Optimized parvovirus H1 production

Abstract

To provide optimized parvovirus H-1 production.SOLUTION: The present invention provides a production method for a single clone Master Cell Bank (MCB) for the production of parvovirus H-1, where the MCB comprises cells that have been deposited according to Budapest Treaty under the accession number DSM ACC3353 with the DSMZ (German Collection of Microorganisms and Cell Cultures), where the method comprises predetermined steps.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Field of Invention The present invention provides a robust single-clonal master cell bank (MCB) for the 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] 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 25 nm diameter non-enveloped icosahedral capsid and contains a single-stranded DNA genome approximately 5 kb long that encodes non-structural proteins—particularly NS1 (83 kDa) and NS2 (25 kDa)—as well as capsid proteins VP1 (81 kDa) and VP2 (65 kDa). Another capsid protein, VP3 (63 kDa), is produced 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 a phase-dependent manner and undergo post-infection lysis cycles in tolerant cells (Burnett et al., 2006). While the natural host of H-1PV is rat, this virus has recently attracted considerable interest because it preferentially replicates in transformed cells, including some human tumor cells. The virus possesses tumor-disintegrating and tumor-suppressing properties, which have been demonstrated in various cell cultures and animal models (Nuesch et al., 2012; Rommelaere et al., 2010). In xenograft models, H-1PV has been shown to suppress 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 this preclinical evidence of the concept, the first clinical trial of H-1PV (Phase I / IIa) was initiated in 2011 in patients with relapsed glioblastoma multiforme (Geletneky et al., 2012).

[0003] To test and ultimately utilize the therapeutic potential of H-1PV, it is necessary to develop efficient, simple, robust, and reproducible processes for virus production and purification. Purification methods have been published for small-scale production using cesium chloride (Halder et al., 2012; Paradiso, 1981) or iodixanol (Wrzesinski et al., 2003; Zolotukhin et al., 1999) density gradient centrifugation.

[0004] Research into tumor-disintegrating protoparvoviruses has progressed to clinical implementation, including the first Phase I / IIa trial of H-1PV in patients with recurrent, resectable gliomas (Geletneky et al., 2012). Furthermore, a Phase I / II clinical trial has been initiated in patients with unresectable 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. Standardized procedures are necessary to generate preclinical data that can provide evidence of concept. Standard operating procedures are also necessary to transfer the technology and standards to accredited facilities responsible for producing clinical batches and establishing their specifications.

[0006] However, the use of well-characterized virus preparation and analysis methods is truly essential and required by regulatory authorities to obtain valid and reproducible evidence of the therapeutic efficacy of tumor-disintegrating protoparvoviruses in oncology.

[0007] H-1PV production is routinely performed in cell cultures of human neonatal kidney cells, such as NB-324K mixed cells (Patent Document 1). [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] WO 2016 / 206807 A1 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] However, there remains a need for novel producible cells for H-1PV production that possess higher mechanical stability, higher transfection efficiency, higher productivity and ammonia-resistant (robustness) compared to NB-324K mixed cells, and produce more infectious H-1PV.

[0010] Therefore, the fundamental technical problem underlying the present invention is to optimize the large-scale production of parvovirus using single-clonal MCB cells. [Means for solving the problem]

[0011] The solution to the technical problem is achieved by providing the embodiments characterized by the claims.

[0012] 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 MEM medium containing fetal bovine serum (FBS), L-glutamine, and gentamicin, under suitable conditions (e.g., 37°C, 5% CO2). Single-cell cloning was performed by seeding one cell per well (theoretically) in a 96-well plate for 20–28 days, preferably about 24 days. Two to five, preferably three-cell clones, were initially selected, showing single colonies per well and rapid growth over time. These were then tested for good cell growth and productivity. Subsequently, two first-round single clones were again seeded as single cells / well in 96 wells for 20–23 days, preferably about 21 days, and tested again for growth and productivity. The best selected single-cell clones were then grown and subculturned in appropriate medium under appropriate conditions for at least 15 passages (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), for example, in T-flasks and Roller bottles. The cells were harvested and the cell suspension was filled into cryovials. The resulting MCBs were identified according to the requirements of the European Pharmacopoeia. This is necessary to ensure that the H-1PV production method conforms to GMP requirements and that the final product satisfies quality requirements.

[0013] For optimal results, a "master cell bank" is characterized by (a) morphology, (b) at least 80% viability, (c) sterility, (d) passage number 15, (e) deletion of mycoplasma and mycobacterial contamination, (f) deletion of SV40 production, (g) deletion of exogenous factors, (f) deletion of exogenous viral contamination, (g) identity, (h) deletion of tumorigenicity, and (i) oncogenity.

[0014] 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 clone cells as described above; (b) transfecting the master cell bank with the sequenced pUC19ΔHindII / H1 plasmid DNA (Kestler et al, 1999) by a suitable method (e.g., calcium phosphate transfection method; Graham and Van der Eb, 1973) (c) at least two rounds of infection of the MCB cells with H-1PV plasmid DNA to generate MSV comprising.

[0015] The present invention further relates to a method for producing a parvovirus H-1PV preparation, the method comprising: (a) providing a master cell bank as described above, (b) pre-seeding the MCB cells (c) seeding the cells at a suitable cell density, e.g., 2.0 - 5.0x10 , , , , -2 , 4 , , 2 , , , , , cells / cm 2 with the master seed virus (MSV) at a suitable MOI, e.g., 0.5 - 5x10 -2 PFU / cell; (d) growing the cells for about 2 - 6 days, harvesting the cells on days 2 - 6 after infection, and obtaining a cell pellet by centrifugation; (e) subjecting the resuspended cell pellet to mechanical, physical or chemical cell lysis methods 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 chromatographic preparation; (i) chromatography for the removal of empty particles and most impurities, e.g., anion exchange chromatography; (j) buffer exchange and concentration by a desalting column or tangential flow filtration; (k)Final formulation in Visipaque / Ringer or other formulation solutions is included.

[0016] The term "cell culture" means 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.

[0017] The term "cultivation" means the in vitro maintenance of cells under favorable conditions for cell growth, differentiation or continued viability in an active or quiescent state.

[0018] The phrase "cell culture medium" refers to a nutritive solution for culturing cells.

[0019] The term "cell bank" refers to a collection of suitable containers whose contents are uniform compositions stored under defined conditions. Each container represents an aliquot of a single pool of cells.

[0020] The term "master cell bank (MCB)" or "master cell seed (MCS)" (both terms can be used interchangeably) refers to a collection of cells of a uniform composition derived from a single source. In particular, it is ScNB-324K (synonym: MCS NB 324K) deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the Budapest Treaty.

[0021] Those skilled in the art know the general conditions for growing master cell strains and the general conditions for infecting cells with parvovirus. Usually, cells are cultured at 37°C, for example, in minimum essential medium (MEM) with heat-inactivated fetal bovine serum (e.g., 5% FBS) 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.

[0022] In a preferred embodiment of the present invention, the cell density is 2.5 x 10 3 ~1x10 5 cells / cm 2 That is the case.

[0023] In a more 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 a carrier system.

[0024] Preferably, for collection, the culture medium is aspirated, and the infected cells are treated with a suitable buffer and / or enzyme or surfactant, e.g., PBS-EDTA, Tris buffer, Tween, or trypsin. The detached cells and, where appropriate, the supernatant of the medium are preferably centrifuged at 5,000 xg for about 5 minutes or filtered to obtain a cell pellet. Those skilled in the art know of suitable mechanical, chemical, or physical methods for releasing parvovirus from producible cells. Preferably, this can be done by freeze / thaw cycles, sonication, and / or enzyme / surfactant treatment. Those skilled in the art also know of suitable methods for sonicating and then DNAse treatment of cells. For example, cells may be sonicated at 30-70 W for a sufficient amount of time, and DNAse treatment may be performed with 10-80 U / ml DNAse, usually at 37°C for 10-60 minutes.

[0025] As described above, the final H-1PV formulation is preferably in Visipaque / Ringer as a carrier. According to a preferred embodiment of the present invention, the carrier is 73.62% Visipaque TM This is iodixanol in Ringer solution, prepared by mixing 320 (GE Healthcare) with a 26.38% Ringer solution. VISIPAQUE TM320 (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 for "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.

[0026] As shown in the following examples, single-clonal MCBs (ScNB-324K) exhibit clear advantages over NB-324K mixed cells: keratin is expressed more profoundly in single-clonal MCB cells, resulting in higher mechanical stability; transfection efficacy using MCB cells is greater, leading to higher infectivity productivity; and single-clonal MCB cells are more resistant to ammonia.

[0027] The following examples are illustrative and not intended to limit the present invention. While these examples are typical of what may be used, other methods known to those skilled in the art may be used instead.

[0028] In other words, the gist of this invention relates to the following: Item 1 A method for producing a single-clonal master cell bank (MCB) for parvovirus H-1 production, wherein the MCB consists of cells deposited in the DSMZ (=German Collection of Microorganisms and Cell Cultures) under accession number DSM ACC3353 in accordance with the Budapest Convention, and the method comprises the following steps: (a) A step of growing NB-324K mixed clones in cell culture medium, (b) A process in which one cell is seeded per well in a 96-well plate and grown for 20-28 days. (c) A step of selecting 2 to 5 cell clones obtained in step (b), wherein the cells show a single colony per well and exhibit the best cell proliferation and productivity. (d) A step in which at least two first-round single-cell clones obtained in step (c) are seeded in 96 wells as single cells / well for 20-23 days and tested for growth and productivity. (e) A step of growing and passing a selected single-cell clone having the best growth and productivity until the number of passages exceeds 15, and (f) The process of collecting and storing the obtained cells. Methods that include... Section 2 The method according to item 1, wherein the cell culture medium in step (a) is MEM medium containing fetal bovine serum (FBS), L-glutamine, and gentamicin. Section 3 The method according to claim 1 or 2, wherein each step of growing and subculturing the subculturing of step (e) is carried out in a T-flask or roller bottle. Section 4 A method for producing a master seed virus (MSV) composition, wherein the method is: (a) A step of providing a master cell bank (MCB) obtained by any of the methods described in item 1 to 3, (b) The MCB from step (a) is transfected with the sequenced pUC19ΔHindIII / H1 plasmid DNA. (c) The process of infecting the MCB cells from step (b) with the pUC19ΔHindIII / H1 plasmid DNA for at least two rounds to generate MSVs. Methods that include... Section 5 The method according to item 4, wherein the transfection in step (b) of item 4 is performed by calcium phosphate transfection. Section 6 A method for producing parvovirus H-1 (H-1PV), the method comprising: Providing a master cell bank (MCB) obtained by the method according to any one of items (a) to (3); Pre-seeding the MCB cells in step (a) with a master seed virus (MSV) obtained by the method according to any one of items 4 and 5; (c) Infecting the cells at a cell density of 2.0 to 5.0 x 10 4 cells / cm 2 with MSV at an MOI of 0.5 to 5 x 10 -2 PFU / cell; Growing the cells for 2 to 6 days, harvesting the cells 2 to 6 days after infection, and obtaining a cell pellet by centrifugation; Subjecting the resuspended cell pellet to mechanical, physical or chemical cell lysis methods to obtain a parvovirus-containing cell lysate; Clarifying the parvovirus harvest by filtration; and Subjecting it to DNAse treatment; (h) A buffer exchange step for chromatographic preparation; (i) A chromatographic step for removing empty particles and most impurities; (j) A buffer exchange and concentration step by a desalting column or tangential flow filtration; and (k) A final formulation step in iodixanol / Ringer or other formulation solutions A method comprising. Item 7 The method according to item 6, wherein the chromatography in steps (h) and (i) of item 6 is anion exchange chromatography.

Advantages of the Invention

[0029] According to the present invention, optimized production of parvovirus H-1 can be provided.

Brief Description of the Drawings

[0030] Brief Description of the Drawings [Figure 1] Figure 1: Generation of master cell bank and master seed virus for H-1PV production. Cells of NB-324K mixed clones underwent two rounds of single colony selection in MEM, 5% FBS, 2% L-glutamine, and 0.2% gentamicin. The selected clone D8-G3 was first used to establish a research cell seed (RCS) and then to establish a master cell bank (MCB; passage 15 or longer) for H1-PV production. For master seed virus (MSV) generation, single-clonal MCB cells were transfected with sequenced pUC19ΔHindII / H1 plasmid DNA. After two rounds of MCB cell infection with H-1PV, the final passage was defined as MSV. For drug production, MCB working cell bank cells were infected with MSV (or the corresponding working seed virus (WSV)). [Figure 2] Figure 2: Two rounds of single-cell clone selection for H-1PV GMP production. Selection in the first round (A). Clones D8 (4.07E+03 ± 2.88E+03 PFU / cell), E6 (4.86E3 ± 3.27E+02 PFU / cell), and C5 (5.0E+03 ± 1.05E+03 PFU / cell) (n=2) exhibit the same productivity as the NB-324K mixed clone (6.9E3 PFU / cell). Furthermore, the production times of the three selected clones (approximately 33-35h) are also similar to the production time of the mixed clone NB-324K (approximately 32h; not shown), where clone D8 has the best production time of 32.9h. As a result, it was decided to work with clone D8 in further experiments. Selection in the second round (B). The production time for both mixed clones D8-G3 and D8-F7 is 50h. The productivity of clone D8-G3 is better than that of clone D8-F7 and similar to that of the mixed clone NB-324K. As a result, we decided to establish a research cell bank and a master cell bank (MCB) using the D8-G3 clone. The established MCB cell line was generated in approximately 30 hours and will be used for H-1PV production. [Figure 3]Figure 3: Immunofluorescence comparison (AC) of mixed-clonal NB-324 and single-clonal MCB cells. NB-324K and MCB cells were stained with CH / HK keratin antibody to mark intermediate keratin and DAPI for nuclear staining. Nuclear counting and total intensity were assessed using ImageJ software (https: / / imagej.nih.gov / ij / ) and evaluated using 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 epithelial tissues, both in single epithelial cells and through cell-cell contact (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 and demonstrated higher mechanical stability compared to NB-324K mixed-clonal cells. This mechanical stability can play a role in a robust upgrade process. [Figure 4A] Figure 4: Higher transfection efficiency of single-clonal MCB cells. NB-324K and MCB cells were transfected with H-1PV-producing plasmid clones using the calcium phosphate method. Single-clonal MCBs show an advantage in terms of higher transfection efficacy compared to mixed NB-324K cells (A). [Figure 4B] Figure 4: Higher transfection efficiency of single-clonal MCB cells. NB-324K and MCB cells were transfected with H-1PV-producing plasmid clones using the calcium phosphate method. The genome-containing particle to infectious particle ratio (GP / PFU) was lower in single-clonal MCBs (B), indicating fewer deficient particles and more infectious particles in the product. [Figure 5]Figure 5: Higher tolerance to ammonia in monoclonal MCB cells compared to NB-324K mixed cells. Cells were treated with different ammonia concentrations and counted after 5 days of proliferation. Monoclonal MCB cells showed higher tolerance up to 20 mM ammonia compared to NB-324K mixed cells. This tolerance indicates an advantage for MCB cells during cell proliferation and H-1PV production processes, as ammonia is the main product of glutamine metabolism. Its accumulation has been shown to reduce proliferation and negatively impact metabolism. Ammonia accumulation can disrupt the electrochemical cell gradient and induce cytoplasmic acidification. Furthermore, 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”). [Modes for carrying out the invention]

[0031] Cells from a specific preferred single-cell MCB ("MCS NB324K Human") were deposited under the Budapest Convention on May 16, 2019, with accession number DSM ACC3353, in the German Collection of Microorganisms and Cell Cultures (DSMZ) in Braunschweig. [Examples]

[0032] Example 1 material and method A. Immunofluorescence test Immunofluorescence assays are based on the use of fluorescently marked antibodies that bind to specific antigens, thereby enabling the identification of specific extracellular and intracellular structures. This principle is used for keratin staining with CH / HK-keratin antibody, which marks intermediate keratin. For this purpose, single-clonal MCB (ScNB-324K) and NB-324K mixed cells were seeded on glass slides in 6 cm cell dishes at 6E5 (Figure 3C) and 1.2E6 (Figure 3A, B) cell / dish ratios. After 24 hours of incubation, the cells on the slides were washed in PBS, fixed in ice-cold methanol for 10 minutes, and then fixed in ice-cold acetone for 5 minutes. For keratin detection, the slides were pre-blocked in PBS with 0.05% BSA and then incubated with CH / HK-keratin antibody for 1 hour. Subsequently, the slides were washed in PBS with 0.1% Triton and then incubated with secondary antibody Cy3gp (Dianova, Germany) for 1 hour. Unbound secondary antibody was removed by three washes in PBS containing 0.1% Triton. Slides were immersed in ethanol and dried. Slides were embedded in Fluoromount™ slide mount medium (SigmaAldrich, Germany) containing 1 μg / ml DAPI (4',6-diamidino-2-phenylindole, SigmaAldrich, Germany) for cell nucleus staining. Analysis was performed using a fluorescence microscope BZ-9000 (Keyence, Germany) (occurring). Nuclear counting and total intensity were evaluated using ImageJ software (https: / / imagej.nih.gov / ij / ).

[0033] B. Culture, transfection, and infection of monoclonal MCB and NB-324K mixed cells Single-clonal MCB (ScNB-324K) and NB-324K mixed cells are cultured at 37°C in VP-SFM medium containing 5% FBS and 4 mM L-glutamine. For transfection, single-clonal MCB and NB-324K mixed cells are cultured at a rate of 3.6E4 cells / 75cm³ in MEM medium containing 5% FBS, 2 mM glutamine, and Pen / Strep. 2 Cells were seeded in flasks and transfected with the puc19ΔHindIII / H1 plasmid using calcium phosphate transfection. Cells were harvested on days 3, 4, and 5.

[0034] To identify the toxic effects of ammonia on single-clonal MCB and mixed NB-324K cells, single-clonal MCB and mixed NB-324K cells were placed in a Corning flask (25 cm). 2 Growth surface and 5% FBS and 4 mM glutamine medium / flask (5 mL VP-SFM) or Nunc flask (75 cm) 2 Cells were cultured on a growth surface in 10 mL VP-SFM flasks containing 5% FBS and 4 mM glutamine medium. Ammonium chloride (Pan Reac AppliChem, Germany) was added to the medium at different concentrations (0, 5, 10, and 20 mM, one flask per setting). On day 5, each setting was counted using a Countess® Cell counter (Life Technologies, Germany).

[0035] For H-1PV production, NB-324K cells and single-clonal MCBs (ScNB-324K) were used at a rate of 3.6E4 cells / cm³. 2Cells were seeded and immediately infected with H-1PV at an infection multiplicity (MOI) of 0.01 plaque-forming units (PFUs) 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 viable cells with 0.4% trypan blue (Invitrogen™, Germany). Cells were counted using a Countess® Cell Counter (Life Technologies, Germany), and their morphology was observed microscopically.

[0036] For cell collection, the culture medium was aspirated and infected cells were treated with PBS / 1 mM EDTA. The supernatant and detached cells were centrifuged at 5,000 x g for 5 minutes. The pellet was washed with PBS and resuspended in Virus Tris / EDTA buffer containing 0.05 M Tris HCl and 0.5 mM EDTA, pH 8.7 (VTE), and subjected to three freeze / thaw cycles. After centrifugation at 5,000 x g for 5 minutes, the cell debris was discarded. The cell lysates were then sonicated at 48 W for 1 minute using a Sonorex Super 10 P ultrasonic homogenizer (Bandelin, Germany) and treated with DNAse (50 U / ml, Sigma, Germany) at 37°C for 30 minutes.

[0037] C. Plaque Formation Assay (PFU) Plaque assays were performed essentially as described in Tattersall and Bratton, 1983. NB-324K cells were grown in monolayer cultures 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 at 37°C for 1 hour. The inoculum was then replaced with a bacto-agar overlay (1.7% in MEM containing 5% FBS). Four days post-infection, viable cells were stained for 18–24 hours with the addition of 0.02% toluene red staining solution (Sigma, Germany) containing bacto-agar (Becton Dickinson, Germany). Dishes were incubated at 37°C under 5% CO2. Plaque-forming units were counted on a lightbox on day 5 after infection, and their concentrations were expressed in PFU / ml.

[0038] D. Determination of genome-containing particles (GPs) The number of genome-containing viral particles (GPs) 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 of template. Q-PCR was performed using a QuantStudio 3 Real-Time PCR System, and the results were processed with QuantStudio 3 Design and Analysis Software 1.4 (Applied Biosystems, Germany).

[0039] Example 2 Comparison of single-cell clone productivity for H-1PV GMP production selection. A. Selection for Round 1 Clones D8 (4.07E+03±2.88E+03), E6 (4.86E3 E+02 ± 3.27E+02), and C5 (5.0E+03±1.05E+03) (n=2) exhibited the same productivity as the NB-324K mixed clone (6.9E3 PFU / cell). Furthermore, the generation times of the three selected clones (approximately 33-35h) were also similar to the generation time of the mixed clone NB-324K (approximately 32h; not shown). Clones D8 had the best generation time at 32.9h.

[0040] As a result, due to the short generation time and similar productivity, we decided to work with clone D8 in round 2 of the selection process.

[0041] B. Selection for Round 2 The generation time for both mixed clones D8-G3 and D8-F7 was 50 hours. The productivity of clone D8-G3 was better than that of clone D8-F7 and similar to that of 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 took approximately 30 hours and was used for H-1PV production.

[0042] Example 3 Comparison of keratin strength between mixed clone NB-324K and single clone MCB cells Comparison of immunofluorescence between mixed clone NB-324 and single clone MCB cells (AC) NB-324K and MCB cells were stained with a CH / HK keratin antibody to mark intermediate keratin and DAPI for nuclear staining. Nuclear counts and total intensity were assessed using ImageJ software (https: / / imagej.nih.gov / ij / ). Single-clonal MCB cells (ScNB-324K) showed 2.7x higher keratin signal intensity / cell, indicating higher mechanical stability of single-clonal MCB cells compared to mixed-clonal NB-324K cells.

[0043] 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. Single-clonal MCBs showed an advantage with approximately 1 log higher transfection efficacy compared to NB-324K mixed cells.

[0044] B. The genome-containing particle to infectious particle ratio (GP / PFU) was lower (approximately 1 log) for single-clonal MCB (ScNB-324K), indicating fewer deficient particles and more infectious particles in the product.

[0045] Example 5 Effects of ammonia on mixed clone NB-234K single clone MCB cells Cells were treated with different ammonia concentrations and counted after 5 days of proliferation. Single-clonal MCB cells (ScNB-324K) showed higher tolerance up to 20 mM ammonia compared to NB-324K mixed cells. This tolerance indicates an advantage for MCB cells during cell proliferation and H-1PV production processes, as ammonia is a major product of glutamine metabolism. It has been shown that its accumulation reduces proliferation and negatively impacts metabolism. Ammonia accumulation can disrupt the electrochemical cell gradient and induce cytoplasmic acidification. Furthermore, it can induce apoptosis in cultured cells.

[0046] List of References [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0047] The following are examples of aspects of the present invention. Item 1 A method for producing a single-clone master cell bank (MCB) for parvovirus H-1PV production, the method comprising the following steps: (a) A step of growing NB-324K mixed clones in cell culture medium, (b) A process in which, ideally, one cell is seeded per well in a 96-well plate and grown for 20-28 days. (c) A step of selecting 2 to 5 cell clones obtained in step (b), where the cells showed a single colony per well. (d) A step in which at least two first-round single clones are seeded in 96 wells as single cells / well for 20-23 days and tested for growth and production. (e) A step of growing and passing selected single-cell clones having the best growth and production characteristics until the number of passages exceeds 15, (f) The process of collecting and storing the obtained cells. Methods that include... Section 2 The method according to item 1, wherein the cell culture medium in step (a) is MEM medium containing fetal bovine serum (FBS), L-glutamine, and gentamicin. Section 3 The method according to claim 1 or 2, wherein the growth and subculturing step (e) is carried out in T-flasks and roller bottles. Section 4 A method for producing a master seed virus (MSV) composition, wherein the method is: (a) A step of providing a master cell bank obtained in any of items 1 to 3, (b) Transfecting the master cell bank with sequenced pUC19ΔHindIII / H1 plasmid DNA. (c) The process of infecting MCB cells with H-1PV plasmid DNA for at least two rounds to generate MSVs. Methods that include... Section 5 The method according to item 4, wherein the transfection in step (b) is carried out by calcium phosphate transfection. Section 6 A method for producing parvovirus H-1 (H-1PV), wherein the method is: (a) A step of providing a master cell bank (MCB) obtained in any of items 1 to 3; (b) A step of pre-seeding 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 0.5~5 x 10 -2 The process of infecting PFU / cells with MSV at MOI; (d) The cells are grown for approximately 2 to 6 days, and the cells are collected 2 to 6 days after infection and a cell pellet is obtained by centrifugation; (e) The step of subjecting the resuspended cell pellet to a mechanical, physical or chemical cell lysis method in order to obtain a parvovirus-containing cell lysate; (f) A process of clarifying the parvovirus sample by filtration; and (g) The process of subjecting it to DNAse treatment; (h) Buffer exchange for chromatography preparation; (i) Chromatography for the removal of empty particles and most impurities; (j) Buffer exchange and concentration by desalting column or tangential flow filtration; (k) Final formulation in Visipaque / Ringer or other formulation solutions Methods that include... Section 7 The method according to item 6, wherein the chromatography in step (h) is anion exchange chromatography. Section 8 A single-clonal master cell bank (MCB) for parvovirus H-1 production, obtainable by any of the methods described in items 1 to 3. Section 9 A single-clonal master cell bank as described in item 8, including cells deposited in the DSMZ (=German Collection of Microorganisms and Cell Cultures) under accession number DSM ACC3353 in accordance with the Budapest Convention.

Claims

1. A method for producing a master seed virus (MSV) composition, wherein the method is: (a) A process of providing a single-clone master cell bank (MCB) consisting of cells deposited in DSMZ (=German Collection of Microorganisms and Cell Cultures) under accession number DSM ACC3353 in accordance with the Budapest Convention. (b) A step of transfecting the MCB cells from step (a) with an H-1PV-producing plasmid clone, and (c) The process of infecting the MCB cells from step (b) with an H-1PV-producing plasmid clone for at least two rounds to generate MSV. Methods that include...

2. The method according to claim 1, wherein the transfection in step (b) of claim 1 is performed by calcium phosphate transfection.

3. A method for producing parvovirus H-1 (H-1PV), wherein the method is: (a) Providing a single-clone master cell bank (MCB) consisting of cells deposited in the DSMZ (=German Collection of Microorganisms and Cell Cultures) under accession number DSM ACC3353 in accordance with the Budapest Convention; (b) A step of pre-seeding the MCB cells of step (a) with a master seed virus (MSV) obtained by the method described in either claim 1 or 2. (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 The process of infecting PFU / cells with MSV at MOI; (d) A step of growing the cells for 2 to 6 days, collecting the cells 2 to 6 days after infection, and obtaining a cell pellet by centrifugation; (e) The step of subjecting the resuspended cell pellet to a mechanical, physical or chemical cell lysis method in order to obtain a parvovirus-containing cell lysate; (f) A process of clarifying the parvovirus sample by filtration; and (g) The process of subjecting it to DNAse treatment; (h) Buffer exchange process for chromatography preparation; (i) A chromatography step to remove empty particles and most impurities; (j) Buffer exchange and concentration steps by desalting column or tangential flow filtration; and (k) Final formulation process in iodixanol / Ringer or other formulation solution Methods that include...

4. The method according to claim 3, wherein the chromatography in steps (h) and (i) of claim 3 is anion exchange chromatography.

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