METHOD FOR PRODUCING RECOMBINANT VIRUSES OR VIRUS-LIKE PARTICLES (VLPs) IN MAMMALIAN CELL LINES

Abstract

The present invention relates to a method for producing higher titers and / or a higher quality of recombinant viruses and / or less or reduced impurities in virus like particle (VLP) production in respective production processes in mammalian cell lines, the method comprising the step of supplementing a suitable mammalian cell line culture media with at least one intermediate substrate of the tricarboxylic acid (TCA) cycle and optionally supplementing the suitable mammalian cell line culture media with further additives and / or further adjusting the conditions of the mammalian cell line culture media. Furthermore, the present invention relates to the balanced suitable mammalian cell line culture media and the use of this balanced suitable mammalian cell line culture media for producing higher titers and / or a higher quality of recombinant viruses and / or less or reduced impurities in virus like particle (VLP) production in respective production processes for producing higher titers and / or quality of recombinant viruses.

Description

[0001] METHOD FOR PRODUCING RECOMBINANT VIRUSES OR VIRUS-LIKE PARTICLES (VLPs) IN MAMMALIAN CELL LINES The present invention relates to a method for producing higher titers and / or a higher quality of recombinant viruses and / or less or reduced impurities in virus like particle (VLP) production in respective production processes in mammalian cell lines, the method comprising the step of supplementing a suitable mammalian cell line culture media with at least one intermediate substrate of the tricarboxylic acid (TCA) cycle and optionally supplementing the suitable mammalian cell line culture media with further additives and / or further adjusting the conditions of the mammalian cell line culture media. Furthermore, the present invention relates to the balanced suitable mammalian cell line culture media and the use of this balanced suitable mammalian cell line culture media for producing higher titers and / or a higher quality of recombinant viruses and / or less or reduced impurities in virus like particle (VLP) production in respective production processes for producing higher titers and / or quality of recombinant viruses. Description Virus-like particles (VLPs) are biological nanoparticles similar to natural virions, but without the genetic material. VLPs are suitable for the analysis of viral infection mechanisms, vaccine production, tissue-specific drug delivery, and as biological nanomaterials (Sánchez-Rodríguez et al., 2012). They have a wide range of applications in biotechnology and medicine. One of the most important applications is to replace traditional vaccines with recombinant and well- defined VLPs, for example in HIV, HCV, and Dengue Virus (Effio, C.L. and Hubbuch, J., 2015). VLPs are considered present, licensed and successful vaccines against prevalent and emergent diseases due to their non-infectious nature, structural similarity with viruses, and high immunogenicity. However, only a few VLP-based vaccines have been commercialized, and these few are either in clinical or preclinical phases. Notably, despite success in the preclinical phase, many VLP-based vaccines are still struggling with small-scale fundamental research owing to technical difficulties. The successful production of VLP-based vaccines on a commercial scale requires a suitable platform and culture mode for large-scale production, optimization of transduction-related parameters, upstream and downstream processing, and monitoring of product quality at each step (Gupta et al., 2023). Le and Müller, 2021 disclose that VLPs are increasingly used for vaccine development and drug delivery. Assembly of VLPs from purified monomers in a chemically defined reaction is advantageous compared to in vivo assembly, because it avoids inclusion of host-derived components and enables loading with added cargoes. Their review provides an overview of ex cellular VLP production methods focusing on capsid protein production, factors that impact the in vitro assembly, and approaches to characterize in vitro VLPs. The uses of in vitro produced VLPs as vaccines and for therapeutic delivery are also described. Furthermore, Zhuo et al., disclose that VLPs are used in gene therapy and clinical tests for diagnostics of immune status or an acute infection. These engineered virus-like particles that efficiently package and deliver therapeutic gene-editing proteins, including base editors (BEs) and Cas9 nuclease, with the ability to overcome cargo packaging, release, and localization bottlenecks, representing potentially promising carriers for delivering gene-editing tools of therapeutic interest. There are different strategies to recombinantly produce VLPs. Usually, VLPs are produced with mammalian expression systems in various organism cell lines, such as CHO or HEK cells, or yeast and microbial systems. The challenge with this kind of production is to achieve a high quality of pure self-assembled VLPs without e.g. oligomerization and aggregations of the VLPs, an instability of product as well as high product yields (Shi et al., 2005). Adeno-associated virus (AAV) is a small, non-enveloped virus that contains a single-stranded DNA genome and belongs to the Parvoviridae family and the Dependoparvovirus genus. It is widely used as a vector in gene therapy due to its ability to deliver genetic material to a variety of cell types with high efficiency and relatively low immunogenicity. Wang, JH et al. (in: Adeno-associated virus as a delivery vector for gene therapy of human diseases. Sig Transduct Target Ther 9, 78 (2024). https: / / doi.org / 10.1038 / s41392-024-01780- w) explore AAV biology with an emphasis on current vector engineering strategies and manufacturing technologies. They discuss how rAAVs are being employed in ongoing clinical trials for ocular, neurological, metabolic, hematological, neuromuscular, and cardiovascular diseases as well as cancers. WO2015031686A1 discloses high titer recombinant AAV vector production in adherent and suspension cells, in particular an in vitro method of producing a recombinant AAV virion in a mammalian host cell (that comprises a functional adenoviral E1A gene, e.g., a HEK 293 cell). The method involves incubating the cell in a transfection medium. The transfection medium can comprise a protein hydrolysate, for example tryptone N1 (T1). Schieferecke AJ, et al. (in: Evolving membrane-associated accessory protein variants for improved adeno-associated virus production. Mol Ther. 2024 Feb 7;32(2):340-351. doi: 10.1016 / j.ymthe.2023.12.015. Epub 2023 Dec 18. PMID: 38115579; PMCID: PMC10861973) describe membrane-associated accessory protein (MAAP) variants containing missense mutations and a frameshifted C-terminal domain that increased overall GFP transgene packaging in AAV2, AAV6, and AAV9 capsids. EP1342780A1 relates to a method for reduction of glucose consumption and / or lactate production during cultivation of animal cells, characterized in that cultivation is performed in the presence of a bi- or tricarbonic acid or a salt thereof at a concentration of about 1 to 50 mmol / l, whereby in the case where said di- or tricarbonic acid is citric acid or citrate this amount of said citric acid or citrate is not bound in a chelate complex with iron or another transition metal ion. It is therefore known to support the TCA with citrate to reduce the lactate level during mammalian cell culture processes. No VLPs or AAVs are described. Baltz JM. (in: Media composition: salts and osmolality. Methods Mol Biol.2012;912:61-80. doi: 10.1007 / 978-1-61779-971-6_5. PMID: 22829369) discusses that the optimum salts levels, osmolality, and amino acid contents of culture media are not independent, but interact strongly because of their roles in cell volume regulation. The main challenge in AAV vector (AAVv) production is to achieve both high yields, in terms of genomic and capsid titers, and a high quality virus particle, such as a high percentage of “full”, i.e. containing a desired vector genome, preferably comprising at least one desired transgene, for example in a suitable expression cassette, virus capsids. A big disadvantage of the current production processes of VLPs is the formation of oligomers and / or aggregates leading to a lower quality of VLPs, because less pure self-assembled VLPs are present after the production process. Several purification steps are necessary to filter the oligomers and / or aggregates resulting in a more cost and time intensive handling of the downstream processes and to a lower yield of VLPs after the purification. A heterogeneous VLP population that requires extensive down-streaming workup in order to collect a pure / homogenous and correctly assembled VLP fraction is also described in the prior art (See, for example, Figure 1, which is Figure 1b of Ding et al., 2010), showing an asymmetrical flow field flow fractogram for a mixture of unreacted capsomeres, virus-like particles and aggregates in an assembly product. It is an object of the present invention to provide a method for producing recombinant viruses and / or virus like particles (VLPs) in a mammalian cell line, while reducing impurities, such as aggregates, and for producing higher titers and / or a higher quality of recombinant viruses, such as reducing insufficiently complete or loaded recombinant virus particles, and to furthermore provide a balanced suitable mammalian cell line culture medium. Other objects and advantages of the present invention will become apparent to the person skilled in the art when studying the following more detailed description of the present invention, including the figures and examples. The present invention provides a novel approach for overcoming the extensive formation of impurities, such as oligomers and aggregations, of VLPs that are developing during the state of the art production processes for VLPs in cell culture. It was furthermore surprisingly found that the approach of the present invention also provides for the improved production of recombinant virus particles, such as AAV vector (AAVv) particles, i.e. while achieving both high yields, in terms of genomic and capsid titers, and high quality, such as a high percentage of “full”, i.e. containing a desired vector genome, preferably comprising at least one desired transgene, for example in a suitable expression cassette, capsids. According to a first aspect of the present invention the above object is solved by providing a method for recombinantly producing virus like particles (VLPs) in a mammalian cell line, comprising the step of suitably culturing the mammalian cell line in a culture medium supplemented with an intermediate substrate of the tricarboxylic acid (TCA) cycle selected from the group consisting of between 0.5 to 25 mM, preferably of between 1 to 20 mM, and most preferably of between 2 to 10 mM, or between 0.5 to 10 mM, preferably of between 1 to 8 mM, and most preferably of between 2 to 6 mM of sodium citrate monobasic, cis-aconitate, iso-citrate, alpha ketoglutarate, succinate, fumarate, malate, and oxaloacetate, preferably between 2 to 8 mM sodium citrate monobasic, more preferably between 4 to 7 mM sodium citrate monobasic, most preferably between 2 to 5 mM sodium citrate monobasic. For further clarity, the molar concentrations as indicated thus denote the concentrations of the respective organic acid anion, that is the anion selected from citrate, cis-aconitate, iso-citrate, alpha ketoglutarate, succinate, fumarate, malate, and oxaloacetate. According to the invention, the concentrations as indicated may be present at the start of the culture, are achieved (e.g. once) during the culture and are preferably held within the ranges during the culture. The inventors observed a mixed population of different oligomeric or aggregated states of VLPs during regular production processes. In order to obtain homogeneous “pure” VLPs, i.e. that are not clustered, a size-exclusion chromatography followed by narrow fractionation is required. This kind of purification leads to a discard of large amounts of VLPs in the oligomeric or aggregated state, and consequently to a drastic reduction of the expression yield of the “pure” VLPs. In the context of recombinant viruses, such as AAV, the inventors observed that it was challenging to achieve both high yield in terms of genomic and capsid titers and high quality, such as a high percentage of “full” virus particles, i.e. containing a desired vector genome, preferably comprising at least one desired transgene, for example in a suitable expression cassette. Also, the undesirable aggregation of HEK293 cells in the presence of high salt concentrations was observed. AAV vectors (AAVv) are typically produced using mammalian cells, such as human embryonic kidney (HEK293) cells. Preferred is a method according to the present invention, wherein the cells, such as the HEK293 or CHO cells, are grown as adherent or suspension cells in the culture. One preferred production method also involves the prior transfection of the cells with plasmids, e.g. with three plasmids: a transfer plasmid containing the transgene, a helper plasmid providing adenoviral functions, and a packaging plasmid encoding AAV rep and cap genes. This technique is commonly designated as “triple transfection” (see, for example, Merten OW, AAV vector production: state of the art developments and remaining challenges Cell Gene Therapy Insights 2016; 2(5), 521-55110.18609 / cgti.2016.067). Another alternative of the method according to the present invention involves the use of inducible packaging and producer cell lines (see, for example, Merten OW. Development of Stable Packaging and Producer Cell Lines for the Production of AAV Vectors. Microorganisms. 2024 Feb 13;12(2):384. doi: 10.3390 / microorganisms12020384. PMID: 38399788; PMCID: PMC10892526). Packaging cell lines are engineered to provide all the essential viral proteins required for the assembly and packaging of recombinant adeno-associated virus (rAAV) particles. These cell lines do not contain the rAAV vector genome itself, but supply packaging / helper functions needed for rAAV production. Typically, these cell lines are stably transfected with plasmids encoding the AAV replication (rep) and capsid (cap) genes, which are essential for replication and encapsulation of the AAV genome as well as for capsid formation. Additionally, they may contain helper virus genes (such as those from adenovirus (Ad)) that provide necessary functions to produce rAAV particles. Producer cell lines contain all the necessary components for rAAV production, including the recombinant vector genome, rep and cap genes, and any required viral helper function. In addition to the viral AAV and Ad genes, these cell lines are engineered to include the recombinant AAV vector genome, which contains the therapeutic gene of interest expression cassette flanked by AAV inverted terminal repeats (ITRs). The production according to the present invention can be performed in suspension-based and adherent-based cell cultures, and these therefore also constitute specific preferred uses of the media according to the present invention. Furthermore, the media according to the present invention can be used in perfusion, batch, fed batch, N / production stage, and seed train culture methods (for the latter see, for example, Frahm B. Seed train optimization for cell culture. Methods Mol Biol. 2014;1104:355-67. doi: 10.1007 / 978-1-62703-733-4_22. PMID: 24297426). Obviously, the production of VLPs or recombinant virus particles, such as AAV, is a highly intensive process for mammalian cells, leading to a high demand for substrates for protein and lipid synthesis. If there is a lack in one of these steps, the mammalian cells generally switch to different (and often less efficient) synthesis pathways. Glucose or a feed (see below) is often fed as bolus or via perfusion in order to avoid limitation during the process. The basal media has the disadvantage of generating low titers during AAVv production process under optimal production conditions. The inventors identified that the lack of the necessary substrates has a pronounced influence on the development of oligomers and aggregates of VLPs and the insufficient production of quality recombinant virus particles, such as AAV. Therefore, the inventors investigated how to avoid a lack of substrates in relevant pathways of intermediate products, e.g. the lipid and amino acid synthesis in the cells as used for the production of VLPs and recombinant virus particles, such as AAV. The inventors also found that the production rate should match the self-assembly rate of the product in the cells. Surprisingly, the inventors found that the use of citrate and TCA intermediates, such as cis- aconitate, iso-citrate, alpha ketoglutarate, succinate, fumarate, malate, and oxaloacetate, as substrates support the TCA and the downstream amino acid and lipid synthesis steps following the TCA, and generates more usable energy, e.g., ATP. As a preferred example, citrate was identified to have a pronounced effect on the non-formation of oligomers and aggregates of VLPs during the VLPs production method and the production of recombinant virus particles, such as AAV during the virus production method. As known to the person of skill and as described in the literature, citrate and the other TCA intermediates can be added to the medium in physiological concentrations (e.g. between 0.5 and 10 mM) and suitable physiological salts (e.g. sodium) and / or in physiologically acceptable buffers or solutions. Furthermore, the inventors identified that the addition of iron, which is an important cofactor in various cellular processes such as the TCA, lipid metabolism, and oxygen transport, respiration and DNA synthesis, also improves the quality of VLP and recombinant viral production, such as rAAV, in particular when supplied as transferrin. Consequently, the inventors analyzed several culture conditions and additives or supplements in order to reduce VLP oligomerization and / or aggregation, and to increase both the genomic and capsid titer and preferably also the quality (%full) of recombinant viral production, such as rAAV. Furthermore, the inventors found that the addition of citrate to the cell culture also significantly reduces the formation of aggregates of the cells as cultured (“cell clumps”). This further helps to maintain a healthy and efficient cell culture and avoids adding anti-clumping agents like “Anti-clumping agent“ (ThermoFisher) or “Scattering reagent CHO” (Gmep International) to the culture medium, which may adversely affect cell behavior. Preferred is the method for recombinantly producing virus like particles (VLPs) or recombinant virus particles, such as rAAVs, in a mammalian cell line, wherein the culture medium is further supplemented with at least one compound selected from the group consisting of transferrin, FeSO4, and CuSO4. Preferred is the method for recombinantly producing virus like particles (VLPs) in a mammalian cell line, wherein the culture medium is further supplemented with at least one vitamin feed, such as a putrescine / pyridoxine feed, preferably vitamin feed, and is preferably not supplemented with an additional amount of choline chloride feed or asparagine feed in the medium as produced or used. It was found that the further addition of either a choline chloride feed or asparagine feed in addition to the original feeding as provided did not provide substantial benefits to the culture. In the context of the present invention, the term “CuSO4” shall relate to all hydrate forms of copper sulfate, and in particular includes copper (II) sulfate pentahydrate (MW 249.69 g / mol) or copper sulfate (MW 159.61 g / mol). The target concentrations as indicated need to be calculated accordingly. In the context of the present invention, the term “FeSO4” shall relate to all hydrate forms of iron sulfate, and particularly includes iron (II) sulfate heptahydrate. The target concentrations as indicated need to be calculated accordingly. In the context of the present invention, a “feed” shall mean a pre-configured and chemically- defined cell culture supplement. A feed may provide lipids, amino acids, and vitamins to support mammalian cell cultures (e.g. fed batch, perfusion) cultures in research and bioprocessing applications. See, for example, Hossler P, et al. Improvement of mammalian cell culture performance through surfactant enabled concentrated feed media. Biotechnol Prog. 2013 Jul-Aug;29(4):1023-33. doi: 10.1002 / btpr.1739. Epub 2013 May 11. PMID: 23554412, Seo-Young Park, et al. Characterizing Basal and Feed Media Effects on Mammalian Cell Cultures by Systems Engineering Approaches, IFAC-PapersOnLine, Volume 55, Issue 7, 2022, Pages 31-36, ISSN 2405-8963, https: / / doi.org / 10.1016 / j.ifacol.2022.07.418, and the like. One example for a vitamin feed as disclosed herein is a vitamin feed, such as a putrescine / pyridoxine feed. In case of a method comprising perfusion, feeds may also be added. When using a triple transfection, the feeds may be added into the pre- or posttransfection preparation or added into the transfection preparation, and then the process is run as a batch process. In general, any suitable mammalian cell line for the production of VLPs can be used for the methods according to the invention. Further preferred is the method according to the present invention, wherein the mammalian cell line is selected from CHO cell lines or HEK, such as HEK293 cell lines. Preferably, the cell line is grown as an adherent or in suspension culture. Further preferably, in the case of recombinant viruses, such as AAV, the cells are transiently or stably transfected (see also above). Also, in general, any suitable cell culture medium for the production of VLPs or recombinant virus particles, such as rAAVs in mammalian cells can be used as a basis for the methods according to the invention. Further preferred is the method according to the present invention, wherein the mammalian cell culture medium is DMEM or a medium equivalent to DMEM. In the context of the present invention, a “medium equivalent to DMEM” relates to a medium that is a functional equivalent thereof, containing the ingredient compounds of the standard medium DMEM and pyridoxine, but is free from any proteins, peptides, and hormones, and which does not contain the additives specifically mentioned herein, e.g. in the claims (such as citrate, transferrin, FeSO4,CuSO4, and feeds) appended herewith. DMEM and a (functional) equivalent of DMEM both denote media compositions which are serum-free. In an optional but not essential specific embodiment, the functional equivalent of DMEM further includes in physiological concentrations pyridoxine, biotin, vitamin B12, and other micronutrients as the case may be. DMEM and a (functional) equivalent of DMEM within the meaning for this invention both support the growth of an exemplary culture of at least CHO cells or human embryonic kidney cells, such as HEK293. Dulbecco's Modified Eagle Medium (DMEM) is modified to contain four times the amino acid and vitamin concentration of original Eagle's Minimal Essential Medium (Dulbecco R, Freeman G. Plaque production by the polyoma virus. Virology. 1959 Jul;8(3):396-7. doi: 10.1016 / 0042-6822(59)90043-1. PMID: 13669362). DMEM is a widely used basal medium for supporting the growth of many different mammalian cells. Cells successfully cultured in DMEM include primary fibroblasts, CHO cells, neurons, glial cells, HUVECs, and smooth muscle cells, as well as cell lines such as HeLa, 293, Cos-7, and PC-12. In the context of the present invention, the suitable mammalian cell line culture media is balanced (sometimes also called “optimized”, “adjusted” or “modified”) in order to promote the growth of the cells as used, in particular for the growth of HEK293 or CHO cells, in order to allow a high titer production of recombinant viruses and / or virus-like particles in mammalian cells. As mentioned below, the medium is based on DMEM or a functional equivalent thereof which contains the ingredient compounds of DMEM, and pyridoxine, and is free from any proteins, peptides, and hormones, and which does not contain the additives specifically mentioned herein, for example in the claims (such as citrate, transferrin, FeSO4, CuSO4, and feeds) appended herewith. High salt concentrations in cell culture media are associated with the undesirable aggregation of cells. As another important aspect of the present invention, the use of citrate in the methods and media of the present invention at the same time allows for high salt concentrations without the occurrence of cell aggregation. As a specific embodiment of the methods according to the invention, the transferrin concentration in the suitable mammalian cell line medium at the start of the culture may be between 0 to 15 mg / l, preferably between 5 to 10 mg / l, more preferably at about 9.4 mg / l or at about 5 mg / l, and preferably is held within these values during the culture. As a specific embodiment of the methods according to the invention, the FeSO4 concentration in the suitable mammalian cell line medium at the start of the culture may be at between 1 to 20 mg / l, preferably between 5 to 15.9 mg / l, and more preferably at about 13.9 mg / l or at about 9.7 mg / l, and preferably is held within these values during the culture. As a specific embodiment of the methods according to the invention, the CuSO4 concentration in the suitable mammalian cell line medium at the start of the culture may be at between 50 to 400 µg / l, preferably at between 99.8 to 350 µg / l, and more preferably at about 250 µg / l and preferably is held within these values during the culture. Also, preferred is a method and medium according to the present invention, wherein the osmolality in the medium at the start of the mammalian cell line culture is at between 290 to 370 mOsm (determined herein as mOsm / kg H2O), preferably at about 295 mOsm. The osmolality in the medium during the culture is preferably kept within these values as well, preferably by replacing consumed medium components and / or by adding feeds. The term virus-like particle (VLP) shall herein refer to particles that self-assemble as a result of the expression of proteins encoding capsids, cores or envelops of viruses or even preparations of monolayered particles derived from a multilayered virus. Additionally, VLPs self-assemble into particles that resemble or mimic the structure, size, and symmetry of original viruses, however VLPs cannot replicate as they lack a genome and replicases. Therefore, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In the context of the present invention, any suitable kind of VLPs can be used for the methods according to the invention that is capable of forming oligomeric or aggregated states of VLPs during regular production processes. Examples of common VLPs, for example as used herein, are HBV-, HPV-, Qβ-, CuMV-, AP205-, CCMV-, MS2-, PP7-, RHDS-, and CPV-derived (see, for example, Mohsen, M.O., Bachmann, M.F. Virus-like particle vaccinology, from bench to bedside. Cell Mol Immunol 19, 993–1011 (2022). https: / / doi.org / 10.1038 / s41423-022-00897-8). Also, AAV virus-like particles (sometimes referred to as AAV empty capsids) are well-characterized AAV capsids devoid of any viral genomic material that are widely used as reference materials during the development of AAV-based gene therapies. Le, D.T., et al. (Adeno-associated virus capsid protein expression in Escherichia coli and chemically defined capsid assembly. Sci Rep 9, 18631 (2019). https: / / doi.org / 10.1038 / s41598-019-54928-y) explored a new route to generate AAV capsids with the aim to analyze capsid assembly in a chemically defined setting and pave the way for new production methods and applications based on AAV virus-like particles (VLPs). These VLPs internalized into human HeLa cells. The term “recombinant virus” shall herein generally refer to viruses that contain a single- or double-stranded RNA or DNA genome, and can be used as vectors for recombinant nucleic acid vector genomes preferably comprising at least one desired transgene, such as a gene, for example in a suitable expression cassette, to be transferred into a cell infected by the virus, and in particular used as a vector in gene therapy due to the ability to deliver genetic material to a cell, ideally with high efficiency and relatively low immunogenicity. The term “recombinant virus” shall herein particularly refer to recombinant adeno-associated virus (AAV) particles, also termed rAAV. The term includes all serotypes and pseudotypes, such as, for example, AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, and AAV2 / 5, and the like. The term shall also include capsid variants, self-complementary AAV (scAAV), and rAAV with increased packaging capacity (see, for example, Duan D, Yue Y, Engelhardt JF. Expanding AAV packaging capacity with trans-splicing or overlapping vectors: a quantitative comparison. Mol Ther.2001 Oct;4(4):383-91. doi: 10.1006 / mthe.2001.0456. PMID: 11592843). A “transgene” in the context of the present invention generally refers to a protein or small ribonucleotide encoding region of a nucleic acid, an “expression cassette” shall designate the functional unit of nucleic acid sequences required for expression of a transgene, such as promoter, coding region, and terminator. As mentioned above, preferred is a method and medium according to the present invention, wherein the recombinantly producing comprises expression of the virus like particles (VLPs) or recombinant virus particles, such as rAAVs, comprising a suitable vector genome, such as an expression construct, preferably wherein the recombinantly producing comprises a triple transfection-based production or inducible producer cell lines or packaging cell lines (see above) for the recombinant virus particles, such as rAAV. Respective expression constructs are known to the person of skill, and disclosed in the art. As mentioned above, the method according to the present invention produces a lower amount of oligomerized and / or aggregated VLPs or higher genomic titer and / or capsid titer quality recombinant virus particles, such as rAAVs, when compared to a culture comprising non- supplemented mammalian cell line medium. Further preferred is the method according to the present invention, wherein the serotype of the rAAV is selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, and pseudotypes thereof. Yet another aspect of the present invention then relates to a mammalian cell line culture medium suitable for recombinantly producing virus like particles (VLPs) ) or recombinant virus particles, such as rAAVs, in a mammalian cell line, comprising a supplement of an intermediate substrate of the tricarboxylic acid (TCA) cycle selected from the group consisting of between 0.5 to 10 mM, preferably of between 1 to 8 mM, and most preferably of between 2 to 6 mM of sodium citrate, cis-aconitate, iso-citrate, alpha ketoglutarate, succinate, fumarate, malate, and oxaloacetate, preferably between 2 to 8 mM sodium citrate, more preferably between 4 to 7 mM sodium citrate in the medium. As a preferred example, citrate was identified to have a pronounced effect on the non-formation of oligomers and aggregates of VLPs during the VLPs production method or provides higher genomic titer and / or capsid titer recombinant virus particles, such as rAAVs, during the recombinant virus particle production method. As known to the person of skill and as described in the literature, citrate and the other TCA intermediates can be added to the medium in physiological concentrations (e.g. between 0.5 and 10 mM, preferably of between 1 to 8 mM, and more preferably of between 2 to 8 mM sodium citrate monobasic, most preferably between 4 to 7 mM sodium citrate monobasic) and as suitable physiologically acceptable salts (e.g. sodium, potassium or magnesium) and / or in physiologically acceptable buffers or solutions. Furthermore, the inventors identified that the addition of iron, which is an important cofactor in various cellular processes such as the TCA, lipid metabolism, and oxygen transport, respiration and DNA synthesis, also improves the higher genomic titer and / or capsid titer recombinant virus particle production, such as rAAVs, in particular when supplied as transferrin. Consequently, the inventors analyzed several culture conditions and additives or supplements in order to reduce VLP oligomerization and / or aggregation. Preferred is the culture medium according to the present invention, wherein the culture medium is further supplemented with at least one selected from the group consisting of transferrin, FeSO4, CuSO4, and vitamin feed, preferably vitamin feed, such as a putrescine / pyridoxine feed, preferably vitamin feed, and is preferably not supplemented with an additional amount of choline chloride feed or asparagine feed in the medium as produced or used. It was found that the further addition of a choline chloride feed or asparagine feed did not provide substantial benefits to the VLP culture. Preferred is the culture medium according to the present invention, wherein the culture medium is any suitable medium for recombinantly producing virus like particles (VLPs) or recombinant virus particles, such as rAAVs, in a mammalian cell line and for reducing oligomerized and / or aggregated VLPs in a method for recombinantly producing virus like particles (VLPs) or for producing a higher titer and higher quality recombinant virus particles, such as rAAVs, in a mammalian cell line, such as, for example DMEM or a medium equivalent to DMEM. Preferred is the culture medium according to the present invention, wherein the osmolality in the medium at the start of the mammalian cell line culture is at between 290 to 370 mOsm, more preferably at about 295 mOsm. The osmolality in the medium during the culture is preferably kept at these values as well, preferably by replacing consumed medium components and / or adding respective feeds. Yet another aspect of the present invention then relates to the use of the mammalian cell line culture media according to the present invention for recombinantly producing virus like particles (VLPs) or recombinant virus particles, such as rAAVs, in a mammalian cell line and for reducing oligomerized and / or aggregated VLPs or for producing a higher titer and / or higher quality recombinant virus particles, such as rAAVs, in a method for recombinantly producing virus like particles (VLPs) or recombinant virus particles, such as rAAVs in a mammalian cell line according to the present invention. Taken together, the inventor´s findings allow for the provision of a suitable mammalian cell line media for reducing impurities in a production process in mammalian cell lines for virus like particles (VLP) or for improved genomic and capsid titers for recombinant virus particles, such as rAAVs. In the context of the present invention, the term ”about” shall include a deviation of + / - 10% from the value as given. The present invention relates to the following items: Item 1. A method for recombinantly producing virus like particles (VLPs) or recombinant virus particles, such as rAAVs, in a mammalian cell line, comprising: the step of suitably culturing the mammalian cell line in a culture medium supplemented with an intermediate substrate of the tricarboxylic acid (TCA) cycle selected from the group consisting of between 0.5 to 10 mM, preferably of between 1 to 8 mM, and most preferably of between 2 to 6 mM of sodium citrate monobasic, cis-aconitate, iso-citrate, alpha ketoglutarate, succinate, fumarate, malate, and oxaloacetate, preferably between 1 to 8 mM sodium citrate monobasic, more preferably between 4 to 7 mM sodium citrate monobasic. Item 2. The method according to Item 1, wherein the culture medium is further supplemented with at least one selected from the group consisting of transferrin, FeSO4, CuSO4, and vitamin feed, preferably vitamin feed, and is preferably not supplemented with an additional amount of choline chloride feed or asparagine feed in the medium as produced or used. Item 3. The method according to Item 1 or 2, wherein the mammalian cell line is selected from CHO cell lines or HEK cell lines, wherein preferably the cell line is grown as an adherent or in suspension culture. Item 4. The method according to any one of Items 1 to 3, wherein the mammalian cell culture medium is DMEM or a medium equivalent to DMEM. Item 5. The method according to any one of Items 2 to 4, wherein the transferrin concentration in the suitable mammalian cell line medium at the start of the culture is between 0 to 15 mg / l, preferably between 5 to 10 mg / l, more preferably at about 9.4 mg / l or at about 5 mg / l, and preferably is held at more preferably at about 9.4 mg / l, and preferably is held at a preferred value during the culture. Item 6. The method according to any one of Items 1 to 5, wherein the osmolality of the mammalian cell line culture medium at the start of the culture is at between about 295 to about 370 mOsm, more preferably at about 300 to about 350 mOsm, and preferably is held within these values during the culture. Item 7. The method according to any one of Items 2 to 6, wherein the FeSO4 concentration in the suitable mammalian cell line medium at the start of the culture is between 1 to 20 mg / l, preferably between 5 to 15.9 mg / l, and more preferably at about 13.9 mg / l or at about 9.7 mg / l, and preferably is held within these values during the culture. Item 8. The method according to any one of claims 2 to 7, wherein the CuSO4 concentration in the suitable mammalian cell line medium at the start of the culture is between 50 to 400 µg / l, preferably at between 99.8 to 350 µg / l, and more preferably at about 250 µg / l, and preferably is held within these values during the culture. Item 9. The method according to any one of Items 1 to 8, wherein the recombinantly producing comprises expression of the virus like particles (VLPs) or recombinant virus particles, such as rAAVs, comprising a suitable genetic expression construct., wherein the recombinantly producing preferably comprises a production for the recombinant virus particles, such as rAAVs, comprising at least one of triple transfection, packaging, and producer cell lines. Item 10. The method according to any one of Items 1 to 9, wherein the serotype of the rAAV is selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, and pseudotypes thereof. 11. A mammalian cell line culture medium suitable for recombinantly producing virus like particles (VLPs) or recombinant virus particles, such as rAAVs, in a mammalian cell line, comprising a supplement of an intermediate substrate of the tricarboxylic acid (TCA) cycle selected from the group consisting of between 0.5 to 10 mM, preferably of between 1 to 8 mM, and most preferably of between 2 to 6 mM of sodium citrate monobasic, cis-aconitate, iso- citrate, alpha ketoglutarate, succinate, fumarate, malate, and oxaloacetate, preferably between 2 to 6 mM sodium citrate monobasic, more preferably 4 mM sodium citrate monobasic in the medium. Item 12. The cell line culture medium according to Item 11, wherein the culture medium is further supplemented with at least one selected from the group consisting of transferrin, FeSO4, CuSO4, and vitamin feed, preferably vitamin feed, and is preferably not supplemented with an additional amount of choline chloride feed or asparagine feed. Item 13. The cell line culture medium according to Item 11 or 12, wherein the mammalian cell culture medium is DMEM or a medium equivalent to DMEM. Item 14. The cell line culture medium according to any one of Items 11 to 13, wherein the osmolality of the mammalian cell line culture medium, preferably at the start of the culture, is between 290 to 370 mOsm, preferably at about 295 mOsm. Item 15. Use of the mammalian cell line culture media according to any one of Items 11 to 13 for recombinantly producing virus like particles (VLPs) or recombinant virus particles, such as rAAVs, in a mammalian cell line and for reducing oligomerized and / or aggregated VLPs or for producing higher titer- and / or quality-recombinant virus particles, such as rAAVs, in a method for recombinantly producing virus like particles (VLPs) or recombinant virus particles, such as rAAVs in a mammalian cell line according to any one of Items 1 to 9. The invention will now be described further in the following examples with reference to the accompanying figures, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited are incorporated by reference in their entireties. Figure 1 shows the result of VLP production according to the state of the art. Ding et al., 2010 shows in its Figure 1b an asymmetrical flow field flow fractogram for a mixture of unreacted capsomeres, virus-like particles and aggregates in an assembly product. Root-mean-square radius, (r. m. s. radius on the right scale, was determined with multiangle light scattering at UV 280 nm. Figure 2 shows the results of a Prediction Profiler analysis (unconstrained). The DoE responses titer (log), quality and desirability were measured as a function of the DoE factors osmolality, the transferrin and sodium citrate concentrations, and for the VLPs an additional supplement with choline chloride, asparagine (Asn) and / or vitamin feed. Solid black lines represent the average response, and the dark gray lines indicate the confidence interval at a 95% confidence level. Figure 3 shows a chromatogram of immunoprecipitated VLPs A as obtained at a wavelength of UV 280 nm. The elution of the pure assembled VLPs was observed at 9.1 minutes. Figure 4 shows a chromatogram of VLP A expression in a culture medium for mammalian cell lines, CDM2-Medium (Thermo Fisher) as obtained at a wavelength of UV 280 nm. The elution of the pure assembled VLPs was observed at approximately 9 - 10 min. The large oligomer peak at smaller retention times (< 9.0 min) are contaminations, such as oligomerized and / or aggregated VLPs. The peaks at the retention times > 10 minutes are components of the culture supernatant medium. Figure 5 shows a chromatogram of VLP A expression in a non-balanced culture medium for mammalian cell lines (here: CHO cells) equivalent to the standard medium DMEM as obtained at a wavelength of UV 280 nm. The elution of the pure assembled VLPs is at approximately 9 – 10 min. Figure 6 shows a chromatogram of VLP A expression in the balanced culture medium for mammalian cell lines (here: CHO cells) equivalent to the standard medium DMEM according to the present invention, obtained at a wavelength of UV 280 nm. The elution of the pure assembled VLPs is at approximately 9 – 10 min. The large oligomer peak at smaller retention times (< 9.0 min) is completely missing in the balanced culture medium for mammalian cell lines (equivalent to the standard medium DMEM), when compared to the CDM2 medium. Figure 7 shows a chromatogram of pure assembled VLPs B at three different wavelengths (260, 280, and 300 nm). The elution of the pure assembled VLPs is at 9.1 minutes. Every shade of gray represents one type of wavelength. Figure 8 shows a chromatogram of VLP B expression in a non-balanced culture media for mammalian cell lines (here: CHO cells) equivalent to the standard medium DMEM, obtained at a wavelength of UV 280 nm. The elution of the pure assembled VLPs is at approximately 9 – 10 min. Figure 9 shows a chromatogram of VLP B expression in a balanced culture medium for mammalian cell lines (here: CHO cells) equivalent to the standard medium DMEM according to the invention, obtained at a wavelength of UV 280 nm. The elution of the pure assembled VLPs is at approximately 9 – 10 min. The large oligomer peak at smaller retention times (< 9.0 min) is missing completely in the balanced culture media for mammalian cell lines (equivalent to the standard medium DMEM), when compared to the medium equivalent to the standard medium DMEM. Figure 10 shows a chromatogram of VLP C expression in a balanced culture medium for HEK293 cells according to the invention (black line, lowest left of peak at 9.068 min, highest at the right side of the peak), obtained at a wavelength of UV 280 nm. The elution of the pure assembled VLPs is at 9.068 min. The balanced culture medium shows significantly less aggregates compared to commonly used media (light gray and dark gray lines). The sample was taken at day 4 of the culture. Figure 11 shows the results of AAV production in HEK293 cells using the Ambr ® 15 Cell Culture System and 1.2 mM, 2.4 mM, 4.8 mM, and 9.6 mM sodium citrate monobasic. A triple- transfection-based AAV production of serotype AAV2 with mGreenLantern® was performed. A) Viable cell density (VCD); B) cell viability: C) cell aggregation; D) fold change genomic titer; E) fold change capsid titer; and F) fold change of %full particles. Figure 12 shows the results of AAV production in HEK293 cells in shake flasks and 1.2 mM, 2.4 mM, 4.8 mM, and 9.6 mM sodium citrate monobasic. A triple-transfection-based AAV production of serotype AAV9 with mGreenLantern® was performed. A) Viable cell density (VCD); B) cell viability: C) cell aggregation; D) fold change genomic titer; E) fold change capsid titer; and F) fold change of %full particles. Examples In the context of the present invention, the inventors have identified various culture media conditions and supplements in order to compensate for a lack of substrates for the cells in amino acid and lipid synthesis. This leads to a reduction of VLP oligomerization and / or aggregation in mammalian cell lines during the present VLP production method, as well as the low production of recombinant virus particles, such as AAVs, and insufficient quality, i.e. low titer, and / or missing a high percentage of “full” virus particles, i.e. containing a desired vector genome, preferably comprising at least one desired transgene, for example in a suitable expression cassette. Materials and Methods Cell lines Mammalian cell lines require a richer medium and feed for their growth, and more stringent growth conditions and sustained growth periods than microorganisms. Chinese hamster ovary cell line K1 (CHO-K1) was derived as a subclone from the parental CHO cell line, which was initiated from a biopsy of an ovary of an adult, female Chinese hamster in 1957. The cell line is commonly used for industrial biotechnology and toxicology research (CCL-61TM) (https: / / www.atcc.org / products / ccl-61). Human embryonic kidney (HEK) 293 cells can be commercially obtained from, for example, the ATCC cell type collection (CRL-1573). Herein, HEK293 cells were grown as adherent or suspension cells. Culture Medium CDM2 OPT 1.1 (https: / / www.thermofisher.com / order / catalog / product / ME110158P1). State of the art production method Common VLP production methods use a standard culture medium for mammalian cell lines that can also be used for other product types, such as antibody production, such as CDM2. These types of standard basic culture media have the disadvantage of the generation of oligomers / aggregates of VLPs during normal production conditions of 37°C and pH 7.0, due to a lack of optimal substrate conditions (Figure 1, which is Figure 1b of Ding et al., 2010). Figure 1 relates to the yield of a VLP production with the state of the art production method for VLPs, showing an asymmetrical flow field flow fractogram for a mixture of unreacted capsomeres, virus-like particles and aggregates in an assembly product. The asymmetricalflow field-flow fractionation was used to characterize the self-association reaction products quantitatively based on differences in size. The size and quantity of species presented in a mixed sample were determined by multiangle light scattering (MALS) and ultra-violet (UV) absorbance, respectively. Figure 1 shows a typical fractogram for products of VLP self- association, with the major peaks corresponding to unreacted capsomeres, VLPs (root-mean- square radius, r = 20–35 nm) and large aggregates (r > 80 nm). A shoulder to the right of the VLP peak including particles morphologically different to the majority of VLPs obtained (Chuan et al., 2008a), which are the undesirable aggregates. Typically, recombinant virus production, such as AAV production, is conducted via transient transfection or with packaging and producer cell lines. Glucose or a feed is often fed as bolus to avoid limitation during the process. The basal media has the disadvantage of generating low titers during AAVv production process under optimal production conditions. AAV depends on a co-infecting helper virus (usually adenovirus) for productive infection to occur. Additionally, the recombinant AAV genome has two essential genes removed to prevent integration and replication to make AAV a safe and effective tool for gene delivery. Therefore, for AAV production, essential genes must be provided in trans. In the experiments, the triple transfection method was used, involving co-transfecting a cell line (usually HEK293) with the recombinant AAV plasmid containing the gene of interest or vector genome or transgene (here GFP), a plasmid containing the essential rep and cap genes, and a third adenovirus-derived helper plasmid supplying genes needed for replication. As the person of skill will be aware, in the context of the present invention concentrations of citrate and other salts are given as sodium citrate monobasic and can be readily converted for the other salt-forms of citrate. See also above. The addition of citrate to the cell culture also significantly reduces the formation of aggregates of the cells as cultured (“cell clumps”). This further helps to maintain a healthy and efficient cell culture and avoids adding anti-clumping agents like “Anti-clumping agent“ (ThermoFisher) or “Scattering reagent CHO” (Gmep International) to the culture medium, which may adversely affect cell behavior. Design and analysis of the experiment for an improved production method Experiment 1 (embodiment VLPs) The development of a method for reducing oligomerization and / or aggregation of virus-like particles or recombinant AAV in a production method in mammalian cell lines started with the design of the experiment (DoE) space for VLPs to investigate the best culture medium conditions, and the conditions were then used for the AAV assays as well. A definitive screening design was created based on six continuous factors (process parameters) and two responses. The tested process parameters (factors) and responses were as follows: The production of VLPs in mammalian cell lines (here, HEK293 or CHO) is an intensive process demanding a high level of substrates for the lipid and protein synthesis pathways. A saturated culture condition leads to less aggregation of the VLPs during the production of VLPs in cell lines. Therefore, the inventors screened various culture conditions and additives to minimize the VLP aggregation in cell lines. In order to support the amino acid and lipid synthesis behind the TCA and to generate more energy in form of ATP, the following process parameters / substrate concentrations (DoE factors) were screened: - Sodium citrate (monobasic) was tested from a range of 0 to 10 mM, in particular at 0 to 4 mM for VLPs. - The osmotic concentration at the start of the culture, also known as osmolality, was tested in a range 290 to 370 mOsm. - A high level of oligomerization of the VLPs is found, when the osmolality is 420 mOsm or higher (data not shown). - Human holo-transferrin was tested in a range of between 0 to 10 mg / l. - Choline chloride, asparagine and vitamin feeds were either supplemented or not. Titer [µg / ml] and the quality of the VLPs were measured as DoE responses. The following additional parameters were tested: - The FeSO4-concentration was tested in a range of between 9.6 to 13.9 mg / l. - The CuSO4-concentration was tested in a range of between 99.8 to 250 µg / l. Analytic process during the production process - embodiment VLP Similar to the rAAV protocol, during the fermentation process, different at-line parameters were monitored to evaluate the culture ingredients. Glucose, glutamine, ammonia and lactate were measured with a Cedex BioHT (Roche) and the cell amount was measured with a Cedex HiRes (Roche). The daily pH recalibration was performed with an analysis module (Sartorius), which is directly connected to the Ambr15 system. The production process was performed with an Ambr1548cc bioreactor system (Sartorius). The bioreactors of this system have a working volume between 10 to 15 ml. The culture media conditions were identical for each experimental setup, and comprised at pH 7.0, stirring between 800 to 1200 rpm, and a temperature of 37°C. Experiment 2 (embodiment rAAV) (see Figure 11) The production of rAAVs in mammalian cell lines (here, HEK293 or CHO) is also an intensive process demanding a high level of substrates for the lipid and protein synthesis pathways. A saturated culture condition leads to insufficient production of rAAV particle titers and an insufficient quality, i.e. a low percentage of “full” virus particles, i.e. containing a desired vector genome, preferably comprising at least one desired transgene, for example in a suitable expression cassette. Therefore, the inventors screened various culture conditions and additives to minimize these issues in cell lines. In order to support the amino acid and lipid synthesis behind the TCA and to generate more energy in form of ATP, the following process parameters / substrate concentrations were screened: The Ambr ® 15 Cell Culture System was used for 48 parallel cultures. A triple transfection-based AAV production was used. Serotype AAV2 was used with mGreenLantern® (Campbell BC, et al.: mGreenLantern: a bright monomeric fluorescent protein with rapid expression and cell filling properties for neuronal imaging. Proc Natl Acad Sci U S A.2020 Dec 1;117(48):30710-30721. doi: 10.1073 / pnas.2000942117. Epub 2020 Nov 18. PMID: 33208539; PMCID: PMC7720163.) as a vector genome or transgene. Sodium citrate (monobasic) was tested from a range of 0 to 10 mM, in particular 1.2 mM, 2.4 mM, 4.8 mM, and 9.6 mM for rAAV The osmolality at the start of the culture (inoculation of bioreactor) was about 290 mOsm / kg. Glucose was fed as a bolus feed. During the culture process, different at-line parameters were monitored in order to analyze and evaluate the culture. Glucose (Glc), glutamine, ammonia and lactate was measured with a Cedex BioHT (Roche), and the cell count was detected with a Cedex HiRes (Roche). A daily pH recalibration was performed with an analysis module (Sartorius), which is directly connected to the Ambr15 system. The production process was performed with an Ambr1548 bioreactor system (Sartorius, see also above). The bioreactors of this system had a working volume of between 10 to 15 mL. The culture was performed at pH 7.0, with stirring at 1000 rpm, and a temperature of 36.5°C. Genomic titer and capsid titer were measured by ddPCR and ECLIA, respectively. “%full” (i.e. loaded capsids) was calculated as ratio of genomic to capsid titer. Experiment 3 (embodiment rAAV) (see Figure 12) The production of rAAVs in mammalian cell lines (here, HEK293 or CHO) is also an intensive process demanding a high level of substrates for the lipid and protein synthesis pathways. A saturated culture condition leads to insufficient production of rAAV particle titers and an insufficient quality, i.e. a low percentage of “full” virus particles, i.e. containing a desired vector genome, preferably comprising at least one desired transgene, for example in a suitable expression cassette. Therefore, the inventors screened various culture conditions and additives to minimize these issues in cell lines. In order to support the amino acid and lipid synthesis behind the TCA and to generate more energy in form of ATP, the following process parameters / substrate concentrations (DoE factors) were screened: The culture was performed in shake flasks in parallel. A triple transfection-based AAV production was used. Serotype AAV9 was used with mGreenLantern® (Campbell BC, et al.: mGreenLantern: a bright monomeric fluorescent protein with rapid expression and cell filling properties for neuronal imaging. Proc Natl Acad Sci U S A.2020 Dec 1;117(48):30710-30721. doi: 10.1073 / pnas.2000942117. Epub 2020 Nov 18. PMID: 33208539; PMCID: PMC7720163.) as a desired vector genome or transgene. Sodium citrate (monobasic) was tested from a range of 0 to 10 mM, in particular 1.2 mM, 2.4 mM, 4.8 mM, and 9.6 mM for rAAV The osmolality at the start of the culture (inoculation of bioreactor) was about 290 mOsm / kg. No glucose feed was added. During the culture process, different at-line parameters were monitored in order to analyze and evaluate the culture. Glucose (Glc), glutamine, ammonia and lactate was measured with a Cedex BioHT (Roche), and the cell count was detected with a Cedex HiRes (Roche). Cultivation of cell cultures was conducted in an orbital shaker (36.5°C, 180 rpm, 5 cm amplitude, 5% CO2). Cells were cultivated in a working volume of between 40 to 47.5 mL. Genomic titer and capsid titer were measured by ddPCR and ECLIA, respectively. “%full” (i.e. loaded capsids) was calculated as ratio of genomic to capsid titer. The media used for experiments 2 and 3 were: Medium 1 DMEM (equivalent medium) Osmolality of about 290+-15 mOsm / kg (at the start of culture). Transferrin concentration (Tf) of 5 mg / l. CuSO4 concentration of 334 µg / l FeSO4concentration of 9.7 mg / L It was found that the osmolality can rise over 400 mOsm / kg in the culture media during culture. Screening and optimization of a suitable cell culture media For the screening experiments, a culture media for mammalian cell lines equivalent to the standard medium DMEM was used, i.e. a functional equivalent thereof which contains the ingredient compounds of DMEM, pyridoxine but is free from any proteins, peptides, and hormones, and which does not contain the additives specifically mentioned herein, e.g. in the claims (such as citrate, transferrin, FeSO4 , CuSO4, and feeds) appended herewith. DMEM and a (functional) equivalent of DMEM both denote media compositions which are serum-free. In an optional but not essential specific embodiment, the functional equivalent of DMEM further includes in physiological concentrations pyridoxine, biotin, vitamin B12, and other micronutrients as the case may be. DMEM and a (functional) equivalent of DMEM within the meaning for this invention both support the growth of an exemplary culture of at least CHO cells or human embryonic kidney cells, such as HEK293. For the optimization of DoE, this starting medium was supplemented with substrates and feeds and different osmolality values were used in 36 different experimental set ups with six DoE factors (Table 1). The prioritized attribute targets / DoE responses were the titer and the quality of the VLPs. The prioritized attribute targets / DoE responses for the rAAV were titers as produced, and “%full” (i.e. loaded capsids), calculated as ratio of genomic to capsid titer. Table 1: Design of Experiment optimization used for screening – embodiment VLPs

[0002] Osmolality (mOsm) was determined as mOsm / kg H2O. The screening data shown in Table 1 was analyzed in a stepwise regression analysis to describe each of the two DoE responses as a function of the DoE factors as a “Prediction Profiler”. Figure 2 shows a Prediction Profiler of each DoE response. Based on the prediction profiler analysis, the inventors surprisingly identified that the addition of 2 to 4 mM sodium citrate, optionally supplemented with 4 to 10 mM transferrin and / or combined with a vitamin feed supplement reduced the aggregate formation below the detection limit (zero). Furthermore, the Prediction Profiler analysis (as shown in Figure 2) showed that it was beneficial to provide a medium with an osmolality of about 300 mOsm, and to have the medium supplemented with sodium citrate, optionally transferrin, and optionally a vitamin feed supplement. The analysis further showed that for the VLPs a supplementation with an additional amount of choline chloride feed or asparagine feed in the medium as produced or used should be avoided. Therefore, the quality for the desired VLPs could be significantly improved without additional preparation steps for reprocessing the usual aggregates generated in the common mammalian cell line media without additional supplement. Design and analysis of further experiments for the improved production method Experiment 4: Further improvement of the cell culture media composition Based on the results of experiment 1, the inventors further balanced the VLP culturing conditions as follows. Screening and optimization of cell culture media composition The screening conditions for this experiment were an osmolality of about 295 mOsm, a sodium citrate concentration of about 4 mM, the addition of transferrin (optional for quality, but increases yield), and an addition of FeSO4 or CuSO4 for increased quality. For the optimization of the DoE, a culture media for mammalian cell lines equivalent to DMEM was balanced. The following DoE factors were chosen to gain prioritized attribute targets such as quality of the VLPs: Medium 2: - Osmolality (Osmo) of 295 mOsm (at the start of the culture) - Transferrin concentration (Tf) of 9.4 mg / l - Sodium citrate concentration of 4 mM (SC4) - Supplemented during the culturing process with a vitamin feed (VitFeed) - start concentration of FeSO4at between 9.6 to 15.9 mg / l (giving different variants of medium 2) Medium 3: - Osmolality (Osmo) of 295 mOsm (at the start of the culture) - Transferrin concentration (Tf) of 9.4 mg / l - Sodium citrate concentration of 4 mM - Supplemented with a vitamin feed during the culturing process - start concentration of CuSO4 at between 99.8 to 350 µg / l (giving different variants of medium 3) Medium 4: - Osmolality of 295 mOsm (at the start of the culture) - Sodium citrate concentration of 4 mM - Supplemented with a vitamin feed during the culturing process - start concentrations of FeSO4 at between 9.6 to 15.9 mg / l (giving different variants of medium 4) Analysis of the quality of the product as produced For analyzing the quality of the VLP process product, size exclusion chromatography (SEC) was applied. Chromatography of a reference: As a reference for pure assembled VLPs, a chromatogram of the immunoprecipitated VLPs was generated. The elution of the pure assembled VLPs was found at 9.1 minutes (see Figure 3). Chromatography of the expression of VLPs in a common base culture media for mammalian cell lines: A chromatography of the expression of VLPs in the common base culture media for mammalian cell lines CDM2-Medium (Thermo Fisher) was performed as a comparison for the pure assembled VLPs (Figure 3). The experimental set up for the fermentation was performed in 1000 l medium. The elution of the pure assembled VLPs was found at approximately 9 – 10 min. A large oligomer peak is clearly visible at smaller retention times (< 9.0 min). These contaminations must be removed through complex downstream processing including immunoprecipitation. Peaks with a retention time > 10 minutes are caused by components of the culture supernatant medium and can be easily removed (Figure 4). The problem of these kinds of contamination is solved by the present invention through an optimization of the medium composition, leading to a minimization of the oligomer species and / or to a complete absence of contamination. Chromatography of the expression of VLPs in a culture medium for mammalian cell lines equivalent to DMEM A comparative chromatography of the expression of VLPs in a culture medium for mammalian cell lines equivalent to the standard medium DMEM was produced for the pure assembled VLPs (Figure 3) and the expression of VLPs in a common base culture media for mammalian cell lines (Figure 4). The elution of the pure assembled VLPs was found at approximately 9 – 10 min. The proportion of oligomer species to the pure assembled VLPS was reduced, but there are still oligomers present in the culture medium (Figure 5). Chromatography of the expression of VLPs in a balanced culture media for mammalian cell lines (balanced equivalent to DMEM) A chromatography of the expression of VLPs in a balanced culture media for mammalian cell lines (equivalent to DMEM) according to the invention was produced. The balanced equivalent to DMEM was supplemented according to the composition Medium 1 (above). A peak of the pure assembled VLPs was found at a retention time of approximately 9 to 10 min. The large oligomer peak “shoulder” at smaller retention times (< 9.0 min) was completely absent (Figure 6). The optimization of the equivalent to DMEM medium according to the composition of Medium 1 as above led to a complete lack of contamination by, for example, VLP oligomers and aggregates. This lack of contamination significantly simplifies the down-stream processing of VLPs production. The production process of VLPs in the balanced culture media for mammalian cell lines provides a higher yield of pure assembled VLPs. The balanced culture media and conditions for mammalian cell lines can be used in the production of other types of VLP particles as well. Chromatography of a reference for three different types of VLP particles: As a reference for the pure assembled VLPs, a chromatography of the immunoprecipitated VLPs was conducted. The elution of pure assembled VLPs was found at 9.1 minutes. Every color represents one wavelength as used (Figure 7). Chromatography of the expression of VLPs in a culture media for mammalian cell lines equivalent to DMEM A chromatography of the expression of VLPs in a culture media for mammalian cell lines equivalent to DMEM was performed as a comparison to the pure assembled VLPs (Figure 7). The elution of the pure assembled VLPs was at approximately 9 to 10 minutes. A large oligomer peak was again visible at smaller retention times of 9.0 minutes. These contaminations must be removed using complex downstream processing, such as immunoprecipitation. Peaks with a retention time > 10 minutes are, as explained above, caused by components of the culture supernatant medium and can be easily removed (Figure 8). Chromatography of the expression of VLPs in a balanced culture media for mammalian cell lines (balanced equivalent to DMEM) A chromatography of the expression of VLPs in balanced culture media for mammalian cell lines (equivalent to DMEM) according to the invention was performed. The balanced equivalent to DMEM was supplemented according to the composition Medium 1. A peak of the pure assembled VLPs was found at a retention time of approximately 9 to 10 min. The large oligomer peak “shoulder” at smaller retention times (< 9.0 min) was completely absent, as seen before (Figure 9). The optimization of the equivalent to DMEM medium according to Medium 1 as indicated above led to a complete lack of contaminations, such as VLP oligomers and aggregates. This lack of contamination significantly simplifies the down-stream processing of VLPs production. The production process of VLPs in the balanced culture medium for mammalian cell lines allows for a higher yield of pure assembled VLP particles. The above examples show that supplementing a suitable mammalian cell line culture medium with at least 2 to 4 mM of the TCA intermediate sodium citrate, and optionally supplementing the suitable mammalian cell line culture medium with further additives and / or further adjusting the conditions (e.g. mOsm / kg H2O) in the mammalian cell line culture medium causes a reduction of impurities in a virus like particle (VLP) production method in mammalian cell lines. Consequently, the reduction of impurities leads to a higher production yield of the pure assembled VLPs and to a more efficient production process of VLPs without further cost and time intensive handling of the downstream processes to remove the impurities, such as oligomers and / or aggregates of the VLPs. References as cited Sánchez-Rodríguez, S. P.; et al. Human parvovirus B19 virus-like particles: In vitro assembly and stability. Biochimie 2012, 94(3): 870–878. Effio, C. L. and Hubbuch, J. Next generation vaccines and vectors: Designing downstream processes for recombinant protein-based virus-like particles. Biotechnol J.2015 May; 10(5): 715-727. Gupta, R; et al. Platforms, advances, and technical challenges in virus-like particles-based vaccines. Front Immunol.2023 Feb 9; 14:1123805. Le, D. T. and Müller K. M., In Vitro Assembly of Virus-Like Particles and Their Applications. Life (Basel) 2021 Apr 10; 11(4): 334. Zhuo, C.; Tao, Y. and Li M. Engineered virus-like particles: paving the way for effective somatic genome editing. Signal Transduction and Targeted Therapy 2022, 7:279. Shi, L.; et al. Stabilization of human papillomavirus virus-like particles by non-ionic surfactants. J Pharm Sci.2005 Jul; 94(7): 1538-51. Ding, Y.; Chuan, Y. P.; He, L.; Middelberg A. P. J. Modeling the competition between aggregation and self-assembly during virus-like particle processing. Biotechnol. Bioeng.2010, 107: 550-560. Chuan Y. P.; Fan Y. Y.; Lua L. H. L.; Middelberg A. P. J. Quantitative analysis of virus-like particle size and distribution byfield-flow fractionation. Biotechnol Bioeng 2008a, 99(6): 1425–1433.

Claims

Claims 1. A method for recombinantly producing virus like particles (VLPs) or recombinant virus particles, such as rAAVs, in a mammalian cell line, comprising: the step of suitably culturing the mammalian cell line in a culture medium supplemented with an intermediate substrate of the tricarboxylic acid (TCA) cycle selected from the group consisting of between 0.5 to 10 mM, preferably of between 1 to 8 mM, and most preferably of between 2 to 6 mM of sodium citrate monobasic, cis-aconitate, iso-citrate, alpha ketoglutarate, succinate, fumarate, malate, and oxaloacetate, preferably between 1 to 8 mM sodium citrate monobasic, more preferably between 2 to 7 mM sodium citrate monobasic.

2. The method according to claim 1, wherein the culture medium is further supplemented with at least one selected from the group consisting of transferrin, FeSO4, CuSO4, and vitamin feed, preferably vitamin feed, and is preferably not supplemented with an additional amount of choline chloride feed or asparagine feed in the medium as produced or used.

3. The method according to claim 1 or 2, wherein the mammalian cell line is selected from HEK cell lines or CHO cell lines, wherein preferably the cell line is grown as an adherent or in suspension culture.

4. The method according to any one of claims 1 to 3, wherein the mammalian cell culture medium is DMEM or a medium equivalent to DMEM.

5. The method according to any one of claims 2 to 4, wherein the transferrin concentration in the suitable mammalian cell line medium at the start of the culture is between 0 to 15 mg / l, preferably between 5 to 10 mg / l, more preferably at about 9.4 mg / l or at about 5 mg / l, and preferably is held at more preferably at about 9.4 mg / l, and preferably is held at a preferred value during the culture.

6. The method according to any one of claims 1 to 5, wherein the osmolality of the mammalian cell line culture medium at the start of the culture is at between about 295 to about 370 mOsm, more preferably at about 300 to about 350 mOsm, and preferably is held within these values during the culture.

7. The method according to any one of claims 2 to 6, wherein the FeSO4concentration in the suitable mammalian cell line medium at the start of the culture is between 1 to 20 mg / l, preferably between 5 to 15.9 mg / l, and more preferably at about 13.9 mg / l or at about 9.7 mg / l, and preferably is held within these values during the culture.

8. The method according to any one of claims 2 to 7, wherein the CuSO4 concentration in the suitable mammalian cell line medium at the start of the culture is between 50 to 400 µg / l, preferably at between 99.8 to 350 µg / l, and more preferably at about 250 µg / l, and preferably is held within these values during the culture.

9. The method according to any one of claims 1 to 8, wherein the recombinantly producing comprises expression of the virus like particles (VLPs) or recombinant virus particles, such as rAAVs, comprising a suitable genetic expression construct., wherein the recombinantly producing preferably comprises a production for the recombinant virus particles, such as rAAVs, comprising at least one of triple transfection, packaging, and producer cell lines.

10. The method according to any one of claims 1 to 9, wherein the serotype of the rAAV is selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, and pseudotypes thereof.

11. A mammalian cell line culture medium suitable for recombinantly producing virus like particles (VLPs) or recombinant virus particles, such as rAAVs, in a mammalian cell line, comprising a supplement of an intermediate substrate of the tricarboxylic acid (TCA) cycle selected from the group consisting of between 0.5 to 10 mM, preferably of between 1 to 8 mM, and most preferably of between 2 to 6 mM of sodium citrate monobasic, cis-aconitate, iso- citrate, alpha ketoglutarate, succinate, fumarate, malate, and oxaloacetate, preferably between 2 to 6 mM sodium citrate monobasic, more preferably 4 mM sodium citrate monobasic in the medium.

12. The cell line culture medium according to claim 11, wherein the culture medium is further supplemented with at least one selected from the group consisting of transferrin, FeSO4, CuSO4, and vitamin feed, preferably vitamin feed, and is preferably not supplemented with an additional amount of choline chloride feed or asparagine feed.

13. The cell line culture medium according to claim 11 or 12, wherein the mammalian cell culture medium is DMEM or a medium equivalent to DMEM.

14. The cell line culture medium according to any one of claims 11 to 13, wherein the osmolality of the mammalian cell line culture medium, preferably at the start of the culture, is between 290 to 370 mOsm, preferably at about 295 mOsm.

15. Use of the mammalian cell line culture media according to any one of claims 11 to 13 for recombinantly producing virus like particles (VLPs) or recombinant virus particles, such as rAAVs, in a mammalian cell line and for reducing oligomerized and / or aggregated VLPs or for producing higher titer- and / or quality-recombinant virus particles, such as rAAVs, in a method for recombinantly producing virus like particles (VLPs) or recombinant virus particles, such as rAAVs in a mammalian cell line according to any one of claims 1 to 9.

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