Optimized Parvovirus H-1 Production

Abstract

The present invention provides a robust monoclonal master cell bank (MCB) for optimized production of H-1 parvovirus (H-1 PV), which is suitable for increasing infectious parvovirus production compared to the standard producer NB-324K mixed cells.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention provides a robust monoclonal master cell bank (MCB) for optimized production of H-1 parvovirus (H-1 PV), which is suitable for increasing the production of infectious parvovirus compared to the standard producer NB-324K mixed cells. [Background technology]

[0002] 2. Background of the Invention H-1PV belongs to the genus Protoparvovirus within the subfamily Parvovirinae of the family Parvoviridae (Cotmore et al., 2014). It consists of a non-enveloped icosahedral capsid with a diameter of 25 nm and contains a single-stranded DNA genome of approximately 5 kb length that encodes nonstructural proteins - notably NS1 (83 kDa) and NS2 (25 kDa) - as well as the capsid proteins VP1 (81 kDa) and VP2 (65 kDa). Another capsid protein, VP3 (63 kDa), is generated by post-translational cleavage of VP2 (Faisst et al., 1995; Halder et al., 2012; Hanson and Rhode, 1991; Toolan et al., 1960). Protoparvoviruses replicate in an S-phase-dependent manner and undergo a lytic cycle after infection of permissive cells (Burnett et al., 2006). The natural host of H-1PV is the rat, but the virus has recently attracted considerable interest because it replicates preferentially in transformed cells, including some human tumor cells. The virus has oncolytic and tumor suppressive properties, which have been shown in various cell culture and animal models (Nuesch et al., 2012; Rommelaere et al., 2010). In xenograft models, H-1PV has been shown to inhibit several human tumors, such as cervical tumors (Faisst et al., 1998; Li et al., 2013), pancreatic tumors (Angelova et al., 2009b; Grekova et al., 2011), breast cancer (Dupressoir et al., 1989), gliomas (Geletneky et al., 2010; Kiprianova et al., 2011), and lymphomas (Angelova et al., 2009a). Based on these preclinical proofs of concept, the first clinical trials (phase I / IIa) of H-1PV were initiated in 2011 for patients with recurrent glioblastoma multiforme (Geletneky et al., 2012).

[0003] To test and ultimately exploit the therapeutic potential of H-1PV, an efficient, simple, robust and reproducible virus production and purification process needs to be developed. Purification methods have been published for small-scale production by cesium chloride (Halder et al., 2012; Paradiso, 1981) or iodixanol (Wrzesinski et al., 2003; Zolotukhin et al., 1999) density gradient centrifugation.

[0004] Research into oncolytic protoparvoviruses has reached a stage of moving towards clinical implementation with the first Phase I / IIa trial of H-1PV in patients with recurrent resectable malignant gliomas (Geletneky et al., 2012). In addition, a Phase I / II clinical trial has been initiated in patients with inoperable metastatic pancreatic cancer (ClinicalTrials.gov Identifier: NCT02653313; manuscript in preparation).

[0005] These developments depend on the availability of robust procedures for protoparvovirus production and characterization. On the one hand, standardized procedures are necessary to generate preclinical data that can provide a proof of concept. On the other hand, standard operating procedures are also necessary to transfer the techniques and standards to certified facilities in charge of generating clinical batches and establishing their specification.

[0006] However, the use of well-characterized virus preparations and analytical methods is indeed essential to obtain valid and reproducible evidence of the therapeutic efficacy in oncology of oncolytic protoparvoviruses and is required by regulatory authorities.

[0007] H-1PV production is routinely performed in cell cultures such as human neonatal kidney cell NB-324K mixed cells (WO 2016 / 206807 A1).

[0008] However, there remains a need for new producer cells for H-1PV production that have higher mechanical stability, higher transfection efficiency, result in higher productivity and higher robustness against ammonia compared to NB-324K mixed cells, and result in more infectious H-1PV.

[0009] Therefore, the technical problem underlying the present invention is to optimize large-scale production of parvovirus by monoclonal MCB cells.

[0010] The solution to the technical problem is achieved by providing the embodiments characterized in the claims. Summary of the Invention [Means for solving the problem]

[0011] Detailed Description of the Invention The present invention relates to a method for preparing a single clone master cell bank (MCB) for parvovirus H-1PV production by growing NB-324K mixed clones (Shein & Enders, 1962; Solon L. Rhode, 1976; Tattersall and Bratton, 1983) in a suitable medium, preferably in MEM medium containing fetal bovine serum (FBS), L-glutamine and gentamicin, under suitable conditions (e.g., 37°C, 5% CO2). Single cell cloning occurred by seeding one cell (theoretical) per well in 96-well plates for 20-28 days, preferably about 24 days. 2-5, preferably 3 cell clones whose cells showed a single colony per well and rapid growth over time were initially selected. These were tested for good cell growth and productivity. The two first round single clones were then seeded again in 96 wells as single cells / well for 20-23 days, preferably about 21 days, and again tested for growth and productivity. The best selected single cell clone was then grown and passaged, for example in T-flasks and Roller bottles, under appropriate conditions in appropriate medium until passage 15 or more (e.g., passages 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41). The cells were harvested and the cell suspension was filled into cryovials. The resulting MCB was specified according to the requirements of the European Pharmacopoeia. This is necessary to ensure that the H-1PV production method complies with GMP requirements and that the final product meets quality requirements.

[0012] For optimal results, the "Master Cell Bank" is characterized by (a) morphology, (b) at least 80% viability, (c) sterility, (d) passage number 15, (e) lack of mycoplasma and mycobacterial contamination, (f) lack of SV40 production, (g) lack of adventitious factors, (f) lack of adventitious viral contamination, (g) identity, (h) lack of tumorigenicity, and (i) oncogenity.

[0013] The present invention also relates to a method for preparing a Master Seed Virus (MSV) composition, the method comprising: (a) preparing a master cell bank from NB-324K mixed clonal cells as described above; (b) transfecting the master cell bank with sequenced pUC19ΔHindIII / H1 plasmid DNA (Kestler et al., 1999) by an appropriate method (e.g., the calcium phosphate transfection method; Graham and Van der Eb, 1973). (c) To generate MSV pUC19ΔHindIII / H1 At least two rounds of infection of MCB cells with plasmid DNA Includes.

[0014] The present invention further relates to a method for producing a parvovirus H-1PV formulation, the method comprising: (a) providing a master cell bank as described above; (b) Preseeding MCB cells (c) Appropriate cell density, e.g., 2.0-5.0x10 4 cells / cm 2 The cells were transfected with a master seed virus (MSV) at an appropriate MOI, for example, 0.5 to 5x10 -2 infecting with PFU / cell; (d) growing the cells for about 2-6 days, harvesting the cells 2-6 days post-infection and obtaining a cell pellet by centrifugation; (e) subjecting the resuspended cell pellet to a mechanical, physical or chemical cell lysis method to obtain a parvovirus-containing cell lysate; (f) clarifying the parvovirus harvest by filtration; and (g) subjecting it to DNAse treatment; (h) buffer exchange for chromatography preparation; (i) Chromatography for the exclusion of empty particles and most impurities, e.g., anion exchange chromatography; (j) buffer exchange and concentration by desalting columns or tangential flow filtration; (k) Final formulation in Visipaque / Ringer or other formulation solution Includes.

[0015] The term "cell culture" refers to the maintenance of cells in an artificial in vitro environment. The medium of the present invention can be used to culture adherent NB-324K cells.

[0016] The term "cultivation" refers to the maintenance of cells in vitro under conditions that favor the proliferation, differentiation or continued viability of the cells in an active or quiescent state.

[0017] The phrase "cell culture medium" refers to a nutrient solution for culturing cells.

[0018] The term "cell bank" refers to a collection of suitable containers whose contents are of homogenous composition stored under defined conditions, with each container representing an aliquot of a single pool of cells.

[0019] The term "Master Cell Bank (MCB)" or "Master Cell Seed (MCS)" (both terms may be used interchangeably) refers to a collection of cells of uniform composition derived from a single source. In particular, it is ScNB-324K (synonym: MCS NB 324K) deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under the Budapest Treaty.

[0020] Those skilled in the art know the general conditions for growing master cell lines and for infecting cells with parvoviruses. Usually, cells are cultured in minimal essential medium (MEM) with heat-inactivated fetal bovine serum (e.g., FBS 5%) at 37°C, e.g., in a 5% CO2 atmosphere. Preferably, the medium should be supplemented with antibiotics (e.g., penicillin, streptomycin, gentamicin) and / or nutrients, e.g., L-glutamine.

[0021] In a preferred embodiment of the present invention, the cell density is 2.5x10 3 ~1x10 5 cells / cm 2 It is.

[0022] In a further preferred embodiment of the method of the present invention, virus production is carried out in a single-use cell culture system, preferably a 10-layer cell STACK® (CS) chamber. Further upscaling can be achieved, for example, using a 40-layer CS chamber or carrier system.

[0023] Preferably, for harvesting, the culture medium is aspirated and the infected cells are treated with a suitable buffer and / or enzyme or detergent, e.g. PBS-EDTA, Tris buffer, Tween or trypsin. The detached cells and, if appropriate, the medium supernatant are preferably centrifuged, preferably at 5,000xg, for about 5 minutes or filtered to obtain a cell pellet. The skilled person knows suitable mechanical, chemical or physical methods for releasing the parvovirus from the producer somatic cells. Preferably, this can be done by freeze / thaw cycles, sonication and / or enzyme / detergent treatment. The skilled person also knows suitable methods for sonicating the cells followed by DNAse treatment. For example, the cells can be sonicated for a sufficient time at 30-70 W and DNAse treatment is usually performed for 10-60 minutes at 37°C with 10-80 U / ml DNAse.

[0024] As mentioned above, the final H-1PV formulation is preferably in Visipaque / Ringer as the carrier. According to a preferred embodiment of the invention, the carrier is 73.62% Visipaque / Ringer. TM 320 (GE Healthcare) and iodixanol in Ringer's solution, which is prepared by mixing 26.38% Ringer's solution. TM 320 (GE Healthcare) contains 652 mg / ml iodixanol (= 65.2% iodixanol), so the iodixanol concentration after mixing with Ringer's solution is 48%. "Iodixanol" is a synonym of "Visipaque" (for human injection use) or "Iodixanolum" (research grade). The IUPAC name is 5-[acetyl-[3-[N-acetyl-3,5-bis(2,3-dihydroxypropylcarbamoyl)2,4,6,-triiodoanilino]2-hydroxypropyl]amino]-1-N,3,N-bis(2,3-dihydroxypropyl)-2,4,6-triiodobenzene-1,3-dicarboxamide. The CAS number is 92339-11-2. It is also a well-known contrast agent for CT imaging.

[0025] As shown in the examples below, the single clone MCB (ScNB-324K) exhibits distinct advantages over NB-324K mixed cells: keratins are more profoundly expressed in the single clone MCB cells, resulting in higher mechanical stability, transfection efficacy with MCB cells is greater, resulting in higher infectious productivity, and the single clone MCB cells are more resistant to ammonia.

[0026] The following examples are intended to illustrate, but not limit, the invention. While such examples are typical of those that might be used, other methods known to those skilled in the art may alternatively be used. [Brief description of the drawings]

[0027] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1: Generation of master cell bank and master seed virus for H-1PV production. NB-324K mixed clone cells were subjected to two rounds of single colony selection in MEM, 5% FBS, 2% L-glutamine and 0.2% gentamicin, and the selected clone D8-G3 was first used to establish a research cell seed (RCS) and then a master cell bank (MCB; passage 15 or more) for H1-PV production. For master seed virus (MSV) generation, single clone MCB cells were transfected with sequenced pUC19ΔHindIII / H1 plasmid DNA. After two rounds of MCB cell infection with H-1PV, the final passage was defined as MSV. For pharmaceutical production, MCB working cell bank cells are infected with MSV (or the corresponding working seed virus (WSV)). [Diagram 2] Figure 2: Two rounds of single-cell clone selection for H-1PV GMP production. First round of selection (A). Clone D8 (4.07E+03 ± 2.88E+03 PFU / cell), clone E6 (4.86E3 ± 3.27E+02 PFU / cell) and C5 (5.0E+03 ± 1.05E+03 PFU / cell) (n=2) show the same productivity, comparable to the NB-324K mixed clone (6.9E3 PFU / cell). Moreover, the generation time of the three selected clones (approximately 33-35 h) is also similar to that of the mixed clone NB-324K (approximately 32 h; not shown), where clone D8 has the best generation time of 32.9 h. As a consequence, in further experiments it was decided to work with clone D8. Second round of selection (B). The generation time of both mixed clones D8-G3 and D8-F7 is 50 h. The productivity of clone D8-G3 is better than clone D8-F7 and similar to the mixed clone NB-324K. As a result, it was decided to establish a research cell bank and a master cell bank (MCB) using the D8-G3 clone. The generation of the established MCB cell line is about 30 h and is used for H-1PV production. [Diagram 3]Figure 3: Immunofluorescence comparison of mixed clonal NB-324 and single clonal MCB cells (AC). NB-324K and MCB cells were stained with CH / HK keratin antibodies to mark intermediate keratins and DAPI for nuclear staining. Nuclei counting and evaluation of total intensity were performed with ImageJ software (https: / / imagej.nih.gov / ij / ) and evaluated with a custom-developed macro (provided by Dr. Damir Krunic, DKFZ Light Microscopy Facility). Keratin plays a major functional role in the integrity and mechanical stability of both single epithelial cells and epithelial tissues via cell-cell contacts (Moll et al, 2008; The human keratins: biology and pathology, Histochem Cell Biol, 129:705-733). Single clonal MCB cells showed 2.7x higher keratin signal intensity / cell, indicating higher mechanical stability of single clonal MCB cells compared to NB-324K mixed clonal cells. This mechanical stability may play a role in a robust upgrading process. [Figure 4A] Figure 4: Higher transfection efficiency of single clone MCB cells. NB-324K and MCB cells were transfected with H-1PV producing plasmid clones using the calcium phosphate method. Single clone MCB shows the advantage of higher transfection efficacy compared to mixed NB-324K cells (A). [Figure 4B] Figure 4: Higher transfection efficiency of single clone MCB cells. NB-324K and MCB cells were transfected with H-1PV producing plasmid clones using the calcium phosphate method. The genome-containing particles to infectious particles ratio (GP / PFU) was lower in single clone MCB (A), indicating fewer defective particles and more infectious particles in the product. [Diagram 5]Figure 5: Higher tolerance to ammonia of single clone MCB cells compared to NB-324K mixed cells. Cells were treated with different ammonia concentrations and counted after 5 days of growth. Single clone MCB cells show higher tolerance up to 20 mM ammonia compared to NB-324K mixed cells. This tolerance indicates an advantage of MCB cells during cell growth and H-1PV production process, since ammonia is the main product from glutamine metabolism. Its accumulation has been shown to reduce growth and negatively affect metabolism. Ammonia accumulation can disrupt the electrochemical cellular gradient and induce cytoplasmic acidification. Moreover, it can induce apoptosis in cultured cells (Cruz et al., 2000, “Effects of ammonia and lactate on growth, metabolism, and productivity of BHK cells”; Hassell et al., 1990, “Growth inhibition in Animal cell culture”). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Cells of a particular preferred single-cell MCB ("MCS NB324K human") were deposited under the Budapest Treaty at the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, on May 16, 2019 under the accession number DSM ACC3353. EXAMPLES

[0029] Example 1 material and method A. Immunofluorescence Test Immunofluorescence tests are based on the use of fluorescently marked antibodies that bind to specific antigens, by which specific extracellular and intracellular structures can be demonstrated. This principle is used for keratin staining with CH / HK-keratin antibodies, which mark intermediate keratins. For this, single clone MCB (ScNB-324K) and NB-324K mixed cells were seeded on glass slides in 6 cm cell dishes at 6E5 (Fig. 3C) and 1.2E6 (Fig. 3A, B) cells / dish. After an incubation time of 24 h, the cells on the slides were washed in PBS and fixed for 10 min in ice-cold methanol and then for 5 min in ice-cold acetone. For keratin detection, the slides were preblocked in PBS with 0.05% BSA and then incubated with CH / HK-keratin antibodies for 1 h. The slides were then washed in PBS with 0.1% Triton and then incubated with the secondary antibody Cy3gp (Dianova, Germany) for 1 h. Unbound secondary antibodies were removed by three washes in PBS with 0.1% Triton. Slides were immersed in ethanol and dried. Slides were embedded with FluoromountTM slide mounting medium (SigmaAldrich, Germany) containing 1 μg / ml DAPI (4',6-diamidino-2-phenylindole, SigmaAldrich, Germany) which stains the nuclei of cells. Analysis occurred with a fluorescence microscope BZ-9000 (Keyence, Germany). Nuclei counting and evaluation of total intensity were performed with ImageJ software (https: / / imagej.nih.gov / ij / ).

[0030] B. Cultivation, transfection and infection of monoclonal MCB and NB-324K mixed cells The single clone MCB (ScNB-324K) and NB-324K mixed cells are cultured in VP-SFM medium with 5% FBS and 4 mM L-glutamine at 37 °C. For transfection, the single clone MCB and NB-324K mixed cells are cultured at 3.6E4 cells / 75 cm in MEM medium with 5% FBS, 2 mM Glutamine, Pen / Strep. 2 Cells were seeded in flasks and transfected with the puc19ΔHindIII / H1 plasmid using the calcium phosphate transfection method. Cells were harvested on days 3, 4 and 5.

[0031] For identification of the toxic effects of ammonia on the single clone MCB and mixed NB-324K, single clone MCB and mixed NB-324K cells were cultured in Corning flasks (25 cm 2 5 mL VP-SFM with growth surface and 5% FBS and 4 mM glutamine medium / flask) or Nunc flasks (75 cm 2 The cells were cultured in 10 mL VP-SFM with a growth surface and 5% FBS and 4 mM glutamine medium / flask. Ammonium chloride (Pan Reac AppliChem, Germany) was added to the medium at different concentrations (0, 5, 10 and 20 mM, one flask per each set-up). On day 5, each set-up was counted in a Countess® Cell counter (Life Technologies, Germany).

[0032] For H-1PV production, NB-324K cells and a single clonal MCB (ScNB-324K) were cultured at 3.6E4 cells / cm 2Cells were seeded at 1000 x 1000 and immediately infected with H-1PV at a multiplicity of infection (MOI) of 0.01 plaque-forming units (PFU) per cell. Infected cells were incubated at 37°C under 5% CO2 for 4 days until the cytopathic effect (CPE), measured as the percentage of dead and detached cells observed under a light microscope, reached at least 30%. Cell density and viability were measured by staining live cells with 0.4% trypan blue (InvitrogenTM, Germany). Cells were counted using a Countess® Cell counter (Life Technologies, Germany) and morphology was observed microscopically.

[0033] For harvesting, the medium was aspirated and the infected cells were treated with PBS / 1 mM EDTA. The medium supernatant and detached cells were centrifuged at 5,000xg for 5 min. The pellet was washed with PBS, resuspended in Virus Tris / EDTA buffer containing 0.05 M Tris HCl, 0.5 mM EDTA, pH 8.7 (VTE) and subjected to three freeze / thaw cycles. After centrifugation at 5,000xg for 5 min, the cell debris was discarded. The cell lysate was then sonicated for 1 min at 48 W in a Sonorex Super 10 P ultrasonic homogenizer (Bandelin, Germany) and treated with DNAse (50 U / ml, Sigma, Germany) for 30 min at 37°C.

[0034] C. Plaque Formation Assay (PFU) Plaque assays were performed essentially as described by Tattersall and Bratton, 1983. NB-324K cells were grown in monolayer culture in MEM medium containing 5% FBS, 100 μg / ml penicillin, 100 μg / ml streptomycin, and 2 mM L-glutamine. They were infected with serial dilutions of H-1PV at 60% confluence and incubated for 1 h at 37 °C. The inoculum was then replaced with a bacto-agar overlay (1.7% in MEM containing 5% FBS). Four days after infection, viable cells were stained for 18-24 h by addition of 0.02% toluylene red staining solution (Sigma, Germany) with bacto-agar (Becton Dickinson, Germany). Dishes were incubated at 37 °C under 5% CO2. Plaque-forming units were counted on day 5 postinfection on a light box and their concentrations were expressed as PFU / ml.

[0035] D. Determination of genome-containing particles (GPs) The number of genome-containing viral particles (GP) was determined by Q-PCR essentially as previously described (Lacroix et al., 2010). Each well received 20 μl of reaction mixture containing 1x Premix Ex Taq™ (TaKaRa, France), 0.3 μM labeled NS1-TaqMan™ probe, 0.3 μM of each primer, and 3 μl template. Q-PCR was performed in a QuantStudio 3 Real-Time PCR System and results were processed with QuantStudio 3 Design and Analysis Software 1.4 (Applied Biosystems, Germany).

[0036] Example 2 Comparison of productivity of single cell clones for H-1PV GMP production selection A. First Round Selections Clone D8 (4.07E+03 ± 2.88E+03), clone E6 (4.86E3 E+02 ± 3.27E+02) and C5 (5.0E+03 ± 1.05E+03) (n = 2) show the same productivity comparable to the NB-324K mixed clone (6.9E3 PFU / cell). Moreover, the generation time of the three selected clones (approximately 33-35 h) was also similar to that of the mixed clone NB-324K (approximately 32 h; not shown). Clone D8 has the best generation time, which is 32.9 h.

[0037] As a result, it was decided to work with clone D8 in the 2. selection round due to its short generation time and similar productivity.

[0038] B. Second Round Selections The generation time of both mixed clones D8-G3 and D8-F7 is 50 h. The productivity of clone D8-G3 is better than clone D8-F7 and similar to the mixed clone NB-324K. As a result, it was decided to generate a master cell bank (MCB) using the D8-G3 clone. The generation of the established MCB cell line is about 30 h and is used for H-1PV production.

[0039] Example 3 Comparison of keratin intensity between mixed clonal NB-324K and single clonal MCB cells Comparison of immunofluorescence of mixed clonal NB-324 and single clonal MCB cells (AC) NB-324K and MCB cells were stained with CH / HK keratin antibody to mark intermediate keratins and DAPI for nuclear staining. Nuclei counting and evaluation of total intensity were performed with ImageJ software (https: / / imagej.nih.gov / ij / ). Single clone MCB cells (ScNB-324K) showed 2.7x higher keratin signal intensity / cell, indicating higher mechanical stability of single clone MCB cells compared to mixed clone NB-324K cells.

[0040] Example 4 Measurement of H-1PV transfection efficiency of NB-324K and MCB cells A. NB-324K and MCB cells were transfected with H-1PV producing plasmid clones using the calcium phosphate method. The single clone MCB shows an advantage over NB-324K mixed cells with approximately 1 log higher transfection efficiency.

[0041] B. The genome-containing particles to infectious particle ratio (GP / PFU) is lower (about 1 log) for the single clone MCB (ScNB-324K), indicating fewer defective particles and more infectious particles in the preparation.

[0042] Example 5 Effects of ammonia on mixed clone NB-234K and single clone MCB cells Cells were treated with different ammonia concentrations and counted after 5 days of growth. Single clone MCB cells (ScNB-324K) show higher intensity up to 20 mM ammonia compared to NB-324K mixed cells. This intensity indicates an advantage of MCB cells during cell growth and H-1PV production process, since ammonia is the main product of glutamine metabolism. Its accumulation has been shown to reduce growth and negatively affect metabolism. Ammonia accumulation can destroy the electrochemical cellular gradient and induce cytoplasmic acidification. Moreover, it can induce apoptosis in cultured cells. The aspects of the present invention include the following. Item 1 A method for producing a monoclonal master cell bank (MCB) for the production of parvovirus H-1PV, the method comprising the steps of: (a) growing NB-324K mixed clones in cell culture medium; (b) seeding cells into 96-well plates, ideally one cell per well, and growing them for 20-28 days; (c) selecting 2 to 5 cell clones obtained in step (b), wherein the cells show a single colony per well; (d) seeding at least two first round single clones into 96 wells as single cells / well for 20-23 days and testing for growth and production; (e) expanding and passaging the selected single cell clones with the best growth and production characteristics until they have been passaged for more than 15 passages; (f) harvesting and storing the obtained cells A method comprising: Section 2 The method of item 1, wherein the cell culture medium in step (a) is a MEM medium containing fetal bovine serum (FBS), L-glutamine and gentamicin. Section 3 The method of paragraph 1 or 2, wherein the expanding and passaging step of step (e) is carried out in T-flasks and roller bottles. Section 4 1. A method for producing a master seed virus (MSV) composition, the method comprising: (a) providing the master cell bank obtained in any one of items 1 to 3; (b) Transfecting the master cell bank with sequenced pUC19ΔHindIII / H1 plasmid DNA (c) infecting MCB cells with H-1PV plasmid DNA for at least two rounds to generate MSVs; A method comprising: Section 5 Item 5. The method according to item 4, wherein the transfection in step (b) is carried out by calcium phosphate transfection. Section 6 1. A method for producing parvovirus H-1 (H-1PV), the method comprising: (a) providing a master cell bank (MCB) obtained in any one of items 1 to 3; (b) preseeding MCB cells with the master seed virus (MSV) obtained in either of items 4 and 5; (c) 2.0~5.0 x 10 4 cells / cm 2 The cells with a cell density of 0.5 to 5 x 10 -2 infecting with MSV at an MOI of PFU / cell; (d) growing the cells for about 2-6 days, harvesting the cells 2-6 days post-infection, and centrifuging to obtain a cell pellet; (e) subjecting the resuspended cell pellet to a mechanical, physical or chemical cell lysis method to obtain a parvovirus-containing cell lysate; (f) clarifying the parvovirus harvest by filtration; and (g) subjecting it to DNAse treatment; (h) buffer exchange for chromatography preparation; (i) Chromatography for the exclusion of empty particles and most impurities; (j) buffer exchange and concentration by desalting columns or tangential flow filtration; (k) Final formulation in Visipaque / Ringer or other formulation solution A method comprising: Section 7 Item 7. The method according to item 6, wherein the chromatography in step (h) is anion exchange chromatography. Section 8 A monoclonal master cell bank (MCB) for producing parvovirus H-1, obtainable by the method according to any one of items 1 to 3. Section 9 9. A single clone master cell bank according to paragraph 8, comprising cells deposited at the DSMZ (=German Collection of Microorganisms and Cell Cultures) under the accession number DSM ACC3353 according to the Budapest Treaty.

[0043] List of References [Table 1-1] [Table 1-2]

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Claims

[Claim 1] A monoclonal master cell bank (MCB) for parvovirus H-1 production consisting of cells deposited at the DSMZ (=German Collection of Microorganisms and Cell Cultures) under the accession number DSM ACC3353 according to the Budapest Treaty.

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