Method for enhancing viral vector production in cell culture using glycyl-glutamine

Abstract

The present invention relates to a method for producing RNA or DNA containing viral particles in cell culture, a supplement for the culture medium, and a culture medium for use in the production of RNA or DNA containing viral particles, the method comprising the step of:

Description

[Technical field]

[0001] The present invention relates to dipeptides and their use in cell culture. Furthermore, the present invention relates to biotechnological production processes. More specifically, the present invention relates to a method for enhancing viral vector production in cell culture using glycyl-glutamine. [Background technology]

[0002] Viral vectors are increasingly being used for therapeutic applications. Prominent examples approved for marketing authorization in the European and / or US markets include vaccine applications such as viral vector-based Ebola vaccines (ERVEBO®, based on vesicular stomatitis virus [1]), COVID-19 vaccines (Vazxevria™, Ad26.COV2.S, based on adenovirus (Ad) [2,3]) or gene therapy (Luxturna®, Zolgensma®, based on adeno-associated virus (AAV)2 resp. AAV9 [4,5]). In addition, various clinical trials based on viral vectors are ongoing, with over 200 approved clinical trials for adeno-associated viruses alone [6]. As biopharmaceutical development progresses from the clinical to commercial stages, there is an increasing demand for efficient viral vector production processes.

[0003] Viral vectors are produced by cell culture processes. Liquid media is necessary to support cell growth, transfection and ultimately viral vector production. While the use of serum and serum-derived (mainly fetal bovine serum or FBS) proteins remains common in academic settings and small-scale clinical trials, industrial production in stirred tank bioreactors is primarily serum-free, limiting supply chain, regulatory and quality control risks. A key challenge is to replace serum without losing performance in terms of cell density and viability as well as productivity [7]. Current research is focused on a better understanding of individual media components while attempting to replace animal and human-derived serum to increase reproducibility in each production batch and reduce blood-derived components in therapeutic manufacturing processes [8].

[0004] Glutamine is a particularly interesting component in mammalian cell culture media. For optimal growth of cells, the recommended glutamine level is 3-10 times higher compared to other amino acids in the medium [9]. It must be added directly before use, since it is poorly soluble and unstable at high temperatures (e.g., during heat sterilization), thereby forming cytotoxic pyroglutamate and ammonia. In contrast, glutamine-dipeptides such as alanyl-glutamine or glycyl-glutamine not only offer high solubility (568 and 154 g / L at 20 °C in water for Aln-Gln and Gly-Gln compared to 36 g / L for Gln)

[10] , but have also been found to be relatively stable during autoclaving and storage. As a result, they can accommodate high growth rates indicated by short doubling times even after sterilization

[11] . An approach to further increase the thermal stability of glutamine-dipeptides included n-acylation of such dipeptides, which resulted in further improvements in thermal stability while maintaining the positive effect on cell growth similar to the corresponding nonacylated dipeptides

[12] .

[0005] State-of-the-art animal component-free, chemically defined media formulations optimized for the growth and transfection of the respective viral vector production in HEK293 cells are still formulated without Gln to avoid the formation of degradation products. These require the addition of either Gln or glutamine dipeptide before use. Standard recommendations issued by manufacturers for media use typically include the addition of Gln or Ala-Gln to a final concentration of 4 mM–8 mM before use [13,14].

[0006] Standard recommendations include Ala-Gln, but the specific differences between Gly-Gln and Ala-Gln have not been investigated so far in viral vector production. Christie et al.

[15] showed that high cell yields could be obtained with antibody-secreting mouse hybridoma (CC9C10) cultures containing either Ala-Gln or Gly-Gln, but monoclonal antibody production was comparable in all three cultures regardless of glutamine source (Gln, Ala-Gln, Gly-Gln). This remained effective despite an even higher concentration of Gly-Gln (20 mM) compared to Ala-Gln (6 mM) used in the study. Neither HEK cell lines nor viral vector production nor medium-free of animal origin were part of the evaluation.

[0007] A similar observation is made in WO2011133902

[16] , which describes a method for producing nucleic acid-containing viral particles in animal cell culture containing different dipeptides. The document mentions the publication on glutamine peptides cited above, but focuses on the use of cysteine- and tyrosine-dipeptides. Furthermore, the referenced patent application only discloses that Gln is unstable and can be replaced by Ala-Gln or Gly-Gln, but does not describe an increase in viral vector titer.

[0008] In contrast, WO2019195729

[17] focuses on AAV production and discloses that Ala-Gln is used, resulting in at least comparable full / empty ratios (determined based on TEM or analytical ultracentrifugation), but does not describe a similar effect for Gly-Gln. Despite the use of HEK cells, this study also used DMEM as an exemplary medium instead of animal origin-free media as described in this study. As a result, a change in Gln source (e.g., from Ala-Gln to Gly-Gln) is not found to have a positive effect on viral vector productivity or quality in HEK cell-based production processes. Although cell culture media described in the prior art containing glutamine, glutamine-dipeptide or derivatives of glutamine-dipeptide are sufficient to enhance cell growth and thereby enhance cell culture-based processes that rely solely on cell proliferation and the resulting density, there is still a need for media or media supplements that enhance product quality, for example by improving the yield of a specific component of interest compared to its by-products.

[0009] Critical quality attributes (CQAs) related to the strength and potency of viral vector production include the number of viral genomes (vg) and capsids, as well as the ratio of vg / capsid titers or the full / empty ratio calculated based on other methods. The vg titer, typically assessed by quantitative polymerase chain reaction (qPCR), is commonly used to control dosing, as it is directly related to the therapeutic effect

[18] . The capsid or viral particle (vp) titer is typically assessed by a serotype-specific enzyme-linked immunosorbent assay (ELISA), measuring viral particles such as intact virions and empty capsids. The determination of both titers as well as the ratio is recommended for gene therapy products by the European Medicines Agency (EMA)

[19] as well as the United States Food and Drug Administration (US FDA)

[20] . Depending on the product control strategy, empty capsid particles are either removed, resulting in more complex downstream purification, or remain in the final product. There, they can reduce transduction efficiency by competing with fully packaged vector particles for binding to / uptake by target cells

[21] and can cause dose-limiting side effects based on immunogenicity due to capsid-specific CD8+ T cell responses, thereby negatively impacting therapeutic success

[22] . Downstream purification to eliminate empty capsids is associated with challenges such as poor scalability or variable efficiency depending on the vector / serotype in the case of ultracentrifugation and increased complexity combined with industrial waste from column resin regeneration in the case of ion exchange chromatography

[23] . [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2011133902 Brochure [Patent Document 2] International Publication No. 2019195729 Brochure

[0011] Summary of the Invention The above shortcomings are addressed by the present invention, which is defined by the terms of the accompanying independent claims. Preferred embodiments of the invention are defined by the dependent claims.

[0012] Laboratory studies were carried out in HEK293 cells to evaluate the impact of glutamine-dipeptide on viral vector production (AAV8). Evaluation of transfection efficiency by fluorescence measurement of green fluorescent protein positive cells, capsid titer in the supernatant determined by ELISA, as well as genome titer determined from the supernatant by qPCR, surprisingly showed a more efficient transfection, as well as an increase in genome and capsid titer, for cultures supplemented with glycyl-glutamine (glycyl-L-glutamine hydrate, Gly-Gln), compared to other variants. Compared to the alanyl-glutamine supplement, which resulted in a higher capsid titer while featuring a genome titer comparable to other processes, the higher genome titer was remarkable, indicating a more efficient production and addressing the challenges related to the empty capsid / full / empty ratio mentioned above. A direct impact on the production phase was found, independent of cell number, compared to the state-of-the-art glutamine supplement, which contributes to a higher yield based on the promotion of cell growth.

[0013] Thus, the invention relates to a method for producing RNA or DNA containing viral particles in cell culture, comprising the steps of: providing a cell capable of producing said viral particles; contacting said cells with a culture medium containing one or more dipeptides or derivatives thereof, one dipeptide being glycyl-glutamine (Gly-Gln); - obtaining said virus particle product from said culture medium or said cells.

[0014] Another aspect of the invention relates to a supplement for a culture medium for use in the production of RNA or DNA containing viral particles, comprising one or more dipeptides or derivatives thereof, one dipeptide being glycyl-glutamine (Gly-Gln), and a culture medium for use in the production of RNA or DNA containing viral particles, comprising one or more dipeptides or derivatives thereof, one dipeptide being glycyl-glutamine (Gly-Gln).

[0015] The present invention further relates to the use of the dipeptide glycyl-glutamine (Gly-Gln) or a derivative thereof for the production of viral particles in cell culture.

[0016] Preferred embodiments of the present invention are described in further detail in the following detailed description of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] "Peptide" is a compound consisting of an alpha-peptide bond (R 1 -CO-NH-R 2 It should be understood that the term "amino acid" refers to a molecule comprising at least two amino acids covalently bonded to each other by a cyclic alkyl group.

[0018] "Dipeptide" is a peptide bond (R 1 -CO-NH-R 2 ) and may exist in the form of a salt or a hydrate.

[0019] In the context of the present invention, an "amino acid" is defined as an amino functional group (-NH 2 ) and a carboxylic acid functional group (-COOH). In the context of the present invention, both alpha-amino acids and beta-amino acids are included. Preferred amino acids of the present invention are the alpha-amino acids, in particular the 20 "natural amino acid" acids, including cystine: Alanine (Ala / A) Arginine (Arg / R) Asparagine (Asn / N) Aspartic acid (Asp / D) Cysteine ​​(Cys / C) Cystine (Cys / C2) Glutamic acid (Glu / E) Glutamine (Gln / Q) Glycine (Gly / G) Histidine (His / H) Isoleucine (Ile / I) Leucine (Leu / L) Lysine (Lys / K) Methionine (Met / M) Phenylalanine (Phe / F) Proline (Pro / P) Serine (Ser / S) Threonine (Thr / T) Tryptophan (Trp / W) Tyrosine (Tyr / Y) Valin (Val / V)

[0020] In the context of the present invention, the expression "natural amino acids" should be understood to include both the L- and D-forms of the 20 amino acids listed above. However, the L-form is preferred. In one embodiment, the term "amino acid" also includes analogs or derivatives of those amino acids.

[0021] A "free amino acid" (e.g., "free cysteine") according to the present invention is understood to be an amino acid having its amino and its (alpha-) carboxylic acid functional groups in free form, i.e. not covalently attached to other molecules, e.g. not forming a peptide bond. Free amino acids may also exist as salts or in hydrate form. When referring to an amino acid that is part of or in a dipeptide, this should be understood to refer to that part of the respective dipeptide structure that is derived from the respective amino acid according to known mechanisms of biochemistry and peptide biosynthesis.

[0022] A "growth factor" according to the present invention should be understood as any naturally occurring substance capable of stimulating cell growth, proliferation and cell differentiation. Preferred growth factors are in the form of proteins or steroid hormones. According to one embodiment of the present invention, the expression "growth factor" should be interpreted as relating to a growth factor selected from the list consisting of fibroblast growth factors (FGF), including acidic and basic FGF, insulin, insulin-like growth factors (IGF), epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and transforming growth factors (TGF), including TGF-alpha and TGF-beta, cytokines such as interleukins 1, 2, 6, granulocyte stimulating factor, and leukocyte inhibitory factor (LIF).

[0023] An "oligopeptide" according to the present invention is to be understood as a peptide compound consisting of 2 to 20 amino acids. More preferred oligopeptides according to the present invention are oligopeptides consisting of 2 to 10 amino acids, 2 to 6 amino acids, 2 to 5 amino acids, 2 to 4 amino acids, or 2 to 3 amino acids. The most preferred oligopeptide according to the present invention is a dipeptide.

[0024] A "culture medium" according to the present invention shall be understood to be a liquid or solid medium containing nutrients, the culture medium being suitable to nourish and support the life and / or product formation of the cells in the culture. The cultured cells according to the present invention may be bacterial cells, yeast cells, fungal cells, animal cells such as mammalian cells or insect cells, and / or plant cells, e.g. algae. Typically, the culture medium provides essential and non-essential amino acids, vitamins, at least one energy source, lipids, and trace elements, all of which are required by the cells to maintain life, growth and / or product formation. The culture medium may also contain components that enhance growth and / or survival beyond a minimum rate, including hormones and growth factors. The culture medium preferably has a pH and salt concentration that supports the life, growth and / or product formation of the cells. The culture medium according to the present invention preferably contains all the nutrients necessary to maintain the life and proliferation of the cell culture. The preferred culture medium is a defined medium.

[0025] A "chemically defined medium" according to the present invention is a medium that does not contain cell extracts, cell hydrolysates, or protein hydrolysates. A chemically defined medium does not contain components of unknown composition. As is generally understood by those skilled in the art, a chemically defined medium usually does not contain animal-derived components. All components of a chemically defined medium have a known chemical structure. Culture media other than defined culture media may be referred to as "complex" culture media.

[0026] A "cell culture medium" should be understood to be a culture medium suitable for sustaining life, growth and / or product formation of animal and / or plant cells.

[0027] "Basal medium" or "basal culture medium" should be understood to be a nutrient-containing solution or substance in which the culture of cells is initiated.

[0028] "Feed medium" should be understood to be a solution or substance that is fed to cells after the start of the culture process. In certain embodiments, the feed medium contains one or more components that are not present in the base medium. The feed medium can also lack one or more components that are present in the base medium. Preferably, the concentration of nutrients in the feed medium exceeds the concentration in the base medium to avoid loss of productivity due to dilution.

[0029] "Perfusion medium" should be understood to be a solution or substance containing nutrients that is continuously added after the initiation of a cell culture from which the harvest is continuously removed.

[0030] A "cell culture supplement" should be understood to be an additive to a culture medium, a chemically defined medium or a cell culture medium that is beneficial for enhancing healthy cell growth, productivity or a specific application. One such application may be the improvement of cell growth, viability and productivity under serum-free or low serum conditions. It may contain dipeptides, amino acids, glucose, vitamins and proteins.

[0031] Examples of derivatives of Gly-Gln may include N-acylated dipeptides, such as N-acetyl-glycyl-L-glutamine, N-formyl-glycyl-L-glutamine, N-propionyl-glycyl-L-glutamine, N-succinyl-glycyl-L-glutamine, N-acyl esters of the dipeptide, or salts of the respective structures.

[0032] The term "N-acylated" with respect to a compound such as an amino acid shall be understood to mean that the N-acylated compound is modified by the addition of an acyl group to the nitrogen functional group of said compound. Preferably, the acyl group is added to the alpha-amino group of the amino acid.

[0033] Surprisingly, it was found that genome and capsid titers in HEK-293 cell-based viral vector production (AAV8) were substantially increased upon glycyl-l-glutamine supplementation.

[0034] The present invention therefore relates to a method for producing RNA or DNA containing viral particles in cell culture, comprising the steps of: providing a cell capable of producing said viral particles; contacting said cells with a culture medium containing one or more dipeptides or derivatives thereof, one dipeptide being glycyl-glutamine (Gly-Gln); - obtaining said virus particle product from said culture medium or said cells.

[0035] In the context of the present invention, "cells capable of producing said viral particles" includes cells that contain integrated helper virus nucleic acid sequences, as well as cells that do not contain integrated helper virus nucleic acid sequences and rely on the transfection of additional nucleic acid constructs to achieve packaging of the (recombinant) viral genome. It is also intended to include all cells that require any combination of helper virus, transfection reagents or plasmids to produce viral particles.

[0036] Viruses naturally introduce their genetic material into target cells as part of their replication cycle, so they can be used to deliver their RNA or DNA to host cells for replication. Engineered RNA or DNA-containing viral particles or viral vectors take advantage of this ability to deliver genetic material of interest to target cells. In some embodiments, this includes the introduction of therapeutic genes into target cells, resulting in the expression of transgenes for therapeutic purposes. To date, several viruses have been used to form engineered RNA or DNA-containing viral particles for therapeutic use.

[0037] The expression "RNA or DNA-containing viral particle" should be understood as an engineered viral particle characterized by its ability to deliver genetic material to a target cell, more specifically, as a composition of capsid and genetic material.

[0038] A "capsid" can be an enveloped or non-enveloped protein shell of 20-100 nm in diameter surrounding an inner RNA- or DNA-containing core. In the context of the present invention, specifically includes AAV capsids of about 22 nm in diameter, Ad capsids of 70-100 nm in diameter, and lentivirus (LV) capsids of 80-100 nm in diameter. Capsids can have different special affinities for certain host cell receptors based on serotype.

[0039] The "genetic material" can be either double-stranded DNA, single-stranded DNA or single-stranded RNA. In preferred embodiments, it consists of linear double-stranded DNA ranging up to 40 kb in length or linear single-stranded DNA up to 5 kb in length or single-stranded RNA up to 10 kb in length.

[0040] In one embodiment of the invention, a method of culturing cells comprises contacting the cells with a basal culture medium under conditions that support the culture of the cells, and supplementing the basal cell culture medium with an enriched medium according to the invention. In a preferred embodiment, the basal culture medium is supplemented with an enriched feed or medium for more than one day.

[0041] Empty capsids compete with full capsids for binding sites on target cells, thus reducing the therapeutically desired delivery of DNA to them, and are therefore considered product-associated impurities. These impurities should be measured and can be reported as ratios, e.g., full / empty ratios or viral particles / infectious units ratios. Both can be considered critical quality attributes (CQAs) in viral particle-based therapeutics.

[0042] In a preferred embodiment, the loading of the viral particles with nucleic acid is a full / empty ratio (ratio of genome titer to capsid titer) of at least 5%, preferably at least 10%, more preferably at least 20%.

[0043] In a preferred embodiment, the "full / empty ratio" shall be defined as the viral genome titer (vg) divided by the viral particle titer (vp). A suitable method for determining vg is quantitative polymerase chain reaction (qPCR). A suitable method for measuring vp titer, including intact virions and empty capsids, is serotype-specific enzyme-linked immunosorbent assay (ELISA).

[0044] Alternative analytical methods for determining viral particle and / or viral genome titers or for directly deriving the full / empty ratio may be based on, among others, anion exchange high performance liquid chromatography (AEX-HPLC), absorbance ratio at wavelengths of 260 / 280 nm, cryo-electron microscopy (cryoEM), size exclusion chromatography multi-angle light scattering (SEC-MALS), charge detection mass spectrometry (CDMS), sedimentation velocity analytical ultracentrifugation (SV-AUC). Other names for said "full / empty ratio" may alternatively be used, such as the packaging ratio or the ratio of capsid titer to genome titer

[24] or their calculable ratios.

[0045] In a preferred embodiment, the viral particle is derived from an adenovirus, lentivirus or adeno-associated virus (AAV), preferably AAV8 or AAV2.

[0046] In the context of the present invention, viral particles include those made from adenoviruses (Ad), adeno-associated viruses (AAV), lentiviruses (LV) herpes simplex viruses and vaccinia viruses. In the case of AAV, they are composed of a protein shell surrounding a single-stranded DNA genome of up to about 5 kilobases (kb), in the case of LV, they are composed of a protein shell containing two copies of a single-stranded RNA genome of up to about 10 kb, and in the case of Ad, they are composed of a protein shell surrounding a double-stranded DNA genome of up to about 40 kb. Viral particles can be used to administer recombinant RNA or DNA to target cells. More than 50 serotypes of Ad

[25] and more than 8 serotypes of AAV

[19] have been identified. These serotypes exhibit a diverse range of tropisms and immune response profiles, resulting in different applications of interest. The efficiency and specificity of AAV gene delivery can be improved using point mutations on the viral capsid [26, 27].

[0047] In a preferred embodiment, the viral particle is derived from an adenovirus (Ad), lentivirus (LV) or adeno-associated virus (AAV), preferably AAV8 or AAV2. However, also included are viral particles containing point-mutated AAV8 and AAV2 capsids, as well as chimeras resulting from the transfer of larger peptide domains from one AAV8 and AAV2 a serotype to another.

[0048] The cultivation of the cells according to the invention can be carried out as a batch culture, a fed-batch culture or a continuous culture.

[0049] In a preferred embodiment, the dipeptide is present in the culture medium at a final concentration of 0.1 mM to 20 mM, or 0.1 mM to 10 mM, or 0.5 mM to 10 mM, or 1 mM to 10 mM, or 5 mM to 10 mM.

[0050] In a preferred embodiment, the culture medium further comprises at least one carbohydrate, at least one free amino acid, at least one inorganic salt, a buffering agent and / or at least one vitamin.

[0051] In a preferred embodiment, said cells are selected from the list consisting of CHO cells, COS cells, VERO cells, BHK cells, HEK cells, HELA cells, AE-1 cells, insect cells, Sf9 cells, TT-D6 cells, BLKCL.4 primary skin fibroblasts, A549 human adenocarcinoma cells, or fibroblasts, muscle cells, neuronal cells, stem cells, skin cells, endothelial cells, immune cells such as NK or T cells and hybridoma cells, preferably HEK cells.

[0052] Another aspect of the invention relates to a supplement for culture media for use in the production of RNA or DNA containing viral particles, comprising one or more dipeptides or derivatives thereof, one dipeptide being glycyl-glutamine (Gly-Gln).

[0053] In a preferred embodiment, the supplement further comprises at least one carbohydrate, at least one free amino acid, at least one inorganic salt, a buffering agent and / or at least one vitamin.

[0054] A further aspect of the invention relates to a culture medium for use in the production of RNA or DNA containing viral particles comprising one or more dipeptides or derivatives thereof, one dipeptide being glycyl-glutamine (Gly-Gln).

[0055] In a preferred embodiment, the supplement is in liquid form, gel, powder, granule, pellet form, or in tablet form.

[0056] In a preferred embodiment, the dipeptide is present in the culture medium at a concentration of 0.1 mM to 20 mM, or 0.1 mM to 10 mM, or 0.5 mM to 10 mM, or 1 mM to 10 mM, or 5 mM to 10 mM.

[0057] In a preferred embodiment, said culture medium is in liquid form, gel, powder, granule, pellet form or tablet form.

[0058] In a preferred embodiment, the culture medium is as an aqueous stock or feed solution.

[0059] In a preferred embodiment, the dipeptide is not N-acylated. N-acylation is known to improve the thermal stability of certain dipeptides. However, it has been found that N-acylated dipeptides can also result in poor viable cell density and viability.

[0060] In a particular configuration, the present invention relates to a cell or tissue culture medium comprising a composition according to the present invention, further comprising at least one carbohydrate, at least one free amino acid, at least one inorganic salt, a buffering agent and / or at least one vitamin. In a particularly preferred embodiment, the culture medium comprises all of at least one carbohydrate, at least one free amino acid, at least one inorganic salt, a buffering agent and at least one vitamin.

[0061] In one embodiment of the present invention, the culture medium does not contain growth factors. According to this embodiment, the dipeptide of the present invention can be used instead of growth factors to promote the growth and / or proliferation of cells in culture. In another embodiment of the present invention, the culture medium does not contain lipids.

[0062] In a preferred embodiment, the culture medium of the present invention is a defined medium or serum-free medium.For example, the composition of intervention can be supplemented with Freestyle™ F17 medium, Freestyle™ 293 medium, Expi293™ medium from ThermoFisher (Waltham, USA), TheraPEAK™ SfAAV™ medium from LONZA (Basel, Switzerland), HEK ViP NB from Xell (Bielfeld, Germany), HyClone™ SFM4HEK293 from Cytiva (Marlborough, USA).The dipeptide of the present invention can be supplemented with DMEM medium (Life Technologies Corp., Carlsbad, USA).However, the present invention is not limited to supplementing the above-mentioned medium.

[0063] In another preferred embodiment, the culture medium is a liquid medium in a 2x, 3x, 3.33x, 4x, 5x or 10x concentrated form (volume / volume) relative to the concentration of said medium in use. This allows the preparation of a "ready-to-use" culture medium by simply diluting the concentrated medium with the respective volume of sterile water. Such concentrated form of the medium of the present invention can also be used by adding it to the culture, for example in fed-batch culture or perfusion process.

[0064] The cell culture medium of the present invention (cell or tissue culture basal medium, feeding medium or perfusion medium) can preferably contain all nutrients necessary for sustained growth and product formation. Recipes for preparing culture media, especially cell culture media, are well known to those skilled in the art (see, for example, Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, Ozturk and Wei-Shou Hu eds., Taylor and Francis Group 2006)

[24] . Various culture media are commercially available from various sources.

[0065] The culture medium of the present invention may preferably contain a carbohydrate source. The main carbohydrate used in cell culture media is glucose, which is usually supplemented at 5 to 25 mM. In addition, any hexose, such as galactose, fructose, or mannose, or a combination, may be used.

[0066] Culture media typically include at least essential amino acids (i.e., His, Ile, Leu, Lys, Met, Phe, Thr, Try, Val) as well as non-essential amino acids. Non-essential amino acids are typically included in cell culture media if the cell line is unable to synthesize the amino acid or if the cell line is unable to produce sufficient amounts of the amino acid to support maximal growth. In addition, mammalian cells can also use glutamine as a primary energy source. Glutamine is often included at higher concentrations than other amino acids (2-8 mM). However, as mentioned above, glutamine can spontaneously decompose to form ammonia, and certain cell lines produce ammonia faster, which is toxic.

[0067] The culture medium of the present invention may preferably contain salts. Salts are added to cell culture media to maintain isotonic conditions and prevent osmotic imbalance. The osmolality of the culture medium of the present invention is about 300 mOsm / kg, although many cell lines can tolerate variations in this value of about 10 percent or more. The osmolality of some insect cell cultures tends to be higher than 300 mOsm / kg, which may be 0.5 percent, 1 percent, 2-5 percent, 5-10 percent, 10-15 percent, 15-20 percent, 20-25 percent, or 25-30 percent higher than 300 mOsm / kg. The most commonly used salts in cell culture media include Na + , K + , Mg 2+ , Ca 2+ , Cl - , S.O. 4 2- , P.O. 4 3- , and HCO 3 -(e.g., CaCl 2 , KCl, NaCl, NaHCO 3 , Na 2 HPO 4 ) are included.

[0068] Other inorganic elements may be present in the culture medium. These include Mn, Cu, Zn, Mo, Va, Se, Fe, Ca, Mg, Si and Ni. Many of these elements are involved in enzyme activity. They include CaCl 2 , Fe(NO 3 ) 3 , MgCl 2 , MgSO 4 , MnCl 2 , NaCl, NaHCO 3 , Na 2 HPO 4 and ions of trace elements such as selenium, vanadium and zinc. These inorganic salts and trace elements can be obtained commercially, for example from Sigma (St. Louis, Mo.).

[0069] The culture medium of the present invention preferably contains vitamins. Vitamins are typically used by cells as cofactors. Although the vitamin requirements of each cell line vary widely, in general, extra vitamins are required when the cell culture medium contains little or no serum or when the cells are grown at high density. Exemplary vitamins preferably present in the medium of the present invention include biotin, choline chloride, folic acid, i-inositol, nicotinamide, D-Ca ++ -Pantothenic acid, pyridoxal, riboflavin, thiamine, pyridoxine, niacinamide, A, B 6 , B 12 , C, D 3 , E, K and p-aminobenzoic acid (PABA).

[0070] The culture medium of the present invention may also contain serum. Serum is the supernatant of clotted blood. Serum components include attachment factors, micronutrients (e.g., trace elements), growth factors (e.g., hormones, proteases) and protective elements (e.g., antitoxins, antioxidants, antiproteases). Serum is available from a variety of animal sources, including human, bovine or horse serum. When included in the cell culture medium according to the present invention, serum is typically added at a concentration of 5-10% (by volume). The preferred cell culture medium is serum-free.

[0071] To promote cell growth in the absence of serum or in serum-reduced medium, one or more of the following polypeptides can be added to the cell culture medium of the present invention.

[0072] In other embodiments, the cell culture medium does not contain a polypeptide.

[0073] One or more lipids, such as linoleic acid, linolenic acid, arachidonic acid, palmitoleic acid, oleic acid, polyenoic acid, and / or fatty acids of 12, 14, 16, 18, 20, 22, or 24 carbon atoms (each carbon atom is branched or unbranched), phospholipids, lecithin (phosphatidylcholine), and cholesterol, can also be added to the cell culture medium of the present invention. One or more of these lipids can be included as supplements in serum-free medium. Phosphatidic acid and lysophosphatidic acid stimulate the growth of certain anchorage-dependent cells, such as MDCK, mouse epithelial and other kidney cell lines, and phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol stimulate the growth of human fibroblasts in serum-free medium. Ethanolamine and cholesterol have also been shown to promote the growth of certain cell lines. In certain embodiments, the cell culture medium does not contain lipids.

[0074] One or more carrier proteins, such as bovine serum albumin (BSA) or transferrin, can also be added to the cell culture medium. Carrier proteins can aid in the transport of certain nutrients or trace elements. BSA is typically used as a carrier for lipids, such as linoleic acid and oleic acid, which are insoluble in aqueous solutions. In addition, BSA can also function as a carrier for certain metals, such as Fe, Cu, and Ni. In protein-free formulations, non-animal-derived alternatives to BSA, such as cyclodextrin, can be used as lipid carriers.

[0075] One or more adhesion proteins, such as fibronectin, laminin and pronectin, can also be added to the cell culture medium to help promote attachment of anchorage-dependent cells to the substrate.

[0076] Cell culture medium may optionally contain one or more buffers. Suitable buffers include, but are not limited to, N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES), MOPS, MES, phosphate, bicarbonate, and other buffers suitable for use in cell culture applications. Suitable buffers provide buffering capacity without substantial cytotoxicity to cultured cells. Selection of suitable buffers is within the scope of those skilled in the art of cell culture.

[0077] Polyanionic or polycationic compounds can be added to the culture medium to prevent cells from clumping and to promote the growth of cells in suspension.

[0078] In a preferred embodiment, the culture medium is in liquid form. However, the culture medium may also be a solid medium, such as a gel-like medium, for example, agar, carrageenan or gelatin-containing medium (powder, agglomerated powder, instantiated powder, etc.). Preferably, the culture medium is in a sterile form.

[0079] The culture medium of the present invention may be in concentrated form. It may be, for example, in a 2- to 100-fold concentrated form (relative to the concentration that supports cell growth and product formation), preferably in a 2-, 3-, 3.33-, 4-, 5-, 10-, 20-, 50- or 100-fold concentrated form. Such concentrated culture media are useful for preparing culture media for use by diluting the concentrated medium with an aqueous solvent such as water. Such concentrated culture media may be used in batch culture, but are also advantageously used in fed-batch or continuous culture, where a concentrated nutrient composition is added to an ongoing culture of cells, for example to replenish nutrients consumed by the cells during the culture.

[0080] In another embodiment of the invention, the culture medium is in a dry form, such as in the form of a dry powder, or in the form of granules, or in the form of pellets, or in the form of tablets.

[0081] The present invention also relates to the use of the culture medium of the present invention for culturing cells. Another aspect of the present invention relates to the use of the culture medium of the present invention for producing a cell culture product.

[0082] Another aspect of the present invention relates to the use of the dipeptide glycyl-glutamine (Gly-Gln) or its derivatives for the production of viral particles in cell culture. This dipeptide can be used as an auxiliary substance in certain cell culture media used for the production of viral particles in cell culture, preferably for the production of viral particles derived from adenovirus (Ad), lentivirus (LV) or adeno-associated virus (AAV), preferably AAV8 or AAV2.

[0083] literature 1:https: / / www.ema.europa.eu / en / documents / assessment-report / ervebo-epar-public-assessment-report_en.pdf 2:https: / / www.ema.europa.eu / en / documents / assessment-report / vaxzevria-previously-covid-19-vaccine-astrazeneca-epar-public-assessment-report_en.pdf 3:https: / / www.ema.europa.eu / en / documents / assessment-report / covid-19-vaccine-janssen-epar-public-assessment-report_en.pdf 4:https: / / www.ema.europa.eu / en / documents / assessment-report / luxturna-epar-public-assessment-report_en.pdf 5:https: / / www.ema.europa.eu / en / documents / assessment-report / zolgensma-epar-public-assessment-report_en.pdf 6:Kuzmin,DA,Shutova,MV,Johnston,NR,Smith,OP,Fedorin,VV,Kukushkin,YS,van der Loo,J.,&Johnstone,EC(2021).The clinical landscape for AAV gene therapies.Nature reviews.Drug discovery,20(3),173-174.https: / / doi.org / 10.1038 / d41573-021-00017-7 https: / / media.nature.com / original / magazine-assets / d41573-021-00017-7 / 18790666 7:Masri,F.,Cheeseman,E.,&Ansorge,S.(2019).Viral vector manufacturing:how to address current and future demands.Cell Gene Ther.Insights,5,949-970. 8:Pennybaker,A.,Pezoa,S.A.,&Alfano,A.(2021).Recent Advances of Chemical Definition of Cell Culture Media and Excipients for Virus and Viral Vector Manufacturing:A Review.Journal of bioprocessing&biotechniques,11,1-4. 9:Eagle,H.(1959).Amino acid metabolism in mammalian cell cultures.Science,130(3373),432-437.https: / / doi.org / 10.1126 / science.130.3373.432 10:Furst,P.(2000)Jonathan E.Rhoads lecture:a thirty year odyssey in nitrogen metabolism:from ammonium to dipeptides.J.Parenter.Enteral Nutr.24,197-209 11:Roth,E.,Ollenschlager,G.,Hamilton,A-,Langer,K.,Fekl,W.and Jakesz,R.(1988).Influence of two glutamine containing dipeptides on growth of mammalian cells.In Vitro Cellular and Developmental Biology 24(7),696-698 12: Drauz, K., Knaup, G., Groeger, F. (1992). New N-acyl-peptide(s) with good solubility and stability. German Patent Application No. 4022267A1 13:Gibco Protocol Pub No.0007831 Rev.1.0 FreeStyleTM F17 Expression medium accessed via https: / / www.thermofisher.com / document-connect / document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FFreeStyle_F17Expression_Medium_man.pdf(2022-03-02) 14:FUJIFILM Irvine Scientific Product User Guide BalanCD HEK293 Media System accessed via https: / / www.irvinesci.com / media / IrvineScientific / Resources / 4 / 1 / 41084_balancd_hek293_media_system.pdf(2022-03-02) 15: Christie, A., & Butler, M. (1994). Glutamine-based dipeptides are utilized in mammalian cell culture by extracellular hydrolysis catalyzed by a specific peptidase. Journal of biotechnology, 37(3), 277-290. https: / / doi.org / 10.1016 / 0168-1656(94)90134-1 16: Barrett, S., & Scott, J. (2011). Cell culture medium comprising small peptides, Life Technologies Corp. International Patent Application Publication No. 2011133902 Pamphlet 17: Girard, V., Truran, R., Tuyen, O, (2019). AAV compositions, methods of making and methods of use WO2019195729A1 18: Tustian, A. D., & Bak, H. (2021). Assessment of quality attributes for adeno - associated viral vectors. Biotechnology and bioengineering, 118(11), 4186 - 4203. https: / / doi.org / 10.1002 / bit.27905 19: EMA. (2018). Guideline on the quality, non - clinical and clinical aspects of gene therapy medicinal products (EMA / CAT / 80183 / 2014). European Medicines Agency https: / / www.ema.europa.eu / en / documents / scientific - guideline / guideline - quality - non - clinical - clinical - aspects - gene - therapy - medicinal - products_en.pdf 20: US FDA, Center for Biologics Evaluation and Research. (2020). Chemistry, manufacturing, control (CMC) information for human gene therapy investigational new drug applications (INDs). https: / / www.fda.gov / media / 113760 / download 21:Gao,K.,Li,M.,Zhong,L.,Su,Q.,Li,J.,Li,S.,He,R.,Zhang,Y.,Hendricks,G.,Wang,J.,&Gao,G.(2014).Empty Virions In AAV8 Vector Preparations Reduce Transduction Efficiency And May Cause Total Viral Particle Dose-Limiting Side-Effects.Molecular therapy.Methods&clinical development,1(9),20139.https: / / doi.org / 10.1038 / mtm.2013.9 22:Pei,X.,Earley,L.F.,He,Y.,Chen,X.,Hall,N.E.,Samulski,R.J.,&Li,C.(2018).Efficient Capsid Antigen Presentation From Adeno-Associated Virus Empty Virions In Vivo.Frontiers in immunology,9,844.https: / / doi.org / 10.3389 / fimmu.2018.00844 23:Qu,W.,Wang,M.,Wu,Y.,&Xu,R.(2015).Scalable downstream strategies for purification of recombinant adeno-associated virus vectors in light of the properties.Current pharmaceutical biotechnology,16(8),684-695.https: / / doi.org / 10.2174 / 1389201016666150505122228 24: Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, Ozturk and Wei-Shou Hu eds., Taylor and Francis Group 2006 25: Daussy, C.F., Pied, N., & Wodrich, H. (2021). Understanding Post Entry Sorting of Adenovirus Capsids; A Chance to Change Vaccine Vector Properties. Viruses, 13(7), 1221. https: / / doi.org / 10.3390 / v13071221 26: Wu, Z., Asokan, A., & Samulski, R.J. (2006). Adeno-associated virus serotypes: vector toolkit for human gene therapy. Molecular therapy, 14(3), 316 - 327. https: / / doi.org / 10.1016 / j.ymthe.2006.05.009 27: Werle, A.K., Powers, T.W., Zobel, J.F., Wappelhorst, C.N., Jarrold, M.F., Lyktey, N.A., Sloan, C.D.K., Wolf, A.J., Adams-Hall, S., Baldus, P., & Runnels, H.A. (2021). Comparison of analytical techniques to quantitate the capsid content of adeno-associated viral vectors. Molecular Therapy-Methods & Clinical Development, 23, 254 - 262. https: / / doi.org / https: / / doi.org / 10.1016 / j.omtm.2021.08.009

Example

[0084] Example 1 HEK293 cells (HEK Expi293F suspension cell line, Thermo Fisher Scientific) were used to study the effect of glutamine-dipeptides compared to L-glutamine on viral vector ("DNA-containing viral particle") production. The virus used was adeno-associated virus 8 (AAV8).

[0085] material and method A laboratory study was performed to evaluate the impact of glutamine-dipeptide on viral vector production (AAV8). HEK293 cells (HEK Expi293F cells, Thermo Fisher Scientific) were pre-cultured in shake flasks in a commercially available culture medium recommended for viral vector production (Freestyle F17 Expression Medium, Gibco, Thermo Fisher Scientific). After thawing the cells in 20 mL of the respective medium variant (final concentration of glutamine or glutamine-dipeptide in the medium variant was 8 mM) in a 125 mL shake flask, they were cultured at 37 °C (185 rpm / 50 mm orbit, 5% CO) after 3-4 days of passaging rhythm. 2 ) for 5 subcultures. For production, cells were cultured at 2 × 10 6 The cells were split into three replicates with a seeding viable cell density of 100000 cells / mL (day 0). They were transfected with polyethyleneimine (PEI MAX, Polysciences) harboring a two-plasmid system for AAV8 (Plasmid Factory) featuring green fluorescent protein (GFP) as the gene of interest 24 hours after inoculation (day 1). Again, the medium variants were supplemented with glutamine (Gln), alanyl-glutamine (Ala-Gln, GlutaMAX™, Gibco, Thermo Fisher Scientific) or glycyl-glutamine hydrate (Gly-Gln, cQrex® GQ, Evonik Operations) via liquid supplements to a final concentration of 8 mM glutamine or glutamine dipeptide (n=3 each).

[0086] To analyze cell culture performance in different medium supplements, viable cell density and viability were determined via an automated cell counter. Ammonium (fluorescence method) and glucose and lactate concentrations (enzymatic amperometric method) were determined for samples from one shake flask per supplement during the preculture and AAV8 production phase. Transfection efficiency was determined by GFP expression by flow cytometry 48, 72 and 96 h after transfection. For AAV8 titration, samples of all replicates (supernatants only) were taken 96 and 120 h after transfection and analyzed by qPCR (genomic titer) and ELISA (capsid titer).

[0087] result Viable cell density and viability during the AAV8 production phase were highly comparable between replicates (Figure 1). For all cultures, viable cell density during AAV production was approximately 2.0 × 10 on day 0. 6 cells / mL, 3.6–4.2 × 10 for three transfections (day 1) 6 From cells, an average of 4.5 × 10 per supplement was obtained on days 3–6. 6 ~5.4×10 6 Viability ranged from 0.1 to 1.0 cells / mL. Viability began at over 98% on the day of transfection and dropped to approximately 75% by the end of the production period (day 6).

[0088] Glucose and lactate concentrations (data not shown) were determined during the AAV8 production phase. Independent of the glutamine compound added, they were characterized by similar trajectories. Glucose levels peaked upon addition of fresh culture medium during transfection and dropped to 0 mg / mL on day 4, while lactate levels remained low throughout the process (all below 2 mg / mL).

[0089] To evaluate the transfection efficiency, all cultures were analyzed by flow cytometry 48 h, 72 h and 96 h after transfection (Figure 5). The transfection efficiency of cells supplemented with Gln, Ala-Gln and Gly-Gln varied between 78% and 86% at 48 h after transfection, between 95% and 97% at 72 h after transfection, and all reached 98% at 96 h after transfection. The highest transfection efficiency was obtained with Gly-Gln. Independent of the medium supplement, the efficiency increased over time. In the case of the measurement of transfection efficiency by fluorescence measurement of GFP-positive cells (Figure 6), the relative fluorescence of all samples was highest 48 h after transfection with 520-572 RFU and decreased over time. At all sampling points, cultures supplemented with Gly-Gln showed the highest relative fluorescence.

[0090] Genomic AAV8 titers of cultures supplemented with Gln, Ala-Gln, and Gly-Gln were determined from supernatants 96 hours post-transfection (Figure 7) and 120 hours post-transfection (Figure 8). They ranged from 4.0 to 7.3 × 10 at 96 hours post-transfection. 10 vg / mL, and 4.4–8.2 × 10 10 Surprisingly, differences between the cultures were observed that were not expected based on similar extracellular glucose and lactate levels during production. Cultures supplemented with Gly-Gln showed significantly higher genome titers at both time points compared to cultures supplemented with Gln or Ala-Gln (7.3 × 10 at 96 h post-transfection). 10 vg / mL, 8.2 × 10 10 vg / mL).

[0091] Capsid titers were determined from the supernatants 96 hours after transfection (Figure 9) and 120 hours after transfection (Figure 10). They ranged from 2.3 to 3.3 × 10 at 96 hours post-transfection. 11vp / mL, and 2.7–3.9 × 10 11 Higher capsid titers were measured for cultures supplemented with Ala-Gln and Gly-Gln compared to cultures supplemented with Gln, in the range of vp / mL.

[0092] The mean values ​​of both sampling points were used to calculate the Full / Empty ratio (ratio of genome titer in vg / mL to capsid titer in vp / mL) (Figure 11). The Full / Empty ratio varied from 14 to 22% at 96 h post-transfection and from 13 to 21% at 120 h post-transfection. A slight decrease between the Full / Empty ratio after 96 h and the Full / Empty ratio after 120 h could be observed in all cultures. The Full / Empty ratio of cultures supplemented with Gly-Gln was significantly higher at both time points (22% at 96 h and 21% at 120 h). [Brief description of the drawings]

[0093] [Figure 1] Figure 1 shows the viable cell density and viability (mean of n=3) of Expi293F™ cells in FreeStyle™ F17 expression medium during AAV8 production in relation to supplemented glutamine compounds. Day 0 is the day of inoculation, day 1 is the day of transfection, and days 2-6 correspond to sampling points from 24 to 120 hours post-transfection. [Diagram 2] Figure 2 shows the effect of supplemental addition on viral vector production (AAV8) using HEK Expi293F cells in Freestyle F17 expression medium 48, 72 and 96 hours after transfection. Plot of transfection efficiency versus supplemented glutamine compound. [Diagram 3] Figure 3 shows the effect of supplemental addition on viral vector production (AAV8) using HEK Expi293F cells in Freestyle F17 expression medium 48, 72 and 96 hours post-transfection. Plot of relative fluorescence versus supplemented glutamine compound. [Figure 4] Figure 4 shows the effect of supplemental addition on viral vector production (AAV8) using HEK Expi293F cells in Freestyle F17 expression medium 96 hours after transfection. Plot of viral genome titer (mean of n=3±SD) against supplemented glutamine compound. [Diagram 5] Figure 5 shows the effect of supplemental addition on viral vector production (AAV8) using HEK Expi293F cells in Freestyle F17 expression medium 120 hours post-transfection. Plot of viral genome titer (mean of n=3±SD) against supplemented glutamine compound. [Figure 6] Figure 6 shows the effect of supplemental addition on viral vector production (AAV8) using HEK Expi293F cells in Freestyle F17 expression medium 96 hours after transfection. Plot of capsid titer (mean of n=3±SD) against supplemented glutamine compound. [Figure 7] Figure 7 shows the effect of supplemental addition on viral vector production (AAV8) using HEK Expi293F cells in Freestyle F17 expression medium 120 hours post-transfection. Plot of capsid titer (mean of n=3±SD) against supplemented glutamine compound. [Figure 8] Figure 8 shows the effect of supplemental addition on viral vector production (AAV8) using HEK Expi293F cells in Freestyle F17 expression medium 96 and 120 hours after transfection. Plot of full / empty ratio (ratio of viral genome titer to viral particle titer) versus supplemented glutamine compound.

[0094] conclusion The AAV8 production phase was monitored for 120 h after transfection. Assessment of transfection efficiency by fluorescence measurement of green fluorescent protein positive cells, capsid titers in the supernatants determined by ELISA, as well as genome titers determined from the supernatants by qPCR, surprisingly showed relatively higher or slightly more efficient transfection, as well as increased genome and capsid titers, for cultures supplemented with Gly-Gln compared to the other variants. The increase in genome titer was more pronounced than the increase in capsid titer, which is reflected in the highest full / empty ratio of cultures supplemented with Gly-Gln. Compared to Ala-Gln supplementation, which resulted in higher capsid titers while featuring comparable genome titers to the other processes, the high genome titer is remarkable, indicating a more efficient production and addressing the challenges associated with empty capsids and the full / empty ratios mentioned above.

Claims

1. 1. A method for producing RNA- or DNA-containing viral particles in cell culture, comprising: - providing a cell capable of producing said viral particles; contacting the cells with a culture medium containing one or more dipeptides, one dipeptide being glycyl-glutamine (Gly-Gln); - Obtaining said viral particle product from said culture medium or said cells.

2. The method described in claim 1, wherein the complete / empty ratio is determined by dividing the genome titer by PCR by the capsid titer by ELISA.

3. 3. The method of claim 1 or 2, wherein the viral particle is derived from an adenovirus, an adeno-associated virus (AAV), or a lentivirus.

4. 3. The method of claim 1 or 2, wherein the dipeptide is present in the culture medium at a concentration of 0.1 mM to 20 mM, or 0.1 mM to 10 mM, or 0.5 mM to 10 mM, or 1 mM to 10 mM, or 5 mM to 10 mM.

5. 3. The method of claim 1 or 2, wherein the culture medium further comprises at least one carbohydrate, at least one free amino acid, at least one inorganic salt, a buffering agent and / or at least one vitamin.

6. 3. The method of claim 1 or 2, wherein the cells are selected from the list consisting of CHO cells, COS cells, VERO cells, BHK cells, HEK cells, HELA cells, AE-1 cells, insect cells, Sf9 cells, TT-D6 cells, BLKCL.4 primary skin fibroblasts, A549 human adenocarcinoma cells, MDCK cells or ender cells, or fibroblasts, muscle cells, nerve cells, stem cells, skin cells, endothelial cells, immune cells such as NK or T cells and hybridoma cells.

7. Use of the dipeptide glycyl-glutamine (Gly-Gln) for the production of viral particles in cell culture.

Images