cells (e.g., HEK293F or HEK293T), Chinese hamster ovary cells (CHO), baby hamster kidney (BHK) cells or Vero cells or bone marrow derived tumor infiltrating cells (BM-TICs). It is preferred to culture the host cells in suspension, for example by adapting the host cells to grow in suspension if they do not already naturally grow in suspension. In a preferred embodiment, the host cells are either HEK293F or HEK293T cells from the human embryonic kidney (HEK) 293 cell line. Preferably, the HEK293F or HEK293T cells are cultured in suspension batch mode in a stirred tank or wave bioreactor system under animal component-free and chemically defined conditions.

[0039] Host cell in the sense of the present invention includes classical packaging cell for producing rhabdovirus from non-replicating vector and producer cell for producing rhabdovirus from replicable vector.Packaging cell usually contains one or more plasmids for expressing the essential genes that are missing in each vector to be packaged and / or are necessary for virus production.Such cells are known to those skilled in the art, who can select suitable cell lineages suitable for desired purpose.

[0040] In one embodiment, the host cell line is HEK293 cell. As used herein, the term "HEK293 cell or HEK293 cell line" refers to an adherent human cell line derived from human embryonic kidney and first immortalized in 1973 by integration of a 4 kbp adenovirus 5 (ad5) genome fragment containing E1A and E1B genes in chromosome 19 (Graham et al., J. Gen. Virol. (1977) 36: 59-72; Malm et al., Nature research, Scientific Reports (220) 10:18996). This cell line can be obtained, for example, from ATCC and DSMZ (ATCC-CRL-1573; DSMZ No:ACC305; RRID:CVCL_0045). In particular, to enable large-scale cultivation and bioproduction of therapeutic proteins or viruses in bioreactors, the parent HEK293 cell line has also been adapted to high-density suspension growth in serum-free medium. These include, but are not limited to, the industrially relevant suspension cell lines HEK293-F, HEK293-H and FreeStyle HEK293-F cells. FreeStyle HEK293-F cells have been adapted to suspension culture in FreeStyle™ 293 expression medium and are available, for example, from ThermoFisher (R79007; RRID:CVCL_D603). HEK293-F and HEK293-H cells are prepared from clonal selection from HEK293 cells due to their rapid growth in serum-free medium (SFM), excellent transfection efficiency and high levels of protein expression, and are available, for example, from ThermoFisher (HEK293-F: 11625019, RRID: CVCL_6642; HEK293-H: 11631017, RRID: CVCL_6643). The HEK293-H line is a mutant that exhibits better adhesion in monolayer cultures when grown in serum-supplemented medium, indicating ease of use in plaque assays and other anchorage-dependent applications. HEK293-F and HEK-293-H are provided to accommodate Gibco® CD 293 medium.Other HEK293 cell lines include, but are not limited to, HEK293.2sus (ATCC CRL-1573.3), HEK293-SF-3F6 (ATCC CRL-12585; RRID: CVCL_4V94), Expi293F (Thermofischer A14527 / A14528 / 100044202 (cGMP banked); RRID: CVCL_D615) and HEK293-S (Ximbio 154155; RRID: CVCL_A784). HEK293 cell lines adapted to suspension growth can also be referred to as "293 cells, SFM adapted."

[0041] For HEK293 cell culture, cells are passaged in a chemically defined medium in a continuous batch mode inoculation step until enough cells are obtained to inoculate, for example, a stirred tank reactor. Several batch passages are performed in a stirred tank before inoculating with the seed virus, with dissolved oxygen, pH and temperature controlled from stirred tank inoculation to virus harvest. The temperature during accumulation of the cell mass may be different from the temperature used after seed virus inoculation, and is usually around 37°C. In a preferred embodiment, the temperature is changed after infection from 37°C to a range of 32°C to 36°C, more preferably to 34°C.

[0042] Generally, the method or process of the present invention is useful for preparing, producing or purifying any rhabdovirus.In a preferred embodiment, the method or process of the present invention is used for preparing, producing or purifying vesiculovirus.The preparation, production or purification of vesicular stomatitis virus is particularly preferred.

[0043] The Rhabdoviridae family includes 18 genera and 134 species with negative-sense single-stranded RNA genomes of approximately 10-16 kb (Walke et al., ICTV Virus Taxonomy Profile: Rhabdoviridae, Journal of General Virology, 99:447-448 (2018)).

[0044] Characteristic features of members of the Rhabdoviridae family include one or more of the following: Bullet- or rod-shaped particles of 100-430 nm in length and 45-100 nm in diameter, composed of a helical nucleocapsid surrounded by a matrix layer and a lipid envelope, although some rhabdoviruses have non-enveloped filamentous viruses; Negative-sense single-stranded RNA of 10.8-16.1 kb, mostly unsegmented; A genome encoding at least five genes encoding the structural proteins nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M) and glycoprotein (G).

[0045] As used herein, a rhabdovirus may belong to the genera almendravirus, curiovirus, cytorhadovirus, dichorhavirus, ephemerovirus, hapavirus, ledantevirus, lyssavirus, nobiladovirus, nucleorhabdovirus, perhabdovirus, sigmavirus, sprivivirus, sripuvirus, tibrovirus, tupavirus, varicosavirus, or vesiculovirus.

[0046] In the genus mentioned herein, rhabdovirus may belong to any of the listed species.The genus Almendravirus includes Arboretum Almendravirus, Balsa Almendravirus, Coot Bay Almendravirus, Puerto Almendras Almendravirus, and Riotico Almendravirus.The genus Curiovirus includes Quirinopolis Curiovirus, Iliricuriovirus, Itacaiunas Curiovirus, and Roshan Beaucuriovirus. The genus of Cytorhabdovirus includes Alfalfa dwarf cytorhabdovirus, Barley yellow dwarf mosaic cytorhabdovirus, Broccoli necrotic yellow cytorhabdovirus, Colocasia bobone disease associated cytorhabdovirus, Festuca leaf streak cytorhabdovirus, Lettuce necrotic yellow cytorhabdovirus, Lettuce yellow spot cytorhabdovirus, Northern wheat mosaic cytorhabdovirus, Sowberry cytorhabdovirus 1, Strawberry crinkle cytorhabdovirus, and Wheat stripe mosaic cytorhabdovirus. The genus of Dichoruhavirus includes Coffee ring spot dicholehavirus, and Orchid necrotic spot dicholehavirus. The genus Ephemerovirus includes Adlade River ephemerovirus, Berryma ephemerovirus, Bovine ephemeral fever ephemerovirus, Kimberly ephemerovirus, Coolpiña ephemerovirus, Cottoncan ephemerovirus, Ovogian ephemerovirus, and Yata ephemerovirus. The genus Hapavirus includes Flanders hapavirus, Grey Lodge hapavirus, Hart Park hapavirus, Joinjakaka hapavirus, Kamese hapavirus, La Joya hapavirus, Landjia hapavirus, Manitoba hapavirus, Marco hapavirus, Mosqueiro hapavirus, Mossir hapavirus, Ngaingan hapavirus, Olde River hapavirus, Parry Creek hapavirus, and Wongabel hapavirus.The genus Redantevirus includes Bauer Dantevirus, Fikirini Redantevirus, Fukuoka Redantevirus, Kanyawara Redantevirus, Kern Canyon Redantevirus, Keuraliba Redantevirus, Corenter Redantevirus, Kumasi Redantevirus, Redante Redantevirus, Mount Elgombat Redantevirus, Nishimuro Redantevirus, Nkolbisson Redantevirus, Oita Redantevirus, Uhan Redantevirus, and Yongjia Redantevirus. The genus Lyssavirus includes Araban rissavirus, Australian bat rissavirus, Bokeloh bat rissavirus, Duvenhage rissavirus, European bat 1 rissavirus, European bat 2 rissavirus, Gunnorwa bat rissavirus, Ikomarissavirus, Irkut rissavirus, Khodzhendrissavirus, Lagos bat rissavirus, Riida bat rissavirus, Mokola rissavirus, Rabies bat rissavirus, Simoni bat rissavirus, and West Caucasian bat rissavirus. The genus Novirhabdovirus includes Flounder novirhabdovirus, Fish novirhabdovirus, Salmon novirhabdovirus, and Snake novirhabdovirus. The genus Nucleorhabdovirus includes Datura vein yellowing nucleorhabdovirus, eggplant mottled dwarf nucleorhabdovirus, corn streak nucleorhabdovirus, Iranian corn mosaic nucleorhabdovirus, corn mosaic nucleorhabdovirus, potato yellow dwarf nucleorhabdovirus, rice yellow dwarf nucleorhabdovirus, sowbuckthorn reticular yellowing nucleorhabdovirus, poppy vein yellowing nucleorhabdovirus, and taro vein whitening nucleorhabdovirus. The genus Perahabdovirus includes eel Perahabdovirus, perch Perahabdovirus, and masperahabdovirus.The sigmavirus genus includes Drosophila affinis sigmavirus, Drosophila ananassae sigmavirus, Drosophila immigrans sigmavirus, Drosophila melanogaster sigmavirus, Drosophila obscura sigmavirus, Drosophila tristis sigmavirus, and Muscina stabulans sigmavirus. The sprivivirus genus includes Koi isprivivirus and Pike juvenile sprivivirus. The sripuvirus genus includes Arunpiba sripuvirus, Chako sripuvirus, Niakha sripuvirus, Sena Madreira sripuvirus, and Sripur sripuvirus. The tiburovirus genus includes Central Congo tiburovirus, Beatrice Hill tiburovirus, Coastal Plain tiburovirus, Ekpoma1 tiburovirus, Ekpoma2 tiburovirus, Sweetwater Branch tiburovirus, and Tiburo Gangan tiburovirus. The genus Tupavirus includes Daram Tupavirus, Klamath Tupavirus, and Tupai Tupavirus. The genus Varicosavirus includes Lettuce Big-Vein-Associated Varicosavirus. The genus Vesiculovirus includes Alagoas vesiculovirus, American bat vesiculovirus, Carajas vesiculovirus, Candipura vesiculovirus, Coccus vesiculovirus, Indiana vesiculovirus, Isfahan vesiculovirus, Jurona vesiculovirus, Malpais Springs vesiculovirus, Maraba vesiculovirus, Morton vesiculovirus, New Jersey vesiculovirus, Perinet vesiculovirus, Piri vesiculovirus, Lardy vesiculovirus, Yug Bogdanovac vesiculovirus, or Musavirus.

[0047] Preferably, the rhabdovirus prepared, produced or purified according to the method or process of the present invention is an oncolytic rhabdovirus.In this respect, oncolytic has its usual meaning known in the art, and refers to the ability of rhabdovirus to infect and lyse (decompose) cancer cells, but not infect (spread significantly) normal cells.Preferably, oncolytic rhabdovirus can replicate in cancer cells.Oncolytic activity can be tested in various assay systems known to those skilled in the art (exemplary in vitro assays are described in Muik et al., Cancer Res., 74(13), 3567-78, 2014).It should be understood that oncolytic rhabdovirus can only infect and lyse certain types of cancer cells.Also, oncolytic activity may vary depending on the type of cancer cells.

[0048] In a preferred embodiment, Rhabdovirus belongs to the genus Vesiculovirus.Vesiculovirus species are mainly defined by serological means combined with phylogenetic analysis of genome.Biological characteristics, such as host range and transmission mechanism, are also used to distinguish virus species within this genus.Thus, the genus Vesiculovirus forms a separate monophyletic group, which is well supported by the maximum likelihood tree inferred from complete L sequence.

[0049] Viruses assigned to different species within the genus Vesiculovirus may have one or more of the following characteristics: A) a minimum amino acid sequence diversity in L of 20%; B) a minimum amino acid sequence diversity in N of 10%; C) a minimum amino acid sequence diversity in G of 15%; D) being distinguishable in serological tests; and E) occupying different ecological niches as evidenced by differences in hosts and / or arthropod vectors.

[0050] Vesicular stomatitis virus (VSV) is preferred. In a most preferred embodiment, the method or process of the invention is used to prepare, produce or purify a vesicular stomatitis virus, in which the glycoprotein G of the vesicular stomatitis virus is replaced by the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), preferably of the WE-HPI strain. Such a VSV (recombinant with the GP of LCMV) is described, for example, in WO 2010 / 040526 and is named VSV-GP.

[0051] The glycoprotein GP of Lymphocytic Choriomeningitis Virus (LCMV) can be GP1 or GP2. Also included are glycoproteins from different LCMV strains. In particular, LCMV-GP can be from wild-type LCMV or LCMV strains LCMV-WE, LCMV-WE-HPI, LCMV-WE-HPlopt. In a preferred embodiment, the gene encoding the glycoprotein GP of LCMV encodes a protein having the amino acid sequence shown in SEQ ID NO:1 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%, 99% sequence identity with the amino acid sequence of SEQ ID NO:1, while maintaining the functional properties of the recombinant rhabdovirus comprising the glycoprotein GP encoding the amino acid sequence shown in SEQ ID NO:1.

[0052] Vesicular stomatitis viruses typically encode at least the vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M) and glycoprotein (G) in their genome.

[0053] In a preferred embodiment, the vesicular stomatitis virus comprises in its genome at least a nucleoprotein (N) of the vesicular stomatitis virus comprising the amino acid sequence set forth in SEQ ID NO:2 or a functional variant thereof at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO:2, an amino acid sequence set forth in SEQ ID NO:3 or a functional variant thereof at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO:3, 5, wherein the amino acid sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO:4; a large protein (L) comprising the amino acid sequence set forth in SEQ ID NO:5 or a functional variant at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO:5; and a matrix protein (M) comprising the amino acid sequence set forth in SEQ ID NO:5 or a functional variant at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO:5.

[0054] It is understood by those skilled in the art that modifications to the sequences of the nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M) or glycoprotein (G) of vesicular stomatitis virus can be made without losing the basic function of these proteins. As used herein, such functional variants retain all or part of their basic function or activity. Protein L, for example, is a polymerase and has an essential function during viral transcription and replication. Its functional variants must retain at least part of this ability. A good indicator for the retention of basic functionality or activity is the successful production of viruses containing these functional variants that are still capable of replicating and infecting tumor cells. Testing for the production of viruses and their infection and replication in tumor cells can be tested in various assay systems known to those skilled in the art (exemplary in vitro assays are described in Muik et al., Cancer Res., 74(13), 3567-78, 2014).

[0055] In a preferred embodiment, the vesicular stomatitis virus encodes at least the vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M) and glycoprotein (G) in its genome, wherein the large protein (L) comprises an amino acid sequence having 80% or more sequence identity to SEQ ID NO:4.

[0056] In a preferred embodiment, the vesicular stomatitis virus encodes at least the vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M) and glycoprotein (G) in its genome, wherein the nucleoprotein (N) comprises an amino acid sequence having 90% or more sequence identity to SEQ ID NO:2.

[0057] In a further preferred embodiment, the vesicular stomatitis virus encodes in its genome at least the vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M) and glycoprotein (G), wherein the large protein (L) comprises an amino acid sequence having 80% or more sequence identity to SEQ ID NO:4, and the nucleoprotein (N) comprises an amino acid sequence having 90% or more sequence identity to SEQ ID NO:2.

[0058] In another embodiment, the glycoprotein G of the vesicular stomatitis virus is replaced by the glycoprotein of the Dandenong virus (DANDV) or Mopela (MOPV) virus. The Dandenong virus (DANDV) is an Old World arenavirus. Today, there is only a single lineage known to those skilled in the art that contains and can utilize the glycoprotein. The glycoprotein of DANDV contained in the vesicular stomatitis virus has seven or more glycosylation sites, in particular seven glycosylation sites. An exemplary preferred glycoprotein is that contained in DANDV, accessible under Genbank number EU136038. In one embodiment, the gene encoding the glycoprotein of DANDV encodes the amino acid sequence shown in SEQ ID NO:6 or a 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:6, while maintaining the functional properties of the vesicular stomatitis virus comprising the glycoprotein encoding the amino acid sequence shown in SEQ ID NO:6. Mopella virus (MOPV) is an Old World arenavirus. There are several strains known to those skilled in the art that contain glycoproteins and can be used as donors for the glycoproteins contained in the vesicular stomatitis virus. The glycoprotein of the MOPV contained in the vesicular stomatitis virus has seven or more glycosylation sites, in particular seven glycosylation sites. Exemplary preferred glycoproteins are those contained in the Mopella virus accessible under Genbank number AY772170. In one embodiment, a gene encoding a glycoprotein of an MOPV encodes an amino acid sequence set forth in SEQ ID NO:7 or a sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:7, while maintaining the functional properties of a vesicular stomatitis virus comprising a glycoprotein encoding the amino acid sequence set forth in SEQ ID NO:7.

[0059] It should be understood that the rhabdovirus, particularly the vesicular stomatitis virus, prepared, produced or purified according to the method or process of the present invention can code additional genes (recombinant rhabdovirus) in its genome.These genes are often called cargo, and include, for example, tumor antigens, chemokines, cytokines or other immune modulating elements.Independent of the type of cargo additionally expressed by rhabdovirus, such cargo-expressing rhabdovirus can be prepared, produced or purified according to the method or process of the present invention.Therefore, in the most preferred embodiment, the vesicular stomatitis virus is prepared, produced or purified according to the method or process of the present invention, in which the glycoprotein G of vesicular stomatitis virus is replaced by the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV). In a further preferred embodiment, a vesicular stomatitis virus is prepared, produced or purified according to the method or process of the invention, in which the glycoprotein G of the vesicular stomatitis virus is replaced by the glycoprotein GP of the lymphocytic choriomeningitis virus (LCMV) and the genome encodes an additional cargo, such as a tumor antigen, a chemokine, a cytokine or other immune modulating element. Preferably, the cargo is CCL21 or a C-terminally truncated CCL21 protein (having amino acids 1-79 of full-length CCL21) characterized by deletion and / or mutation of amino acids in the extended c-terminus of the CCL21 protein, thereby reducing the deletion and / or mutation of amino acids in the extended c-terminus bond to a glycosaminoglycan, such as heparin. In a further preferred embodiment, the cargo is a fusion protein comprising the extracellular domain of CD80 fused to the Fc terminus of IgG1, resulting in a CD80Fc fusion protein. In a most preferred embodiment, the cargo is a CD80 extracellular domain-Fc fusion protein comprising or consisting of SEQ ID NO: 8 or SEQ ID NO: 9. In a further preferred embodiment, the cargo is a CCL21 protein comprising or consisting of SEQ ID NO: 10 or SEQ ID NO: 11.However, for the purposes of the methods or processes of the present invention, the inventors have found that the additional cargo does not affect the performance of the method or process, and therefore the nature of the cargo should not be construed as limiting the applicability of the present invention.

[0060] Rhabdoviruses encoding additional genes can be produced according to methods known to those skilled in the art, including but not limited to: (1) using cDNA transfected into cells, or (2) using a combination of cDNA transfected into helper cells, or (3) using cDNA transfected into cells that are further infected with helper / miniviruses that provide the remaining components or activities required in trans to produce either infectious or non-infectious recombinant rhabdoviruses.The minimum components required to use any of these methods (e.g., helper / miniviruses, helper cell lines, or cDNA transfection only) are (1) a DNA molecule that contains the cis-acting signals for the encapsidation of genomic (or antigenomic) RNA by the N protein, P protein, and L protein of rhabdovirus, and (2) the replication of genome or antigenomic (replicative intermediate) RNA equivalents.

[0061] Replication element or replicon is a strand of RNA that minimally contains the leader and trailer sequences of rhabdovirus at the 5' and 3' ends. In the genomic sense, the leader is at the 3' end and the trailer is at the 5' end. Any RNA placed between these two replication signals will then be replicated. The leader and trailer regions must further contain minimal cis-acting elements for the purpose of encapsidation by N protein and for the polymerase binding required to start transcription and replication. To prepare recombinant rhabdovirus, the minivirus containing G gene will also contain the G gene with the leader region, trailer region and the start and stop signal suitable for producing mRNA of G protein. If the minivirus further contains M gene, the start and stop signal suitable for producing mRNA of M protein must also be present.

[0062] For any gene contained in the recombinant rhabdovirus genome, the gene will be adjacent to the appropriate transcription initiation and termination signal that will allow the expression of the gene and the production of protein products (Schnell et al., Journal of Virology, p.2318-2323, 1996).To produce a "non-infectious" recombinant rhabdovirus, the recombinant rhabdovirus must have the minimum replicon element and N, P and L proteins, and must contain the M gene.This produces virus particles that bud from cells but are non-infectious particles.To produce an "infectious" particle, the virus particle must further contain a protein that can mediate the binding and fusion of the virus particle, for example, by using an attachment protein or a receptor ligand.The native receptor ligand of rhabdovirus is G protein.

[0063] Any cell that will allow the assembly of recombinant rhabdovirus can be used. One method for preparing infectious virus particles includes infecting a suitable cell line with a plasmid that codes for T7 RNA polymerase or other suitable bacteriophage polymerase, such as T3 or SP6 polymerase. The cells can then be transfected with individual cDNAs that contain the genes that code for G, N, P, L and M rhabdovirus proteins. These cDNAs will provide the proteins for constructing recombinant rhabdovirus particles. The cells can be transfected by any method known in the art.

[0064] Also, "polycistronic cDNA" containing rhabdovirus genomic RNA equivalent is transfected into cell lineage. If infectious recombinant rhabdovirus particles are intended to be lytic in infected cells, genes encoding N, P, M and L proteins must be present as well as any heterologous nucleic acid segment. If infectious recombinant rhabdovirus particles are not intended to be lytic, the gene encoding M protein is not included in polycistronic DNA. "Polycistronic cDNA" refers to a cDNA that comprises at least a transcription unit that contains genes encoding N, P and L proteins. The polycistronic DNA of recombinant rhabdovirus can also contain genes encoding protein variants or polypeptide fragments thereof or therapeutic nucleic acids or proteins. Alternatively, any protein or fragment thereof that is originally associated with the initially produced viral particles can be supplied in trans.

[0065] The contemplated polycistronic cDNA can contain either a gene encoding a protein variant, a gene encoding a reporter, a therapeutic nucleic acid and / or an NPL gene or an NPLM gene. The first step of generating recombinant rhabdovirus is the expression of RNA, which is the genome or antigenome equivalent, from cDNA. The RNA is then packaged by N protein and then replicated by P / L protein. The recombinant virus thus produced can be recovered. If G protein is not present in the recombinant RNA genome, it is typically supplied in trans. If both G and M proteins are not present, both are supplied in trans. To prepare "non-infectious rhabdovirus" particles, the procedure can be the same as above, except that the polycistronic cDNA transfected into cells will only contain the N, P and L genes of rhabdovirus. The polycistronic cDNA of non-infectious rhabdovirus particles can further contain genes encoding proteins.

[0066] After transfection of the cell culture, the whole cell culture is usually harvested 1-3 days after infection. Advantageously, the method / process of the present invention allows processing of the whole cell culture for a subsequent harvest step.

[0067] In a first alternative of the method or process of the invention, the rhabdovirus harvest is obtained from the cell culture by directly adding a virus release agent, such as salt or dextran sulfate, to the cell culture. 50 Although the measured virus concentration per unit volume or genome copy number suggested a sufficient yield to meet the demand, a significant drop in virus concentration was observed after harvesting that could not be explained. It was hypothesized that this could be due to the interaction of the virus with either cells, debris and / or other cell culture components, and that these viruses were then lost after the clarification step, e.g., depth filtration, centrifugation or TFF. Without being bound by theory, it is further believed that virus particles that have already budded from the host cell may be electrostatically restricted to the cell surface. For this reason, the virus particles can be released from the cell surface, for example, by increasing the ionic strength of the cell culture with a virus release agent. This hypothesis was tested by adding various virus release agents to the cell culture.

[0068] In this respect, the term "virus releasing agent" or "virus releasing agent" is applied to the cell culture prior to harvesting, thereby determining the amount of virus released into the supernatant (TCID 50 The effect and suitability of a virus releasing agent can be evaluated, for example, by obtaining a sample from a cell culture prior to harvest, treating the sample with a virus releasing agent, and then centrifuging the treated sample and measuring the amount of virus (TCID 50The virus release agent of the present invention can be easily measured by determining the virus harvest (TCID ) obtained from cell cultures treated with a virus release agent compared to untreated cell cultures. Preferably, the virus release agent of the present invention does not (permanently) inactivate or damage the virus, does not interfere with subsequent method / process steps, and / or can be easily removed during subsequent method / process steps. Another aspect is the cost of goods, with preferred virus release agents being cost-effective and easily procured. In one aspect, the virus harvest (TCID ) obtained from cell cultures treated with a virus release agent compared to untreated cell cultures can be easily measured by determining the virus harvest (TCID ) obtained from cell cultures treated with a virus release agent compared to untreated cell cultures. 50 (as measured by TCID 50 (as measured by) is increased by at least about 10-fold, preferably at least about 25-fold, preferably at least about 50-fold, preferably at least about 75-fold, and more preferably at least about 100-fold.

[0069] For the purposes of the present invention, the amount and / or suitability of the virus releasing agent required to achieve the desired effect can be expressed either (i) by the increase in ionic strength in the cell culture after the virus releasing agent is added to the cell culture or alternatively (ii) by the increase in the concentration of the virus releasing agent in the cell culture. For the alternative (i), it will be understood that for the purposes of the present invention, the virus releasing agent then added to the cell culture will result in a suitable increase in ionic strength in the cell culture. In this context, the term "increase in ionic strength" refers to the increase in ionic strength of the cell culture after the addition of the virus releasing agent. For the alternative (ii), the "increase in virus releasing agent concentration" is calculated based only on the specific virus releasing agent added to the cell culture, and the term "increase in salt concentration" applies mutatis mutandis below. This term describes salts as virus releasing agents in more detail.

[0070] Thus, in one embodiment, the virus release agent is preferably an agent in solution. The agent, for example, a salt, amino acid, or sulfated polysaccharide described herein, can increase the ionic strength of the cell culture after it is added to the recruitment culture, thereby promoting the release of virus particles. The increase in ionic strength in the cell culture should be at least about 0.01M or at least about 0.05M. In another embodiment, the increase in ionic strength in the cell culture is about 0.01M to about 1.5M or about 0.05M to about 1.5M, about 0.1M to about 1.5M, about 0.15M to about 1.5M, or about 0.2M to about 1.5M. In another embodiment, the increase in ionic strength in the cell culture is about 0.01M to about 5M or 0.05M to about 5M, about 0.1M to about 5M, about 0.15M to about 5M, or about 0.2M to about 1.5M. In a preferred embodiment, the ionic strength increase in cell culture is at least about 0.1M or more preferably at least about 0.2M.Depending on the type of rhabdovirus, as long as rhabdovirus is not permanently inactivated or damaged, the ionic strength can be increased even higher.Thus, the ionic strength increase can be increased to about 2M, 2.5M, 3M, 3.5M, 4M, 4.5M or 5M in cell culture.

[0071] The ionic strength of a solution is known to be a measure of the concentration of ions in the solution. Ionic compounds dissolve in water and dissociate into ions, resulting in the ionic strength of the solution being a function of the concentration of all ions present.

number

[0072] In this formula, c is the molar concentration of ion i (M, mol / L), z is the number of charges on that ion, and the sum is taken over all ions n in the solution. For sodium chloride, the ionic strength is equal to the concentration, but for MgSO 4For salts such as , the ionic strength is 4 times higher, thus multivalent ions strongly contribute to the ionic strength. Furthermore, it is also known how the desired ionic strength of a (salt) solution, e.g. a buffer, can be adjusted, and the setting of the ionic strength of the (salt) solution can be made depending on the concentration and ionic potency of the agents present, e.g. salts. Furthermore, a huge number of publications and patent documents exist in the prior art, so that specific values ​​or ranges of ionic strength can be found in handbooks, monographs, etc. Thus, the skilled person is able to provide a (salt) solution with the necessary ionic strength to bring about the required increase in ionic strength in cell culture.

[0073] Preferably, by adding the virus releasing agent to the cell culture, the ionic strength of the cell culture is increased by at least about 0.01M or at least about 0.05M, at least about 0.1M or at least about 0.2M or at least about 0.25M or at least about 0.3M or at least about 0.35M or at least about 0.4M or at least about 0.45M or at least about 0.5M, or about 0.01M to about 1.5M or about 0.05M to about 1.5M or about 0.1M to about 1.5M or about 0.15M to about 1.5M or about 0.2M to about 1.5M. In some examples, an increase in ionic strength of up to about 2M, 2.5M, 3M, 3.5M, 4M, 4.5M or 5M can be achieved in the cell culture. It will be understood that any of the above lower limits and ranges can be combined with any of the upper limits or ranges.

[0074] The term "cell culture" should be understood to include host cells, rhabdoviruses and cell culture medium. The volume and / or concentration of the virus release agent, particularly salt or dextran sulfate, is mainly determined by the volume of the cell culture and the final concentration or ionic strength achieved in the cell culture. The salt can be added to the cell culture as solid salt or as an aqueous salt solution. For the salt solution, in some cases it may be appropriate to add a small amount of high-concentration salt solution, while in other cases it may be desirable to add a larger amount of salt solution with a lower salt concentration. The salt solution can be added continuously over time or can be added all at once to the cell culture. In one embodiment, the salt solution is added to the culture at a 4M stock solution with a dilution of 1:20, which results in an additional 0.2M NaCl concentration increase just before harvesting. For dextran sulfate, it may be appropriate to add a small amount of high-concentration dextran sulfate solution, while in other cases it may be desirable to add a larger amount of lower concentration dextran sulfate solution. The dextran sulfate solution can be added continuously over time or can be added all at once to the cell culture. In one embodiment, the dextran sulfate solution is added to the culture just before harvesting, resulting in a concentration of 100 μg / ml in the cell culture. However, in some cases, it may be necessary to add lower or higher concentrations of dextran sulfate to the cell culture.

[0075] The efficacy and suitability of the virus releasing agent, as well as the required concentration, can be readily determined by one of skill in the art, as explained above.

[0076] Apparently, the virus release agent in cell culture, particularly salt or dextran sulfate, helps to dissolve the aggregates of rhabdovirus that may be formed during culture, and also supports the release of rhabdovirus from host cell or host cell membrane.Therefore, by adding the virus release agent, particularly salt or dextran sulfate, more recoverable rhabdovirus is released into the supernatant or released from cells, which can be captured and collected in subsequent steps.

[0077] It is understood that other sulfated polysaccharides, then dextran sulfate, are also preferred. Sulfated polysaccharides having a higher degree of sulfation may be preferred. Sulfated polysaccharides may include, but are not limited to, dextran sulfate, pentosan polysulfate, fucoidan and carrageenan, and their respective salts.

[0078] It has also been observed that the amount of rhabdovirus released into the supernatant can be improved by using arginine as virus release agent.Therefore, in one embodiment, the virus release agent is a solution containing arginine.Preferably, the arginine concentration in cell culture or the ionic strength in cell culture increases by at least about 0.05M, 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M or at least about 0.5M by adding the solution.The use of other amino acids and their respective salts is also contemplated, and in one aspect, amino acids and their respective salts, preferably polar amino acids, more preferably basic or acidic amino acids, are useful as virus release agents.Most preferred amino acids include, but are not limited to, aspartic acid, cysteine, glutamic acid, histidine, lysine or tyrosine. Preferably, the amino acid concentration in the cell culture or the ionic strength in the cell culture is increased by at least about 0.01M, 0.05M, 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M or at least about 0.5M upon addition of the amino acid.

[0079] For purposes of the present invention, it is important that the increased salt concentration is achieved by adding salt to the cell culture.

[0080] In this context, the term "increased salt concentration" always refers to the particular salt that is added to the cell culture and whether the concentration of this particular salt is increased.

[0081] It should be understood that the cell culture may pre-contain a certain level of salt concentration in the medium. The salt that may pre-contain in the cell culture and the salt that is added to the cell culture for harvesting may be the same salt or different salt. If the salt in the cell culture and the salt that is added to the cell culture are the same, a person skilled in the art will understand that a sufficiently concentrated salt or salt solution must be prepared to increase the salt concentration, taking into account the salt present in the medium. As an example, to increase the NaCl concentration to 0.2M in a cell culture that pre-contains a 0.1M NaCl concentration (resulting in a final concentration of 0.3M NaCl in the cell culture), a sufficiently concentrated NaCl salt solution must be prepared, taking into account the existing NaCl concentration in the medium.

[0082] On the other hand, if the cell culture does not contain any salts or contains a different salt than the salt added to the cell culture, the increase in salt concentration will be based only on the salt that will be added to the cell culture. Thus, in both such cases (no salt or different salt), the increase in salt concentration will be based only on the specific salt that is added to the cell culture. That is, the increase in salt concentration will be calculated based only on the specific salt that is added to the cell culture.

[0083] The increase in salt concentration in the cell culture should be at least about 0.01 M or at least about 0.05 M. In another embodiment, the increase in salt concentration in the cell culture is from about 0.01 M to about 1.5 M, from about 0.05 M to about 1.5 M, from about 0.1 M to about 1.5 M, from about 0.15 M to about 1.5 M, from about 0.2 M to about 1.5 M.

[0084] In a preferred embodiment, the salt concentration in cell culture is increased to at least about 0.1M or more preferably at least about 0.2M.The inventors have tested an increase in concentration up to 1.5M without any adverse effect on rhabdovirus.Depending on the type of rhabdovirus, higher salt concentrations can be achieved, as long as rhabdovirus is not permanently inactivated or damaged by salt concentration.Therefore, an increase in salt concentration up to about 2M, 2.5M, 3M, 3.5M, 4M, 4.5M or 5M can be achieved in cell culture.

[0085] Suitable salts include organic and inorganic salts. The inorganic salt is not limited according to the present invention, and any inorganic salt that is soluble in aqueous solution and does not permanently damage the cell culture and / or the virus can be utilized. The inorganic salt is selected from the group consisting of alkali or alkaline earth salts, such as sulfates, nitrates, phosphates, carbonates, halides, borates, silicates, etc. If a pharmaceutically acceptable product will be provided, the inorganic salt and the organic salt will be selected from the group of pharmaceutically acceptable salts known per se. For example, the pharmaceutically acceptable inorganic salt is selected from sodium salts, such as sodium halides, preferably sodium chloride, sodium sulfate, sodium borate; calcium salts, such as calcium halides, preferably calcium chloride, calcium sulfate, calcium borate; magnesium salts, such as magnesium halides, preferably magnesium chloride, magnesium sulfate, magnesium borate, and combinations thereof, as well as other pharmaceutically acceptable inorganic salts.

[0086] Further examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues, such as amines; alkali or organic salts of acidic residues, such as carboxylic acids. For example, such salts include salts from benzenesulfonic acid, benzoic acid, citric acid, ethanesulfonic acid, fumaric acid, gentisic acid, hydrobromic acid, hydrochloric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, 4-methyl-benzenesulfonic acid, phosphoric acid, salicylic acid, succinic acid, sulfuric acid, and tartaric acid. Further pharmaceutically acceptable salts can be formed with cations from ammonia, L-arginine, calcium, 2,2'-iminobisethanol, L-lysine, magnesium, N-methyl-D-glucamine, potassium, sodium, and tris(hydroxymethyl)-aminomethane.

[0087] Preferred salts are NaCl, KCl, MgCl 2 , CaCl 2 , N.H. 4 Cl, sulfate NH 4 , NH acetate 4 or NH bicarbonate 4 In a most preferred embodiment, a NaCl salt solution is used. The salt solution may include a buffer, preferably having a pH of about 5 to about 9, desirably about 6.5 to about 8.5.

[0088] It will be appreciated that the pH of the cell culture following addition of the virus releasing agent will be maintained in a range that preserves the integrity of the cells and / or, more importantly, the virus, for example, in the range of about pH 5 to 9, or more preferably, about 6.5 to 8.5. Also, in some cases, depending on the nature of the virus releasing agent, it may be desirable to adjust the pH to a particular range to affect the charge of the virus releasing agent and thereby promote its properties as a virus releasing agent.

[0089] No specific holding or incubation time is foreseen after the virus releasing agent, in particular salt or dextran sulfate, is added to the cell culture to achieve the desired concentration or ionic strength in the cell culture. Therefore, after achieving the desired concentration or ionic strength in the cell culture, the whole cell culture will be further processed in the next step. It may be desirable to stir or move the cell culture to achieve a uniform distribution of the virus releasing agent in the cell culture. The specific incubation time with the virus releasing agent is essentially achieved by the technical necessity of adding the virus releasing agent, in particular salt or dextran sulfate, and preparing or moving the cell culture to the next process step. In some cases, it may be desirable to incubate the cell culture for a certain period of time after adding the virus releasing agent. The incubation time can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours.

[0090] According to the method or process of the present invention, the cell culture is then subjected to a clarification step.When rhabdovirus is secreted into the supernatant, this clarification step serves to separate most of particulate matter, i.e. cells and cell debris, from the supernatant containing rhabdovirus.Various methods for removing cells from cell culture supernatant are available to those skilled in the art, and preferably, clarification comprises one or more of the following methods: depth filtration, tangential flow filtration and / or centrifugation.In each of these clarification methods, the supernatant is separated from cells and cell debris, and the supernatant containing rhabdovirus is collected for further processing.Other methods known to those skilled in the art for separating particulate matter from the supernatant can be equally applied in this clarification step.In a preferred embodiment, the cell culture is clarified by microfiltration by cross-flow, more preferably by using a hollow fiber cutoff of 0.65 μm.In another preferred embodiment, the cell culture is clarified through a depth filter. In some cases, 0.45 μm / 0.2 μm filtration is connected in series with the depth filter to prevent potential bioburden contamination.

[0091] In the second alternative of the method or process of the present invention, rhabdovirus harvest is obtained from cell culture by first clarifying cell culture through a filtration step, preferably through depth filtration, and then rinsing filter, preferably depth filter, with virus release agent, particularly saline solution or dextran sulfate.When saline solution is used, this salt solution has a concentration or ionic strength of at least about 0.01M or 0.05M, or a concentration or ionic strength of about 0.01M to about 1.5M or about 0.05M to about 1.5M.Then, the supernatant (or in this alternative, the filtrate) and the rinse solution, both of which contain rhabdovirus harvest, are collected.

[0092] Therefore, in this alternative, the virus releasing agent, particularly salt solution or dextran sulfate, is not added directly to the cell culture, but the cell culture is first clarified by a filtration process, preferably by depth filtration, and the filter, preferably by depth filter, is rinsed with a virus releasing agent, preferably salt solution or dextran sulfate.Cells and cell debris are retained by the filter, preferably by depth filter, and the virus releasing agent mainly supports the release of rhabdovirus from the cell or cell membrane that is retained by the filter, preferably by depth filter.Therefore, in this alternative, the effect of adding a virus releasing agent is the same as that of the first alternative.That is, a larger amount of recoverable rhabdovirus is released and collected, which can be captured and collected in a subsequent step.It will be understood that a person skilled in the art can choose from a range of different filters that will allow the removal of cells and cell debris, and will select a suitable filter.

[0093] Also, depending on the type of rhabdovirus, even higher, for example, salt concentration, can be applied here, as long as rhabdovirus is not permanently inactivated or damaged by salt concentration.Suitable salts include organic salts and inorganic salts.Inorganic salts are not limited according to the present invention, and can utilize any inorganic salt that is soluble in aqueous solution and does not interfere with cell culture and / or virus.Inorganic salts are selected from the group consisting of, for example, alkali salts or alkaline earth salts such as sulfates, nitrates, phosphates, carbonates, halides, borates, silicates, etc.When pharmaceutically acceptable products will be provided, inorganic salts and organic salts will be selected from the group of pharmaceutically acceptable salts known per se. For example, the pharma- ceutically acceptable inorganic salt is selected from sodium salts, e.g., sodium halides, preferably sodium chloride, sodium sulfate, sodium borate; calcium salts, e.g., calcium halides, preferably calcium chloride, calcium sulfate, calcium borate; magnesium salts, e.g., magnesium halides, preferably magnesium chloride, magnesium sulfate, magnesium borate, and combinations thereof, as well as other pharma-ceutically acceptable inorganic salts.

[0094] Preferred salts are again NaCl, KCl, MgCl 2 , CaCl 2 , N.H. 4 Cl, sulfate NH 4 , NH acetate 4 or NH bicarbonate 4 Examples of suitable salt solutions include, but are not limited to, NaCl salt solution. In the most preferred embodiment, a NaCl salt solution is used. The salt solution may include a buffer, preferably having a pH of about 5 to about 9, desirably about 6.5 to about 8.5. For dextran sulfate, it may be appropriate to add a small amount of a high concentration dextran sulfate solution, while in other instances it may be desirable to add a larger amount of a lower concentration dextran sulfate solution. The required concentration can be readily determined by one of skill in the art, as explained above.

[0095] Similarly, in the second alternative, arginine can be used as a virus release agent. Thus, in one embodiment, the virus release agent is a solution containing arginine. Preferably, the arginine concentration of the solution or the ionic strength of the solution is at least about 0.01M, 0.05M, 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M or at least about 0.5M. The use of other amino acids and their respective salts is also contemplated, and in one aspect, amino acids, preferably polar amino acids, more preferably basic or acidic amino acids, are useful as virus release agents. Most preferred amino acids include, but are not limited to, aspartic acid, cysteine, glutamic acid, histidine, lysine or tyrosine. Preferably, the amino acid concentration of the solution or the ionic strength of the solution is at least about 0.01M, 0.05M, 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M or at least about 0.5M.

[0096] In another embodiment, both the first and second alternatives can be combined.That is, rhabdovirus harvest is obtained from cell culture by directly adding virus release agent, particularly salt or dextran sulfate, to cell culture, followed by deep filtration of cell culture, and then rinsing the depth filter with virus release agent (preferably salt solution or dextran sulfate).

[0097] Optionally, in some cases, it may be necessary to reduce or remove virus release agent from the obtained rhabdovirus collection, so as not to interfere with subsequent method / process steps or drug substance.For example, salt concentration may interfere with subsequent capture step, DNA decomposition step, or salt concentration may damage or inactivate rhabdovirus when incubated for a longer period of time.Reduction of salt concentration can be preferably achieved by dilution, diafiltration and / or dialysis.In any case, in each of these methods, salt concentration is reduced, and then the rhabdovirus collection with reduced salt concentration is further processed according to the method or process of the present invention.Other methods known to those skilled in the art that are suitable for reducing salt concentration in rhabdovirus collection can be equally applied.

[0098] Further optionally, in some cases, it may be desirable to treat the rhabdovirus collection with a DNA-degrading nuclease, such as benzonase.In a preferred embodiment, the DNA-degrading nuclease is a salt-active nuclease, such as SAN-HQ.

[0099] The rhabdovirus collection collected after the clarification step is then subjected to further purification steps by loading the collection onto a cation exchanger.Collected can be loaded onto a cation exchanger immediately after the clarification step.In some cases, the collected collection is stored for a period of time, for example overnight, and then applied to a cation exchanger the next day.In one embodiment, the material of the cation exchanger is a monolith, a resin, a fiber or a membrane.In a preferred embodiment, the cation exchanger is a monolithic adsorbent.It has been found that the use of cation exchanger capture of rhabdovirus is much more efficient than, for example, anion exchange.Preferably, this capture and purification step is carried out by monolithic cation exchange chromatography in binding / elution mode.

[0100] In one embodiment, the aforementioned purification steps also serve the purpose of concentrating the rhabdovirus harvest. In this regard, the (clarified) virus harvest (TCID50 Preferably, the virus is concentrated by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (TCID 50 The concentration is at least about 10-fold, preferably at least about 25-fold, preferably at least about 50-fold, preferably at least about 75-fold, and more preferably at least about 100-fold (measured in units of 1×10 / mL). 9 TCID 50 A 10-fold concentration of 10 L of virus solution with a concentration of 1 × 10 10 TCID 50 This will yield 1 L of virus solution with a virus concentration of 100 / mL.

[0101] The buffer that is selected for applying rhabdovirus collection onto cation exchanger is generally known to those skilled in the art.The condition that will bring about the maximum binding of rhabdovirus to cation exchanger is selected.Preferably, before applying it to cation exchanger, collection is diluted with conditioning buffer.In one embodiment, conditioning buffer comprises Tris buffer, more preferably, conditioning buffer comprises 100mM Tris buffer that is adjusted to pH 7.5 with HCl.Preferably, collection and conditioning buffer are diluted at a ratio of at least 1:1 or at least 1:2 before applying to cation exchanger.

[0102] The captured rhabdovirus is then eluted from the cation exchanger.Elution is typically carried out by applying an elution buffer with a linearly increasing salt concentration to the cation exchanger or by using a single-step elution process.In a preferred embodiment, the elution buffer further comprises arginine.

[0103] The eluate thus purified is then collected and pooled.Although this rhabdovirus eluate is already highly purified, depending on the intended further use of the rhabdovirus eluate, one or more of the following optional processing steps can be carried out on the rhabdovirus eluate:

[0104] (i) Refining the Rhabdovirus eluate to further remove impurities, preferably by size exclusion, multimodal size exclusion, ion exchange and / or tangential flow filtration.

[0105] In a preferred embodiment, the polishing step is based on flow-through mode, e.g., mixed-mode bead-based size-exclusion and anion-exchange chromatography (Capto™ Core) with an exclusion limit of about 700 kD.

[0106] (ii) changing the buffer of the rhabdovirus eluate or refined rhabdovirus, preferably by ultrafiltration and diafiltration or dialysis.

[0107] In a preferred embodiment, buffer exchange is performed using ultrafiltration / diafiltration followed by bioburden filtration to yield the drug substance.

[0108] (iii) sterile filtering the rhabdovirus.

[0109] Pharmaceutical Compositions For use in treatment, the rhabdovirus prepared, produced and / or purified according to the method or process of the present invention is formulated into a suitable pharmaceutical composition for easy administration to animals or humans.A typical formulation can be prepared by mixing rihabdovirus with physiologically acceptable carrier, excipient or stabilizer in the form of aqueous solution or aqueous or non-aqueous suspension.Carrier, excipient, modifier or stabilizer is non-toxic at the dosage and concentration used. They may contain buffer systems such as phosphate, citrate, acetate and other inorganic or organic acids and their salts; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone or polyethylene. Examples of suitable excipients include polysaccharides such as PEG, glycine, glutamine, asparagine, histidine, arginine or lysine, mono-, di-, oligo- or polysaccharides including glucose, mannose, sucrose, trehalose, dextrin or dextran and other carbohydrates, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt forming counterions such as sodium, metal complexes (e.g. Zn-protein complexes), and / or ionic or non-ionic surfactants such as TWEEN™ (polysorbates), PLURONICS™ or fatty acid esters, fatty acid ethers or sugar esters. Excipients may also have release-modifying or absorption-modifying functionality.

[0110] In one embodiment, the rhabdovirus is formulated into a pharmaceutical composition comprising Tris, arginine and optionally citrate.Tris is preferably used at a concentration of about 1 mM to about 100 mM.Arginine is preferably used at a concentration of about 1 mM to about 100 mM.Citrate can be present at a concentration of up to 100 mM.A preferred formulation comprises about 50 mM Tris and 50 mM arginine.

[0111] The pharmaceutical composition may be provided as a liquid, a frozen liquid or a lyophilized form. The frozen liquid may be stored at a temperature of about 0°C to about -85°C, including temperatures of -70°C to -85°C and about -15°C, -16°C, -17°C, -18°C, -19°C, -20°C, -21°C, -22°C, -23°C, -24°C or about -25°C.

[0112] The rhabdovirus or pharmaceutical composition may, but need not, be formulated with one or more drugs currently used to prevent or treat the disorder of interest.The effective amount of such other drugs depends on the amount of recombinant antibody present in the formulation, the type of disorder or treatment, and other factors discussed above.They are generally used in the same dosage and route of administration as described herein, or about 1-99% of the dosage described herein, or any dosage and route that is empirically / clinically determined to be appropriate.

[0113] For the prevention or treatment of disease, the appropriate dosage of rhabdovirus or pharmaceutical composition (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of rhabdovirus, the severity and course of disease, whether rhabdovirus is administered for prevention or treatment, previous treatment, the clinical history of patient and response to rhabdovirus and the discretion of the attending physician.The rhabdovirus or pharmaceutical composition of the present invention is appropriately administered to patient once or over a series of treatments.

[0114] TCID of rhabdoviruses depending on type and severity of disease 50 Measured by about 10 8~10 13 The infectious particles can be the initial candidate dose for administration to a patient, whether by one or more separate administrations or by continuous infusion. In the case of repeated administration over several days or more, depending on the condition, the treatment will generally be sustained until the desired suppression of disease symptoms occurs. One exemplary dose of rhabdovirus is TCID 50 Measured by about 10 8 ~10 13 Therefore, the TCID 50 Measured by about 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13 One or more doses of the recombinant rhabdovirus (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example every week or every three weeks (e.g., so that the patient receives from about 2 to about 20 or, for example, about 6 doses of the recombinant rhabdovirus). An initial higher loading dose can be administered, followed by one or more lower doses, or vice versa, although other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0115] The effectiveness of rhabdovirus and the composition comprising it can be tested according to the specific disease involved by using any suitable in vitro assay, cell-based assay, in vivo assay and / or animal model known per se, or any combination thereof.Suitable assay and animal model will be clear to those skilled in the art, and include, for example, the assay and animal model used in the following examples.

[0116] The actual pharmacologic effective amount or therapeutic dose will of course depend on factors known to those skilled in the art, such as age and weight of patient, route of administration and severity of disease.In any case, rhabdovirus will be administered in a dosage and manner that allows it to deliver a pharmacologic effective amount based on the unique condition of the patient.

[0117] Alternatively, the rhabdovirus or pharmaceutical composition of the present invention can be delivered in a volume of about 50 μL to about 100 mL, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, and the desired effect of the route and method of administration.

[0118] For intratumoral administration, the volume is preferably about 50 μL to about 5 mL, including volumes of about 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1000 μL, 1100 μL, 1200 μL, 1300 μL, 1400 μL, 1500 μL, 1600 μL, 1700 μL, 1800 μL, 1900 μL, 2000 μL, 2500 μL, 3000 μL, 3500 μL, 4000 μL, or about 4500 μL. In a preferred embodiment, the volume is about 1000 μL.

[0119] For example, for systemic administration by injection of the rhabdovirus, the volume may of course be larger. Alternatively, a concentrated solution of the rhabdovirus can be diluted with a larger volume of injection solution immediately prior to injection.

[0120] In particular, for intravenous administration, the volume is preferably 1 mL to 100 mL, including volumes of about 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 55 mL, 60 mL, 70 mL, 75 mL, 80 mL, 85 mL, 90 mL, 95 mL, or about 100 mL. In a preferred embodiment, the volume is about 5 mL to 15 mL, and more preferably, the volume is about 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, or about 14 mL.

[0121] Preferably, the same formulation is used for intratumoral and intravenous administration. The dose and / or volume ratio between intratumoral and intravenous administration can be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or about 1:20. For example, a dose and / or volume ratio of 1:1 means that the same dose and / or volume is administered intratumorally and intravenously, while a dose and / or volume ratio of, for example, about 1:20 means that the dose and / or volume of intravenous administration is 20 times higher than the dose and / or volume of intratumoral administration. Preferably, the dose and / or volume ratio between intratumoral and intravenous administration is about 1:9.

[0122] The effective concentration of the rhabdovirus is preferably about 10 per milliliter. 8 ~10 14 The range of vector genome (vg / mL). Infectious units can be measured as described in McLaughlin et al., J Virol.;62(6):1963-73 (1988). Preferably, the concentration is about 1.5×10 9 ~Approx. 1.5×10 13 and more preferably about 1.5×10 9 ~Approx. 1.5×10 11 In one embodiment, the effective concentration is about 1.5×10 9In another embodiment, the effective concentration is about 1.5×10 10 In another embodiment, the effective concentration is about 1.5×10 11 In yet another embodiment, the effective concentration is about 1.5×10 12 In another embodiment, the effective concentration is about 1.5×10 13 In another embodiment, the effective concentration is about 1.5×10 14 It may be desirable to use the lowest effective concentration to reduce the risk of undesirable effects. Still other dosages within these ranges can be selected by the attending physician, taking into account the physical condition of the subject, preferably the human, being treated, the age of the subject, the particular type of cancer, and, if the cancer is progressive, the extent to which the cancer has progressed.

[0123] Effective target concentration of rhabdovirus in TCID 50 It can be expressed as follows: TCID 50 can be determined, for example, by using the Spearman-Karber method. Desirably, the range is 1×10 8 / mL~1×10 14 / mL TCID 50 Preferably, the effective target concentration is about 1×10 9 ~1×10 12 / mL, more preferably about 1 × 10 9 ~1×10 11 In one embodiment, the effective target concentration is about 1×10 10 / mL. In a preferred embodiment, the target concentration is about 5×10 10 In another embodiment, the effective target concentration is about 1.5×10 11 In one embodiment, the effective target concentration is about 1×10 12 In another embodiment, the effective target concentration is about 1.5×10 13 / mL.

[0124] The effective target dose of rhabdovirus is also TCID 50Preferably, the range is 1×10 8 ~1×10 14 TCID 50 Preferably, the target dose is about 1×10 9 ~1×10 13 , more preferably about 1×10 9 ~1×10 12 In one embodiment, the effective concentration is about 1×10 10 In a preferred embodiment, the effective concentration is about 1×10 11 In one embodiment, the effective concentration is about 1×10 12 In another embodiment, the effective concentration is about 1×10 13 It is.

[0125] The actual pharmacologic effective amount or therapeutic dose will of course depend on factors known to those skilled in the art, such as age and weight of patient, route of administration and severity of disease.In any case, rhabdovirus will be administered in a dosage and manner that allows it to deliver a pharmacologic effective amount based on the unique condition of the patient.

[0126] Generally, for the treatment and / or alleviation of the diseases, disorders and conditions mentioned herein, depending on the specific disease, disorder or condition to be treated, the effectiveness of the specific rhabdovirus, the specific route of administration and the specific pharmaceutical preparation or composition, the rhabdovirus will generally be administered, for example, twice a week, once a week or once a month in dose, but can vary considerably, especially depending on the above-mentioned parameters.Therefore, in some cases, it may be sufficient to use less than the minimum dose given above, while in other cases, it may be necessary to exceed the upper limit.When administering large amounts, it may be recommended to divide them into many smaller doses that are distributed throughout the day.

[0127] It will be understood that those skilled in the art can select and combine various alternatives in the method or process of the present invention without undue burden.For example, those skilled in the art can freely carry out any process steps as necessary and combine them with various setups, such as the specific method of clarifying different host cells, specific rhabdoviruses or cell cultures.Below, some preferred embodiments are described, but the method and process of the present invention should not be interpreted as being limited by these embodiments.

[0128] In one embodiment, the method or process of the invention allows the preparation, production and / or purification in / from a cell culture of a vesiculovirus, preferably a vesicular stomatitis virus, more preferably a vesicular stomatitis virus in which the glycoprotein G of the vesicular stomatitis virus has been replaced by the glycoprotein GP of Lymphocytic Choriomeningitis Virus (LCMV), the method or process of the invention comprising: (i) a. by adding a virus release agent directly to the cell culture, followed by clarification of the cell culture, preferably by depth filtration, tangential flow filtration or centrifugation, and recovering the virus harvest in the supernatant; or b. subjecting the cell culture to a filtration step, preferably depth filtration, followed by rinsing the filter, preferably the depth filter, with a virus releasing agent and recovering the virus harvest in the supernatant; Obtaining a virus harvest from the cell culture.

[0129] In another embodiment, the method or process of the invention allows the preparation, production and / or purification in / from a cell culture of a vesiculovirus, preferably a vesicular stomatitis virus, more preferably a vesicular stomatitis virus in which the glycoprotein G of the vesicular stomatitis virus has been replaced by the glycoprotein GP of Lymphocytic Choriomeningitis Virus (LCMV), the method or process of the invention comprising: (i) a. adding a virus release agent directly to the cell culture, where the virus release agent is a solid salt or an aqueous salt solution, followed by clarification of the cell culture, preferably by depth filtration, tangential flow filtration or centrifugation, and recovering the virus harvest in the supernatant; or b. subjecting the cell culture to a filtration step, preferably depth filtration, followed by rinsing the filter, preferably a depth filter, with a virus releasing agent, where the virus releasing agent is a saline solution, and recovering the virus harvest in the supernatant; Obtaining a virus harvest from the cell culture.

[0130] In another embodiment, the method or process of the invention allows the preparation, production and / or purification in / from a cell culture of a vesiculovirus, preferably a vesicular stomatitis virus, more preferably a vesicular stomatitis virus in which the glycoprotein G of the vesicular stomatitis virus has been replaced by the glycoprotein GP of Lymphocytic Choriomeningitis Virus (LCMV), the method or process of the invention comprising: (i) a. by adding a virus release agent directly to the cell culture, where the virus release agent is a solid salt or an aqueous salt solution, increasing the salt concentration in the cell culture, followed by clarification of the cell culture, preferably by depth filtration, tangential flow filtration or centrifugation, and recovering the virus harvest in the supernatant; or b. subjecting the cell culture to a filtration step, preferably depth filtration, followed by rinsing the filter, preferably a depth filter, with a virus releasing agent, where the virus releasing agent is a saline solution, and recovering the virus harvest in the supernatant; Obtaining a virus harvest from the cell culture.

[0131] In another embodiment, the method or process of the invention allows the preparation, production and / or purification in / from a cell culture of a vesiculovirus, preferably a vesicular stomatitis virus, more preferably a vesicular stomatitis virus in which the glycoprotein G of the vesicular stomatitis virus has been replaced by the glycoprotein GP of Lymphocytic Choriomeningitis Virus (LCMV), the method or process of the invention comprising: (i) a. adding a virus releasing agent directly to the cell culture, where the virus releasing agent is a solid salt or an aqueous salt solution, by increasing the salt concentration in the cell culture by at least about 0.01M, 0.05M, 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, or 0.5M, followed by clarifying the cell culture, preferably by depth filtration, tangential flow filtration, or centrifugation, and recovering the virus harvest in the supernatant; or b. subjecting the cell culture to a filtration step, preferably depth filtration, followed by rinsing the filter, preferably a depth filter, with a virus releasing agent, where the virus releasing agent is an aqueous salt solution having a concentration of at least about 0.01M, 0.05M, 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, or 0.5M, and recovering the virus harvest in the supernatant; Obtaining a virus harvest from the cell culture.

[0132] In another embodiment, the method or process of the invention allows the preparation, production and / or purification in / from a cell culture of a vesiculovirus, preferably a vesicular stomatitis virus, more preferably a vesicular stomatitis virus in which the glycoprotein G of the vesicular stomatitis virus has been replaced by the glycoprotein GP of Lymphocytic Choriomeningitis Virus (LCMV), the method or process of the invention comprising: (i) a. adding a virus releasing agent directly to a cell culture, wherein the virus releasing agent is a solid salt or an aqueous salt solution, increasing the salt concentration in the cell culture to about 0.01 M to about 5 M, about 0.05 M to about 5 M, about 0.1 M to about 5 M, about 0.15 M to about 5 M, about 0.2 M to about 5 M, about 0.25 M to about 5 M, about 0.3 M to about 5 M, about 0.35 M to about 5 M, about 0.4 M to about 5 M, about 0.45 M to about 5 M, or about 0.5 M to about 5 M, followed by clarifying the cell culture, preferably by depth filtration, tangential flow filtration, or centrifugation, and recovering the virus harvest in the supernatant; or b. subjecting the cell culture to a filtration step, preferably depth filtration, followed by rinsing the filter, preferably the depth filter, with a virus releasing agent, where the virus releasing agent is an aqueous salt solution having a concentration of about 0.01M to about 5M, about 0.05M to about 5M, about 0.1M to about 5M, about 0.15M to about 5M, about 0.2M to about 5M, about 0.25M to about 5M, about 0.3M to about 5M, about 0.35M to about 5M, about 0.4M to about 5M, about 0.45M to about 5M, or about 0.5M to about 5M, and recovering the virus harvest in the supernatant; Obtaining a virus harvest from the cell culture.

[0133] In a further preferred embodiment, any of the preceding embodiments further comprises: (ii) (optionally) reducing the salt concentration of the harvest obtained after step (ia) or (ib), preferably by dilution, diafiltration or dialysis; (iii) (optionally) treating the rhabdovirus collection with a DNA degrading nuclease, preferably benzonase or salt active nuclease; (iv) capturing the rhabdovirus by loading the solution obtained after any of steps (i) to (iii) onto a cation exchanger, preferably a monolith, resin or membrane, more preferably a monolith adsorbent; (v) eluting the rhabdovirus and recovering the eluate; (vi) (optionally) refining the rhabdovirus eluate of step (vii), preferably by size exclusion, multimodal size exclusion, ion exchange and / or tangential flow filtration; (vii) (optionally) exchanging the buffer of the refined rhabdovirus eluate, preferably by ultrafiltration and diafiltration or dialysis; (viii) (optionally) sterile filtering the rhabdovirus.

[0134] In a further related embodiment, any of the preceding embodiments further comprises: (ii) (optionally) reducing the salt concentration of the harvest obtained after step (ia) or (ib), preferably by dilution, diafiltration or dialysis; (iii) treating the rhabdovirus collection with a DNA degrading nuclease, preferably benzonase or salt active nuclease; (iv) capturing the rhabdovirus by loading the solution obtained after any of steps (i) to (iii) onto a cation exchanger, preferably a monolith, resin or membrane, more preferably a monolith adsorbent; (v) eluting the rhabdovirus and recovering the eluate; (vi) (optionally) refining the rhabdovirus eluate of step (vii), preferably by size exclusion, multimodal size exclusion, ion exchange and / or tangential flow filtration; (vii) (optionally) exchanging the buffer of the refined rhabdovirus eluate, preferably by ultrafiltration and diafiltration or dialysis; (viii) (optionally) sterile filtering the rhabdovirus.

[0135] In a further related embodiment, any of the preceding embodiments further comprises: (ii) (optionally) reducing the salt concentration of the harvest obtained after step (ia) or (ib), preferably by dilution, diafiltration or dialysis; (iii) treating the rhabdovirus collection with a DNA degrading nuclease, preferably benzonase or salt active nuclease; (iv) capturing the rhabdovirus by loading the solution obtained after any of steps (i) to (iii) onto a cation exchanger, preferably a monolith, resin or membrane, more preferably a monolith adsorbent; (v) eluting the rhabdovirus and recovering the eluate; (vi) refining the rhabdovirus eluate of step (vii), preferably by size exclusion, multimodal size exclusion, ion exchange and / or tangential flow filtration; (vii) (optionally) exchanging the buffer of the refined rhabdovirus eluate, preferably by ultrafiltration and diafiltration or dialysis; (viii) (optionally) sterile filtering the rhabdovirus.

[0136] In a further related embodiment, any of the preceding embodiments further comprises: (ii) (optionally) reducing the salt concentration of the harvest obtained after step (ia) or (ib), preferably by dilution, diafiltration or dialysis; (iii) treating the rhabdovirus collection with a DNA degrading nuclease, preferably benzonase or salt active nuclease; (iv) capturing the rhabdovirus by loading the solution obtained after any of steps (i) to (iii) onto a cation exchanger, preferably a monolith, resin or membrane, more preferably a monolith adsorbent; (v) eluting the rhabdovirus and recovering the eluate; (vi) refining the rhabdovirus eluate of step (vii), preferably by size exclusion, multimodal size exclusion, ion exchange and / or tangential flow filtration; (vii) exchanging the buffer of the refined rhabdovirus eluate, preferably by ultrafiltration and diafiltration or dialysis; (viii) (optionally) sterile filtering the rhabdovirus.

[0137] In a further related embodiment, any of the preceding embodiments further comprises: (ii) (optionally) reducing the salt concentration of the harvest obtained after step (ia) or (ib), preferably by dilution, diafiltration or dialysis; (iii) treating the rhabdovirus collection with a DNA degrading nuclease, preferably benzonase or salt active nuclease; (iv) capturing the rhabdovirus by loading the solution obtained after any of steps (i) to (iii) onto a cation exchanger, preferably a monolith, resin or membrane, more preferably a monolith adsorbent; (v) eluting the rhabdovirus and recovering the eluate; (vi) refining the rhabdovirus eluate of step (vii), preferably by size exclusion, multimodal size exclusion, ion exchange and / or tangential flow filtration; (vii) exchanging the buffer of the refined rhabdovirus eluate, preferably by ultrafiltration and diafiltration or dialysis; (viii) sterile filtering the rhabdovirus.

[0138] In a further related embodiment, any of the preceding embodiments further comprises: (ii) reducing the salt concentration of the harvest obtained after step (ia) or (ib), preferably by dilution, diafiltration or dialysis; (iii) treating the rhabdovirus collection with a DNA degrading nuclease, preferably benzonase or salt active nuclease; (iv) capturing the rhabdovirus by loading the solution obtained after any of steps (i) to (iii) onto a cation exchanger, preferably a monolith, resin or membrane, more preferably a monolith adsorbent; (v) eluting the rhabdovirus and recovering the eluate; (vi) refining the rhabdovirus eluate of step (vii), preferably by size exclusion, multimodal size exclusion, ion exchange and / or tangential flow filtration; (vii) exchanging the buffer of the refined rhabdovirus eluate, preferably by ultrafiltration and diafiltration or dialysis; (viii) sterile filtering the rhabdovirus.

[0139] In further relation to any of the specific embodiments described above, it is preferred to prepare, produce and / or purify a vesiculovirus, preferably a vesicular stomatitis virus, more preferably a vesicular stomatitis virus in which the glycoprotein G of vesicular stomatitis virus is replaced by the glycoprotein GP of Lymphocytic choriomeningitis virus (LCMV), in / from a cell culture comprising mammalian host cells, preferably mammalian host cells cultured in suspension, more preferably HEK293 cells.

[0140] In a preferred embodiment of any of the foregoing specific embodiments, the RNA genome of the vesicular stomatitis virus consists of a coding sequence that is at least 98%, at least 99%, or 100% identical to SEQ ID NO:12.

[0141] Working Example Example 1: API upstream manufacturing process Overview and batch definition Based on one thawed HEK293 cell vial (UPS01), the cells were expanded and cultivated in larger shake flask volumes (UPS02 to UPS06). UPS06 was subsequently seeded into the first bioreactor (UPS07), followed by the cells being cultivated and expanded in a second seed bioreactor (UPS08) and finally expanded in a production bioreactor (UPS09). One batch of crude harvest containing the drug substance was produced in batch mode by infecting the cells 48 or 56 hours after seeding into the production bioreactor (UPS10). During infection, the culture temperature was kept constant (process variant 1, 48 hours) or varied between 37.0°C and 34.0°C (process variant 2, 56 hours). Infection was performed with VSV-GP (wherein the glycoprotein G of the vesicular stomatitis virus is replaced by the glycoprotein GP of the lymphocytic choriomeningitis virus (LCMV)) or with VSV-GP-cargo, i.e. VSV-GP encoding an additional transgene, in particular a truncated CCL21(1-79) protein or a CD80-Fc fusion protein. In the following, all processes were performed with VSV-GP and with different VSV-GP-cargo variants (CCL21(1-79) and CD80-Fc fusion), which for readability will be referred to by the word VSV-GP(-cargo). Harvesting was performed 34±2 hours post-infection (UPS11). The total working volume of about 200 L was then subjected to downstream processing.

[0142] UPS01-Cell Thawing HEK293 cells from one Master Cell Bank (MCB) or Working Cell Bank (WCB) vial were thawed in a heat block and adjusted to culture medium. After a centrifugation step, the resulting cell pellet was resuspended in medium and incubated in a 125 mL shake flask.

[0143] UPS02~UPS06 - Cell growth in shake flasks HEK293 cells were grown in shake flasks for five subculture steps. When the inoculation criteria was reached, the next step was started, where the next larger shake flask volume was used for each new subculture step. If the maximum culture volume was exceeded, the remaining cells were discarded. For UPS07, as many as 2000 mL shake flasks were used as needed to cover the inoculation criteria of the wave-mixed bioreactor.

[0144] UPS07 - (First seed) Cell growth in bioreactor (optional) The last shake flask subculture (UPS06) was used to inoculate the first seed bioreactor for further cell expansion in batch mode. Wave bioreactors or stirred tank reactors are suitable for cell expansion. Before and during inoculation, the pH value was monitored by CO2 injection via the headspace. 2 The culture was controlled by adding 100% ethanol to the bioreactor. Headspace aeration with air was present during the entire culture period. Dissolved oxygen values ​​were controlled by gasified air and oxygen using an advanced controller in cascade regulation mode. The medium volume to be added was calculated depending on the inoculation dose and the viable cell concentration at the end of UPS06. The bioreactor was filled with the calculated medium volume. The medium was conditioned in the bioreactor for at least 2 hours without aeration and pH control. Just before inoculation, aeration was started and the medium was kept in the bioreactor under active pH control. The total conditioning time should not exceed 24 hours. Once the necessary conditions for inoculation of the bioreactor were met, the cell suspension was transferred to the bioreactor by pumping. If the cell suspension was not completely used during inoculation, the remaining cells are discarded.

[0145] UPS08-Cell Growth in Seed Bioreactor A 50 L disposable stirred tank bioreactor was used for the cultivation. Before and during inoculation, the pH value was controlled by CO via the sparger and headspace. 2was added to the bioreactor. After inoculation, low side pH control by base addition was activated.

[0146] As an alternative, glass bioreactors were used for the cultivation. Before and during inoculation, the pH value was controlled by CO2 injection via the sparger and headspace. 2 was added to the bioreactor. After inoculation, low side pH control by base addition was activated. Headspace aeration with air was present during the entire cultivation period. % DO was controlled by the water oxygen input and regulated by an advanced controller.

[0147] For inoculation, the viable cell concentration of the cell suspension contained in the wavemixed bioreactor was determined. Depending on the required cell concentration in the bioreactor, the inoculation volume and the medium volume that had to be added were calculated. The bioreactor was filled with the calculated medium volume. The medium was conditioned in the seed bioreactor for at least 2 hours without aeration and pH control. At the time of inoculation, aeration was started and the medium was kept in the seed bioreactor under active pH control. The total conditioning time should not exceed 24 hours. Once the necessary conditions for inoculation of the bioreactor were met, the cell suspension was transferred to the bioreactor by pumping. If the cell suspension was not completely used during inoculation, the remaining cells were discarded.

[0148] Antifoam strategy (optional): To prevent foam formation during cultivation, an antifoam agent can be added to the culture using a time-dependent pump profile that starts immediately after inoculation. Thus, the antifoam agent shall be administered directly to the cell suspension every 6 hours, with the pump running for 1 second each time. The volume of antifoam agent added is recorded by the process control system.

[0149] UPS09 - Cell Growth in Production Bioreactors A 200 L disposable stirred tank bioreactor was used for production. Before and during inoculation, the pH value was controlled by CO via the sparger and headspace.2 The pH was controlled by adding 0.1% NaCl. After inoculation, low side pH control by base addition was activated. Headspace aeration with air was present during the entire cultivation period. The % dissolved oxygen was controlled by the water oxygen input and regulated by a 4 gas mixing module using an advanced controller.

[0150] For inoculation, the viable cell concentration of the cell suspension contained in the seed bioreactor was determined. Depending on the required cell concentration in the bioreactor, the inoculation volume and the medium volume that had to be added were calculated. The bioreactor was filled with the calculated medium volume. The medium was conditioned in the production bioreactor for at least 2 hours without aeration and pH control. Just before inoculation, aeration was started and the medium was kept in the seed bioreactor under active pH control. The total conditioning time shall not exceed 24 hours. Once the necessary conditions for inoculation of the bioreactor were met, the cell suspension was transferred to the bioreactor by pumping. If the cell suspension is not used completely during inoculation, the remaining cells are discarded. To prevent foam formation during the culture, an antifoam agent can be added to the culture using a time-dependent pump profile that is started immediately after inoculation. Thus, the antifoam agent shall be administered directly to the cell suspension every 6 hours, with the pump running for 1 second each time. The volume of antifoam agent added was recorded by the process control system.

[0151] UPS10-Virus Infection Variant I Infection with VSV-GP (-cargo) was performed 48 h after cell inoculation in the production bioreactor stage. This resulted in a 1.0 × 10 6 ~2.0×10 6A viable cell concentration of 1000 cells / mL was obtained. The virus was pre-diluted in conditioned cell culture medium. After meeting the criteria for infecting the production bioreactor, the control cells were seeded into two 250 mL shake flasks and the cell suspension was removed from the production bioreactor. The shake flasks were incubated and the control cells were operated in the same way as the production cells until the production bioreactor was harvested. At the time of infection, the culture temperature of the production bioreactor would be constant in the range of 36.0-39.0 °C.

[0152] UPS10-virus infection variant II Infection with VSV-GP(-cargo) was performed 56 h after cell inoculation in the final bioreactor stage, resulting in a 1.0 × 10 6 ~2.0×10 6 A viable cell concentration of 1000 cells / mL was obtained. The virus was pre-diluted in conditioned cell culture medium. After meeting the criteria for infecting the production bioreactor, the control cells were seeded in two 250 mL shake flasks and the cell suspension was removed from the production bioreactor. The shake flasks were incubated and the control cells were operated in the same way as the production cells until the production bioreactor was harvested. During infection, the culture temperature of the production bioreactor was varied between 37.0 and 34.0 °C. In case of excessive foam formation during infection, for example to prevent clogging of the aeration tube, an antifoam agent can be added directly to the bioreactor. The volume of antifoam agent added was recorded by the process control system.

[0153] UPS11-Collection Harvesting was performed 34±2 hours post-infection. The harvesting procedure was initiated by adding 4M-5M NaCl stock solution to the infected cell broth. This resulted in an increased concentration of approximately 0.2M NaCl, followed by an incubation period of at least 10-30 minutes before initiating transfer and cell separation. Unless otherwise stated, all parameters were controlled as in the culture. After the specified incubation time, temperature, DO and pH controls were deactivated. The infected cell broth was then transferred to downstream processing.

[0154] Process Performance Data Exemplary results from process monitoring and in-process analytical control of the VSV-GP-CCL21(1-79) collection produced by the described process are summarized below: Process performance between the different VSV-GP(-cargo) variants (no cargo, CCL21(1-79) or CD80-Fc fusion) was comparable. μ=0.7d in production bioreactor -1 highest cell-specific proliferation rate; In collections with >90% viability, 2–3 × 10 per milliliter 6 Total cell number concentration of cells; The infectious virus content in the final cell culture supernatant was approximately 2×10 9 TCID 50 / mL, and the viral genome content in the cell culture supernatant was approximately 3–3.5 × 10 10 TCID 50 / mL.

[0155] Example 2: Drug Substance Downstream Manufacturing Process Overview and batch definition The production of VSV-GP-(cargo) was based on one batch production. All downstream unit operations were performed in one cycle without any splitting and pooling steps. The salt-treated harvest (UPS11) was clarified by depth filtration or tangential flow microfiltration followed by a nuclease digestion step. The clarified harvest was further diluted and then purified by monolithic cation exchange chromatography in bind-elute mode (DPS03). At this point, a retention step was performed. In this case, the intermediate was stored overnight. On the second downstream processing day, the intermediate was purified by multimodal chromatography in flow-through mode (DPS04). The flow-through was collected and processed by an ultrafiltration / diafiltration step (DPS05). The final filtration and formulation step (DPS06) resulted in a single batch that was dispensed into cell culture bottles, packaged and frozen at -80°C (-86°C to -70°C).

[0156] Collection variant I - Depth filtration A depth filtration step was performed to clarify the bulk harvest. Thus, the VSV-GP (-cargo)-containing crude harvest was pumped from the STR® 200 bioreactor through a 3M™ Zeta Plus™ Encapsulated System depth filtration capsule into a disposable bag. The maximum pressure was 0.9 bar. The clarification was performed at room temperature. The 3M™ Zeta Plus™ Encapsulated System depth filtration capsule was disinfected with 0.5 M NaOH for disposal.

[0157] Collection Variant II - Tangential Flow Microfiltration A tangential flow microfiltration step was performed to clarify the bulk harvest. Therefore, the disposable bioreactor was connected to a microfiltration module equipped with 0.65 μm mPES hollow fiber membranes with an effective length of 41.5 cm and an inner fiber diameter of 0.75 mm. Filtration was performed for 2100 s. -1The microfiltration was carried out at a constant retentate flow rate, with a shear rate of 0.01 mm and a maximum transmembrane pressure of 0.7 bar or less. Microfiltration was carried out until the inlet pressure rose to 0.7 bar or more. 2-5 liters of concentrated cell suspension were expected to remain in the bioreactor vessel. The permeate was collected in a 500 L disposable bag. Temperature and pH were controlled at 37°C and pH 7.1 until the probe was no longer in contact with the collection.

[0158] DPS02-Enzyme Digestion Process step DPS02 is the enzymatic digestion of the filtrate from DPS01, performed with SAN-HQ (ArcticZymes®) endonuclease. Inside the laminar flow in the USP compartment, the SAN-HQ endonuclease stock solution was equilibrated to room temperature and diluted in a separate disposable bag with buffer (50 mM Tris, pH 7.5). Magnesium chloride was added as a cofactor for the enzyme. The digestion buffer was added continuously to the intermediate (clarified harvest). The resulting intermediate was mixed for >10 min and incubated at room temperature for >20 min. After nuclease treatment, the intermediate was diluted 1:2 with 0.1 M Tris buffer to the defined pH and conductivity values. The aim of the enzymatic digestion step is the removal of nucleic acids (DNA and RNA) present in the suspension.

[0159] Clarified harvest level bioburden reduction strategies - Variation I Bioburden reduction filtration using 0.45 μm / 0.2 μm Sartopore® 2 MaxiCaps® Size 1 (filters connected in series) was performed to reduce putative bioburden contamination during the harvesting process. The filter can be connected in series to the permeate port of a depth filter or microfiltration system. The filter was replaced when the transmembrane pressure was 0.9 bar or higher. After filtration, the 0.45 μm / 0.2 μm Sartopore® 2 MaxiCaps® Size 1 filter was removed from the assembly and tested for integrity.

[0160] Clarified harvest level bioburden reduction strategies-Variation II To reduce putative bioburden contamination during the collection process, bioburden reduction filtration using 5 μm polypropylene filters Kleenpak Nova 10'' Profile II 0.5 μm or 0.45 μm / 0.2 μm Sartopore® 2MaxiCaps® Size 1 was performed. Filtration was performed after nuclease treatment and after dilution. Filters were replaced if transmembrane pressure was 0.9 bar or higher. After filtration, filters were removed from the assembly and tested for integrity.

[0161] DPS03 - Cation exchange chromatography Process step DPS03, CIMmultusTM SO 3 - The capture step was performed by cation exchange chromatography on the monolith. The binding buffer contained 50 mM Tris, pH 7.5, optionally supplemented with arginine at various concentrations. Chromatography was performed in bind / elute mode with step elution. VSV-GP (-cargo) purification was performed at room temperature. The eluates were collected in one bag. Loading was performed at a flow rate of 5 or 10 CV / h. The monolith was washed with 10 CV or 50 CV and a volumetric flow rate of 5 CV / h or 10 CV / h. Elution was performed at a flow rate of 120 CV / h or 10 CV / h. The collected eluates were stored overnight at 2-8 °C. The purpose of this step was to concentrate the virus and remove impurities (specifically residual host cell DNA and host cell proteins).

[0162] DPS04-Multimodal Size Exclusion Chromatography Process step DPS04 was a multimodal chromatography process step carried out with an Akta™ preparative gradient using a ReadyToProcess™ Capto® Core 700 with a bed height of 20 cm and a bed volume of 2.5 L. Chromatography was carried out in flow-through mode. The resin was conditioned using the elution buffer of the cation exchange chromatography. Before the polishing step, the capture eluate was incubated at room temperature for at least 30 minutes. VSV-GP (-cargo) polishing was carried out at room temperature and at a constant speed of 42 cm / h. The flow-through was collected in one bag. The purpose of this step was the final removal of trace impurities (specifically nuclease residues, HCPs).

[0163] DPS05-Ultrafiltration / Diafiltration The buffer exchange step was performed by two-step diafiltration, using an ultrafiltration module MiniKros® hollow fiber module (Repligen) with a cut-off of 750 kD. In the first step, constant volume diafiltration was performed, whereby the initial volume of the intermediate was diafiltered 6× against the diafiltration buffer at a constant cross-flow rate. This resulted in a diafiltration time of 3000 s. -1 In step 2, the diafiltered retentate was concentrated to a defined retentate volume at a constant cross-flow velocity. This resulted in a maximum shear stress of 3000 s -1 A maximum shear stress of 100 μg / ml was applied. The concentrated intermediate was discharged into a 5 L disposable bag. The purpose of this step was matrix exchange against the diafiltration buffer.

[0164] DPS06-Filling and freezing The diafiltration retentate was filtered through 0.45 μm / 0.2 μm filters (Sartopore® MidiCaps®) and collected in bags or bottles as a suspension. The final primary packaging material was aseptically connected to the filled bags / bottles by pumping. The bottles were then vacuum sealed and packed in plastic foil. The suspension was then frozen and stored at -80°C. The purpose of the 0.2 μm filter was for final bioburden reduction. The freezing process allowed for storage conditions of the drug substance suspension until further processing.

[0165] Process Performance Data Exemplary results from process monitoring and in-process analytical control of VSV-GP (-cargo) harvest for drug substance manufactured with the described process using variant I at any process step are summarized in Table 1 and Figures 2-3. The impurity reduction meets the high demands and standards of oncolytic virus formulations. The impurity reduction performance from upstream to the final drug substance corresponds to a log reduction value of about 3.5 for host cell proteins (<0.4 μg / mL) and about 5.4 for host cell DNA (<3 ng / mL final concentration). This is far below the typical limits of common impurity restrictions in biopharmaceutical development (e.g. 10 ng hcDNA per dose according to WHO guidelines). The overall yield allows a highly concentrated virus dose sufficient for even the highest demands.

[0166] Table 1: Examples of infectious VSV-GP (-cargo) content in manufacturing unit operations, TCID of (centrifuged and salt-treated) initial culture supernatants 50 Recovery rate calculated based on [Table 1]

[0167] Example 3: Testing of various additives before harvest Figure 4 Lower harvest titers of VSV-GP were observed in the supernatant after clarification by centrifugation compared to the non-centrifuged harvest material. It was hypothesized that VSV-GP interacted with and bound to host cell membrane fragments. These fragments with bound VSV-GP would then be pelleted. Additives were proposed to disrupt the interactions between membrane fragments that would allow pelleting of the membrane fragments and sufficiently clear VSV-GP that remained "free" in the supernatant. The clarified harvest material was aliquoted into 15 mL fractions at 30 hours post-infection and additives were added at the final concentrations indicated in Figure 4. NaCl, CaCl 2 Samples containing 100 mM Tween and Pluronic were incubated at room temperature for 10 minutes and centrifuged at 355 g for 3 minutes. Samples containing benzonase and trypsin were incubated at 37°C for 30 minutes and centrifuged at 355 g for 3 minutes. Samples containing dextran sulfate were incubated under culture conditions for 18 hours and centrifuged at 355 g for 3 minutes. The supernatants were then analyzed using TCID 50 The highest recovery rates were observed for two concentrations of NaCl (0.2M and 1M) and 100 μg / mL dextran sulfate, indicating that these additives successfully released free virus particles into the supernatant. Other additives did not show significant improvement in titer after centrifugation.

[0168] Example 4: Further testing of different additives and concentrations Figure 5 Further additive testing was performed to investigate whether the titer of VSV-GP at harvest could be improved with alternative additives. Additives were selected based on how they interacted with the virus: potassium chloride was used to test alternative monovalent ions, glycine increases solubility and can create exclusion pressure, and L-arginine relieves hydrophobic interactions and has a 2+ / 1- charge at neutral pH. The clarified harvest material was aliquoted into 15 mL fractions at 30 hours post-infection and additives were added at the indicated final concentrations, incubated for 10 minutes at room temperature and centrifuged at 355 g for 3 minutes. The supernatants were then assayed for TCID 50and qPCR. The unprocessed crude harvest, not centrifuged, showed high titers of infectious VSV-GP, and interactions of VSV-GP with host cell debris did not inhibit the ability of VSV-GP to infect new cells, although large amounts of debris are a widely observed impurity that negatively impacts process performance in subsequent downstream processing steps. Of all the additives tested in Figure 5, only KCl showed improved infection recovery over NaCl. It was also observed that increasing the concentration of KCl improved VSV-GP release. However, since NaCl showed good release of VSV-GP and would be used for elution in cation exchange capture and for other process steps, it was decided that NaCl would be used as the additive for VSV-GP harvest.

[0169] Example 5: Testing the minimum NaCl concentration Figure 6 NaCl was chosen as an additive to release VSV-GP at harvest because it exhibits high infection recovery and will be present at various concentrations throughout the process. We investigated whether lower concentrations of NaCl, below 0.2M, could have a comparable virus release effect because it would reduce the amount of dilution buffer required to reduce the conductivity of the feed prior to capture of VSV-G using cation exchange chromatography, a salt-sensitive process step. At 30 hours post-infection, the clarified harvest material was aliquoted into 15 mL fractions, NaCl was added at the final concentrations shown in Figure 6, incubated at room temperature for 10 minutes, and centrifuged at 355 g for 3 minutes. The supernatant was then analyzed by TCID 50 The highest infection recovery was shown to be achieved using 0.2M NaCl, although lower concentrations also showed some efficacy. This concentration was therefore considered to be the most promising for high recovery of VSV-GP at harvest, while minimizing the dilution factor required to achieve capture of VSV-GP in the subsequent cation exchange step.

[0170] Example 6: Exemplary Process Performance Data Figure 7A+Figure 7B Here we present further exemplary performance data for infection of cells with VSV-GP and at a culture volume of 50 L (Figure 7A) or 4 L (Figure 7B) for the process detailed in Examples 1 and 2. Briefly, cells were cultured in suspension for 48 h, VSV-GP was added at an MOI of 0.0005, virus was harvested and dissociated from host cell debris at a TOH of 36 h by adding a final concentration of 0.2 M NaCL. VSV-GP was clarified to remove host cell debris using 0.65 μm hollow fiber microfiltration and treated with SAN-HQ nuclease to reduce hcDNA. The feed was prepared prior to chromatographic capture by 0.5 μm depth filtration and 1:2 dilution in binding buffer (final concentration 50 mM Tris, pH 7.5). The virus suspension was captured and concentrated using a cation exchange monolith, and elution was achieved using a salt step. The captured material was held overnight. This was followed by chromatographic refinement using multimodal size-exclusion CC700 resin. This step further purified VSV-GP from HCP and hcDNA that were not removed during capture. Finally, the refined eluate was diafiltered into formulation buffer and sterile filtered using a 0.2 μm filter.

[0171] Example 7: MgCl as an alternative additive during collection 2 Exam Figure 8 In addition to the excipients previously tested, magnesium chloride was added at harvest for the release of VSV-GP (-cargo) from infected cells. The effect of magnesium chloride as a divalent cation was investigated because it is highly soluble in water and exhibits similar ionic strength compared to sodium chloride at lower concentrations. Furthermore, the magnesium cation (Mg 2+) plays an important role as a cofactor during enzymatic digestion of nucleic acids for the removal of free DNA / RNA. Upon collection, the virus suspension was divided into 5 mL or 40 mL aliquots, incubated (10 min at 34°C) with various amounts of magnesium chloride (added 0.04M to 0.07M) as shown in Figure 8, and centrifuged at 1000g for 5 min. The supernatants were analyzed using a TCID 50 The NaCl-treated samples (0.2M spiked) were analyzed using GC qPCR. 2 (similar ionic strength compared to 100 nm) was performed in the same manner.

[0172] NaCl-treated crude harvest (control, 0.2 M spiked) was 2 Similar virus titers (TCID 50 and GC qPCR). 2 At lower concentrations of 1000 mg / mL, the release of VSV-GP (-cargo) was reduced. Magnesium chloride represents a possible alternative for virus release from HEK293 cells during harvest. The lower conductivity in the salt-treated virus suspension also reduces the amount of dilution buffer required to adjust the conductivity for the subsequent cation exchange chromatography step (capture).

[0173] Example 8: Filter flush after depth filtration for virus release using NaCl Figure 9

[0174] Apart from the addition of sodium chloride for VSV-GP(-cargo) release at harvest, we tested viral elution using filter flushing after depth filtration. In this study, we investigated whether VSV-GP(-cargo) could be released from HEK293 cells after being retained in the filter material.

[0175] At harvest, VSV-GP (-cargo) was incubated with benzonase for 30 min at 37° C. in a shaking incubator. Depth filtration (3M™ Zeta Plus™) for clarification of the bulk harvest was performed at a defined volumetric filter load (200 L / m 2 ), constant volume flux (200 LMH) and maximum pressure (prefilter) of 1.5 bar. After depth filtration, a filter flush with one-fifth (20%) of the filtration volume was performed using Tris buffer containing sodium chloride (0.25M and 0.5M NaCl). Figure 9 shows an excellent example of the interaction of VSV-GP (-cargo) with HEK293 cells when no additive was added at harvest. This is because the infectious virus titer in the clarified harvest is very low. Virus release using the first filter flush with 0.25M NaCl was already sufficient for total infectious virus recovery. A second filter flush with 0.5M NaCl released some additional virus, but most of the virus had already been "eluted" by the first filter flush. The amount of NaCl required for virus release using the filter flush is comparable to the amount of NaCl added at harvest (0.2M added). Despite good recovery of VSV-GP(-cargo) following depth filtration followed by filter flush, the addition of NaCl at harvest confirms the simplicity of the procedure.

[0176] Example 9: TCID 50 Exemplary Methods for Determining Cells and viruses BHK-21 cells (#603126(C13), CLS) were incubated in 5% CO 2 and cultured at 37°C. The medium (GMEM #21710082, Thermo) is supplemented with 8.7% FCS and 4.3% tryptose phosphate broth. BHK-21 cells are washed with PBS and detached from the cell culture flask by incubation with TrypLE™ Select Enzyme for 6-8 min at 37°C. Cells are harvested in medium, counted using Flex2 (nova biomedical), and seeded into 96-well plates.

[0177] TCID 50 Assay In a 96-well plate, 10 4 BHK-21 cells are seeded per well. After 24 hours, adherent cells are inoculated with 0.5 log of virus. 10 or dilution alone (negative control), and then incubated at 37°C, 5% CO 2 The wells are incubated at 4°C for 3 days. Bright field images of the cell culture wells are taken using a Cytation5 Multi-Mode Imaging Reader (BioTek) using a 4× objective. The imaged wells are assessed by eye (i.e., visually) as to whether they are CPE positive or negative. Final titer [TCID 50 The TCID50 / mL is calculated using the Spearman and Karber formula. For each virus sample, infections with serial dilutions are performed on the same day for a total of eight plates. Based on these eight replicates, the TCID50 50 Calculate / mL as above (single measurement).

Claims

1. 1. A method for producing a rhabdovirus in cell culture, comprising: (i) a. By adding a virus release agent directly to the cell culture, followed by clarification of the cell culture, preferably by depth filtration, tangential flow filtration or centrifugation, and collecting the rhabdovirus harvest in the supernatant. or b. subjecting the cell culture to a filtration step, preferably depth filtration, followed by rinsing the filter, preferably the depth filter, with a virus releasing agent and recovering the rhabdovirus harvest in the supernatant; Obtaining a rhabdovirus harvest from the cell culture, method.

2. 2. The method of claim 1, wherein the rhabdovirus is a vesiculovirus, preferably a vesicular stomatitis virus.

3. 3. The method of claim 2, wherein the glycoprotein G of vesicular stomatitis virus is replaced by the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV).

4. 4. The method of any one of claims 1 to 3, wherein the virus releasing agent in step (ia) is a solid salt or an aqueous salt solution and the virus releasing agent in step (ib) is an aqueous salt solution.

5. 5. The method of claim 4, wherein in step (ia) the salt concentration in the cell culture is increased by at least about 0.01 M, 0.05 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M or 0.5 M, and the aqueous salt solution in step (ib) has a concentration of at least about 0.01 M, 0.05 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M or 0.5 M.

6. 5. The method of claim 4, wherein the increase in salt concentration in the cell culture in step (ia) and the concentration of the aqueous salt solution in step (ib) are from about 0.01 M to about 5 M, 0.05 M to about 5 M, from about 0.1 M to about 5 M, from about 0.15 M to about 5 M, from about 0.2 M to about 5 M, from about 0.25 M to about 5 M, from about 0.3 M to about 5 M, from about 0.35 M to about 5 M, from about 0.4 M to about 5 M, from about 0.45 M to about 5 M, or from about 0.5 M to about 5 M.

7. The salts are NaCl, KCl, MgCl ２ , CaCl ２ , N.H. ４ Cl, sulfuric acid NH ４ , NH acetate ４ or NH bicarbonate ４ The method according to any one of claims 4 to 6, wherein

8. The method of any one of claims 1 to 3, wherein the virus releasing agent is an amino acid, preferably a polar, acidic or basic amino acid, more preferably arginine.

9. The method of any one of claims 1 to 3, wherein the virus releasing agent is a sulfated polysaccharide, preferably dextran sulfate.

10. In step (ia), the ionic strength of the cell culture is preferably increased by at least about 0.01 M, or at least about 0.05 M, or at least about 0.1 M, or at least about 0.15 M, or at least about 0.2 M, or at least about 0.25 M, or at least about 0.3 M, or at least about 0.35 M, or at least about 0.4 M, or at least about 0.45 M, or at least about 0.5 M, or from about 0.01 M to about 5 M, or from about 0.05 M to about 5 M, or from about 0.1 M to about 5 M, or from about 0.15 M to about 5 M, or from about 0.2 M to about 5 M, The method of any one of claims 1 to 3, wherein in ib), the filter is rinsed with an aqueous solution containing a virus-releasing agent having an ionic strength of at least about 0.01 M, or at least about 0.05 M, or at least about 0.1 M, or at least about 0.15 M, or at least about 0.2 M, or at least about 0.25 M, or at least about 0.3 M, or at least about 0.35 M, or at least about 0.4 M, or at least about 0.45 M, or at least about 0.5 M, or about 0.01 M to about 5 M, or about 0.05 M to about 5 M, or about 0.1 M to about 5 M, or about 0.15 M to about 5 M, or about 0.2 M to about 5 M.

11. The method according to any one of claims 1 to 10, wherein the rhabdovirus is produced in a mammalian host cell, preferably in a HEK293 cell.

12. The method of claim 11 , wherein the mammalian host cells are cultured in suspension.

13. (ii) (optionally) reducing the salt concentration of the harvest obtained after step (ia) or (ib), preferably by dilution, diafiltration or dialysis; (iii) (optionally) treating the rhabdovirus collection with a DNA-degrading nuclease, preferably benzonase or salt-active nuclease; (iv) capturing the rhabdovirus by loading the solution obtained after any of steps (i) to (iii) onto a cation exchanger; (v) eluting the rhabdovirus and collecting the eluate; (vi) (optionally) refining the rhabdovirus eluate of step (vii), preferably by size exclusion, multimodal size exclusion, ion exchange and / or tangential flow filtration; (vii) (optionally) exchanging the buffer of the refined rhabdovirus eluate, preferably by ultrafiltration and diafiltration or dialysis; (viii) (optionally) sterile filtering the rhabdovirus.

14. 14. The method of claim 13, wherein the cation exchanger is a monolith, a resin or a membrane.

15. The method of claim 14, wherein the cation exchanger is a monolithic adsorbent.

16. The method of any one of claims 1 to 15, wherein the rhabdovirus is formulated into a pharmaceutical composition.

17. 1. A method for purifying a rhabdovirus from a cell culture infected with the rhabdovirus, comprising: (i) a. By adding a virus release agent directly to the cell culture, followed by clarification of the cell culture, preferably by depth filtration, tangential flow filtration or centrifugation, and collecting the rhabdovirus harvest in the supernatant. or b. subjecting the cell culture to a filtration step, preferably depth filtration, followed by rinsing the filter, preferably the depth filter, with a virus releasing agent and recovering the rhabdovirus harvest in the supernatant; Obtaining a rhabdovirus harvest from the cell culture, method.

18. 18. The method of claim 17, wherein the rhabdovirus is a vesiculovirus, preferably a vesicular stomatitis virus.

19. 20. The method of claim 18, wherein the glycoprotein G of vesicular stomatitis virus is replaced by glycoprotein GP of lymphocytic choriomeningitis virus (LCMV).

20. 20. The method of any one of claims 17 to 19, wherein the virus releasing agent in step (ia) is a solid salt or an aqueous salt solution and the virus releasing agent in step (ib) is an aqueous salt solution.

21. 21. The method of claim 20, wherein in step (ia) the salt concentration in the cell culture is increased to at least about 0.01 M, 0.05 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M or 0.5 M, and wherein the aqueous salt solution in step (ib) has a concentration of at least about 0.01 M, 0.05 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M or 0.5 M.

22. 22. The method of claim 21, wherein the increase in salt concentration in the cell culture in step (ia) and the concentration of the aqueous salt solution in step (ib) are from about 0.01 M to about 5 M, from about 0.05 M to about 5 M, from about 0.1 M to about 5 M, from about 0.15 M to about 5 M, from about 0.2 M to about 5 M, from about 0.25 M to about 5 M, from about 0.3 M to about 5 M, from about 0.35 M to about 5 M, from about 0.4 M to about 5 M, from about 0.45 M to about 5 M, or from about 0.5 M to about 5 M.

23. The salts are NaCl, KCl, MgCl ２ , CaCl ２ , N.H. ４ Cl, sulfuric acid NH ４ , NH acetate ４ or NH bicarbonate ４ The method according to any one of claims 20 to 22, wherein

24. The method of any one of claims 17 to 19, wherein the virus releasing agent is an amino acid, preferably a polar, acidic or basic amino acid, more preferably arginine.

25. The method of any one of claims 17 to 19, wherein the virus releasing agent is a sulfated polysaccharide, preferably dextran sulfate.

26. In step (ia), the ionic strength of the cell culture is preferably increased by at least about 0.01 M, or at least about 0.05 M, or at least about 0.1 M, or at least about 0.15 M, or at least about 0.2 M, or at least about 0.25 M, or at least about 0.3 M, or at least about 0.35 M, or at least about 0.4 M, or at least about 0.45 M, or at least about 0.5 M, or from about 0.01 M to about 5 M, or from about 0.05 M to about 5 M, or from about 0.1 M to about 5 M, or from about 0.15 M to about 5 M, or from about 0.2 M to about 5 M, and step (i The method of any one of claims 17 to 19, wherein in b) the filter is rinsed with an aqueous solution containing a virus-releasing agent having an ionic strength of at least about 0.01 M, or at least about 0.05 M, or at least about 0.1 M, or at least about 0.15 M, or at least about 0.2 M, or at least about 0.25 M, or at least about 0.3 M, or at least about 0.35 M, or at least about 0.4 M, or at least about 0.45 M, or at least about 0.5 M, or about 0.01 M to about 5 M, or about 0.05 M to about 5 M, or about 0.1 M to about 5 M, or about 0.15 M to about 5 M, or about 0.2 M to about 5 M.

27. The method according to any one of claims 17 to 26, wherein the rhabdovirus is purified from a mammalian host cell, preferably from HEK293 cells.

28. 28. The method of claim 27, wherein the mammalian host cells are cultured in suspension.

29. (ii) (optionally) reducing the salt concentration of the harvest obtained after step (ia) or (ib), preferably by dilution, diafiltration or dialysis; (iii) (optionally) treating the rhabdovirus collection with a DNA-degrading nuclease, preferably benzonase or salt-active nuclease; (iv) capturing the rhabdovirus by loading the solution obtained after any of steps (i) to (iii) onto a cation exchanger; (v) eluting the rhabdovirus and collecting the eluate; (vi) (optionally) refining the rhabdovirus eluate of step (vii), preferably by size exclusion, multimodal size exclusion, ion exchange and / or tangential flow filtration; (vii) (optionally) exchanging the buffer of the refined rhabdovirus eluate, preferably by ultrafiltration and diafiltration or dialysis; (viii) (optionally) sterile filtering the rhabdovirus.

30. 30. The method of claim 29, wherein the cation exchanger is a monolith, a resin or a membrane.

31. 31. The method of claim 30, wherein the cation exchanger is a monolith adsorbent.

32. The method of any one of claims 17 to 31, wherein the rhabdovirus is formulated into a pharmaceutical composition.

33. Vesicular stomatitis virus, 32. A method according to claim 1, wherein the glycoprotein G of the vesicular stomatitis virus is replaced by the glycoprotein GP of the lymphocytic choriomeningitis virus (LCMV), the method being produced according to any one of claims 1 to 16 or purified according to any one of claims 17 to 32. Vesicular stomatitis virus.

34. 34. Vesicular stomatitis virus produced or purified according to claim 33, wherein the RNA genome of the vesicular stomatitis virus consists of a coding sequence at least 98%, at least 99% or 100% identical to SEQ ID NO:

12.

35. The amount of infectious particles is ５０ At least about 1 × 10 ９ , 2 × 10 ９ , 3×10 ９ , 4 × 10 ９ , 5×10 ９ , 6 × 10 ９ , 7 x 10 ９ , 8 x 10 ９ , 9 x 10 ９ Or at least about 1×10 １０ 35. A vesicular stomatitis virus produced or purified according to claim 33 or 34, wherein

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