WO2020142304A1

WO2020142304A1 - Supplemented serum-containing culture medium for enhanced arpe-19 growth and human cytomegalovirus vaccine production

Info

Publication number: WO2020142304A1
Application number: PCT/US2019/068230
Authority: WO
Inventors: Sianny CHRISTANTI; Esther Wei Yee LIM; Jason Rodriguez; Christopher L. DANIELS
Original assignee: Merck Sharp & Dohme Corp.
Priority date: 2019-01-03
Filing date: 2019-12-23
Publication date: 2020-07-09
Also published as: EP3905880A4; EP3905880A1; US20220090005A1

Abstract

The present invention relates to supplemented serum-containing cell culture media that provides enhances ARPE-19 cell growth and/or improves the yield of human cytomegalovirus (HCMV) grown in ARPE-19 cell cultures. The media of the invention includes two additives, a hormone (e.g., a glucocorticoid hormone such as dexamethasone) and a growth factor (e.g., EGF). The invention further provides methods of producing HCMV in such growth media.

Description

TITLE OF THE INVENTION

SUPPLEMENTED SERUM-CONTAINING CULTURE MEDIUM FOR ENHANCED

ARPE-19 GROWTH AND HUMAN CYTOMEGALOVIRUS VACCINE PRODUCTION

FIELD OF INVENTION

The present invention relates to supplemented serum-containing cell culture media that provides enhanced ARPE-19 cell growth and/or improved yields of human cyto egalovirus (HCMV) grown in ARPE-19 cell cultures. The culture media of the invention includes a glucocorticoid hormone such as dexamethasone, a growth factor such as epidermal growth factor (EGF), additional essential amino acids, nucleosides (e.g., as provided in glutamine synthetase expression medium supplement) and various trace elements. The invention further provides methods of producing HCMV in such culture media.

BACKGROUND OF THE INVENTION

Human cytomegalovirus (HCMV), also known as human herpesvirus 5 (HHV-5), is a herpes virus classified as being a member of the beta subfamily of herpesviridae. HCMV is an enveloped virus, having an outer lipid bilayer envelope containing a variety of glycoproteins including glycoprotein B (gB), gH, gL, gM, gN, and gO, with a double stranded linear DNA core in an icosahedral nucleocapsid. See Crough et al ., 2009, Clinical Microbiology Reviews 22:76- 98.

According to the Centers for Disease Control and Prevention, HCMV infection is found fairly ubiquitously in the human population, with an estimated 40-80% of the United States adult population having been infected. The virus is spread primarily through bodily fluids and is frequently passed from pregnant mothers to the fetus or newborn. In most individuals, HCMV infection is latent, although virus activation can result in high fever, chills, fatigue, headaches, nausea, and splenomegaly. HCMV infections in immunocompromised individuals (such as HIV-positive patients, allogeneic transplant patients and cancer patients) or persons whose immune system has yet to be fully developed (such as newborns) can be particularly problematic. See Mocarski et al ., 2007, Cytomegalovirus, In Knipes and Howley (Eds.), Field Virology pp. 2701-2772. HCMV infection in such individuals can cause severe morbidity, including pneumonia, hepatitis, encephalitis, colitis, uveitis, retinitis, blindness, and neuropathy, among other deleterious conditions. In addition, HCMV infection during pregnancy is a leading cause of birth defects. See, e.g., Adler, 2008, J. Clin Virol 41 :231; Arvin et al., 2004, Clin Infect Dis 39:233; and Revello et al., 2008, J Med Virol, 80: 1415.

To date, there is no available HCMV vaccine. However, a promising candidate based on a live attenuated HCMV is currently in clinical trials. See U.S. Pat. No. 9,546,355 and Wang et al., 2016, Sci Transl Med 8:362ral45. This vaccine is a genetically modified HCMV produced using human retinal pigment epithelial cells (ARPE-19).

For commercial vaccine production based on a live virus, optimization of the production process, including cell culture and its culture media, is critical. Despite potential problems associated with serum-containing culture media for HCMV production, HCMV production in serum-free culture media has failed to provide adequate virus yield. Thus, to date, serum-containing culture media is required for HCMV production.

Serum-free culture media containing a combination of dexamethasone and EGF for the preparation of vaccines has been described in U.S. Pat. No. 6,656,719 (rotavirus vaccine) and Japanese Patent Application Publication No. JP2006158388 (polio vaccine).

Serum-containing media containing glucocorticoids such as dexamethasone have shown mixed results in viral production and infectivity. See Costa et al., 1974, Nature 252:745- 746; Solodushko et al., 2009, Am J Physiol Lung Cell Mol Physio 297U538-45; Tanaka et al., 1984, J Gen Virol. 65: 1759-67; and Tanaka et al., 1984, Virology 136:448-52. Treatment of cells with pharmacological concentrations of adrenal glucocorticoids such as dexamethasone enhanced HCMV replication; treatment with oestrogenic or androgenic hormones did not do so.

SUMMARY OF THE INVENTION

The present invention relates to supplemented serum-containing cell culture media that provides enhanced ARPE-19 cell growth and/or improved yields of human cyto egalovirus (HCMV) grown in ARPE-19 cell cultures. The culture media of the invention includes a glucocorticoid hormone such as dexamethasone, a growth factor such as epidermal growth factor (EGF), additional essential amino acids, nucleosides (e.g., as provided in glutamine synthetase expression medium supplement) and various trace elements.

In certain embodiments, the invention provides a cell culture medium comprising a basal cell culture medium that comprises serum and is further supplemented with between about 0.1 mM and about 10 mM of dexamethasone, between about 0.1 ng/mL and about 100 ng/mL of epidermal growth factor (EGF), IX MEM essential amino acids, 1X-4X glutamine synthetase expression medium (GSEM) supplement and trace elements. In one aspect, the basal cell culture medium is DMEM/F-12. In certain aspects, the serum is fetal bovine serum (FBS), optionally present at a concentration between 2% and 10%, inclusive.

In certain embodiments, the dexamethasone is present at a concentration between about 1 and about 10 mM. In certain embodiments, the EGF is present at a concentration between about 1 and about 40 ng/mL. In certain embodiments, the IX MEM essential amino acids consist of arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine,

phenylalanine, threonine, tryptophan, tyrosine, and valine. In certain embodiments, the IX GSEM supplement consists of a mixture of L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, adenosine, guanosine, cytidine, uridine, and thymidine. In certain embodiments, the trace elements are selected from cupric sulfate, ferric citrate, sodium selenite, and zinc sulfate.

The certain embodiments, the invention provides a composition comprising a cell culture medium of the invention and cells selected from ARPE-19, Vero, MDCK, MRC-5, BHK, CEM, and LL-I cells. In certain aspects of this embodiment, the composition further comprises a virus. In certain sub-aspects of this aspect, the virus is a human herpesvirus, optionally a human cyto egalovirus (HCMV). The HCMV can be a conditional replication defective HCMV mutant or a recombinant genetically modified HCMV. The conditional replication defective HCMV can have the genomic sequence of SEQ ID NO: 1.

The present invention is also directed to a method of increasing virus production in cells, said method comprising culturing any of the compositions described herein comprising a virus, wherein said composition provides increased production of the virus by at least 10% relative to virus grown in a composition lacking the supplements described above.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is

Figure 2 includes a graph demonstrating

Figure 3 includes a graph demonstrating the difference brtween supplemented and non-supplemented media.

Figure 4 includes a graph demonstrating culture passage of various media.

Figure 5A-5C include graphs that demonstration improved ARPE-19 cell growth with addition of select supplements at various sera types Figure 6 includes a graph that compares the volumetric production of virus as measured by Apogee flow cytometry.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the production of HCMV in ARPE-19 cell culture with supplemented serum-containing culture media. The present invention is based on the discovery that supplementation of culture media with dexamethasone, EGF, essential amino acids, nucleosides and various trace elements to a basal DMEM/F-12 culture medium containing serum reduces the ARPE-19 doubling time as well as increases HCMV virus particles by up to 37% for ARPE-19 cells in static cultures and on microcarriers. The effect of these supplements results in increased cell density per growth vessel for a given time and/or increased productivity of virus on a per-cell basis. The culture media of the invention can be used to increase the production efficiency of HCMV, including conditional replication-defective strains that can be used in prophylactic vaccines.

While dexamethasone and EGF have been used as supplements for virus propagation in serum-free media, the additional of these supplements provided comparable virus yields to serum-containing media. When dexamethasone has been used in serum-containing culture medium, results have been conflicting. For example, HSV-2 viral productivity decreased in SME (Swiss mouse embryo) cells, but increased in NIH3T3 cells, and retroviral infectivity was increased in rat pulmonary microvascular endothelial cells. See Costa et al ., 1974, Nature 252:745-746; and Solodushko et al., 2009, Am J Physiol Lung Cell Mol Physio 297:L538-45. Dexamethasone has also been shown to inhibit foot-and-mouth disease virus (FMDV) in infected cattle. See Ilott et al., 1997, Epidemiol Infect 118: 181-7. Applicants have found that the addition of dexamethasone and EGF (as well as MEM essential amino acids, GSEM supplement and trace elements) to serum-containing media improves the yield of HCMV in ARPE-19 cells.

The present invention was first demonstrated in micro-scale culture (96-well plates) and T-flasks, and later extended to microcarrers. The compositions and methods of the invention can also be used in bioreactors for large-scale virus production (e.g., for commercial viral vaccine production). The process is scalable from laboratory scale (<3L) to manufacturing scales (e.g., 50 - 2000L) and capable of ultizing a range of feed streams, including medium from static cell culture (e.g., from cell stacks) and culture in bioreactors.

Applicants believe this is the first time that this particular combination of dexamethasone, EGF, essential amino acids, nucleosides and trace elements has been used for viral propagation in serum-containing media to enhance cell yield and virus productivity. As used herein, purification of“Human cyto egalovirus” or“HCMV” generally refers to purification of HCMV virus particles.

As used herein,“cell culture medium” or“culture medium” refers to the liquid portion of a cell culture having one or more components other than HCMV particles, such as, but not limited to, proteins, nucleic acids, lipids, various cell culture medium components and additives. HCMV particles are released by the cells into the cell culture medium without any intervention or the cells may be lysed or rendered permeable to virus through chemical agents or mechanical means. The cell culture medium can be partially purified using centrifugation, clarification or other methods. In certain embodiments, the cell culture medium contains HCMV through its release into the cell culture.

Cell Culture Media

The invention provides serum-containing cell culture media that is supplemented with a glucocorticoid hormone such as dexamethasone, a growth factor such as EGF, additional essential amino acids, nucleosides (from glutamine synthetasae expression medium supplement), and a trace element mixture and is capable of increasing ARPE-19 cell growth and/or the viral yield in the production of HCMV. These supplements can be added to any basal cell culture medium suitable for the culture of cells, particularly ARPE-19 cells, which support virus growth. Exemplary basal culture media include OptiPro™ SFM (ThermoFisher Scientific), VP-SFM AGT™ (ThermoFisher Scientific), SFM4MegaVir (GE Healthcare), Roswell Park Memorial Institute medium (RPMI) 1640 medium, Minimal Essential Medium (MEM), Dulbecco's modified Eagle medium (DMEM), DMEM/F-12 (ThermoFisher Scientific), Eagle's MEM (EMEM), and any other medium in which the cells and virus can grow, as can be tested using standard methods.

The basal cell culture medium is formulated with serum (e.g., fetal bovine serum (FBS)), which is standard for most cell culture applications and provides additional nutrients. Generally, serum will be added at about 1% to about 15%. Suitable serum-containing culture media includes, but is not limited to, DMEM/F12 medium supplemented with about 2% to about 10% fetal bovine serum (FBS), DMEM supplemented with about 2% to about 10% FBS, and EMEM (Eagle’s Minimum Essential Medium) supplemented with about 1% to about 15% FBS.

Generally, the basal culture medium will contain inorganic salts for regulation of the osmolality, preferably in the range of 200-400 milliOsmoles (mOss), and more preferably in the range of 290-350 mOsm. Osmolality regulators are generally salts. Inorganic salts used in the basal medium of the present invention include, for example, salts of Ca²⁺, K⁺, Mg² , Na⁺, CO3² , PO4³ . Any salt of each of these moieties can be present in the basal cell culture media. For example, the following salts can be used: CaCl₂, KC1, KNO3, MgS0₄, NaCl, NaHCCh, and NaH₂P0₄ 2H2O.

The basal cell culture medium will also contain buffering agents, which act to stabilize the hydrogen ion concentration and therefore the pH of a solution by neutralizing, within limits, both acids and bases. Buffering agents commonly used in the cell cloning medium maintain the pH in the range about 6.5-7.5, preferably about pH 7.0, and include carbonates such as NaHCCb; chlorides, sulphates and phosphates such as CaCl₂ 2H₂0, MgSCE 7H₂0,

NaH₂P0₄ 2H₂0, NazFtPCE 7H₂0, beta-glycerol -phosphate, bicarbonate or sodium pyruvate.

Such buffers are generally present in an amount from about 50-3000 mg/liter. Other buffers, such as N-[2-hydroxyethyl]piperazine-N'-[2-ethanesul-phonic acid] otherwise known as HEPES and 3-[N-Morpholino]-propanesul-fonic acid otherwise known as MOPS can be present in an amount from about 1000-10,000 mg/liter.

The basal cell culture medium will further contain an energy source, such as a carbohydrate. The energy source of use in the cell cloning medium is preferably a

monosaccharide, such as mannose, fructose, galactose or maltose, preferably glucose, and particularly D-glucose, which is generally present in an amount from about 1000-10,000 mg/liter.

Vitamins and enzyme co-factor vitamins (co-factors), such as Vitamin B6

(pyridoxine), Vitamin B 12 (cyanocobalamin) and Vitamin K (biotin) may be present in the basal cell culture medium, preferably in an amount of about 0.01-0.5 mg/liter. Vitamin C (ascorbic acid) may be present in an amount of aboutl0-30 mg/liter, and Vitamin B2 (riboflavin) may be present in an amount of about 0.1 -1.0 mg/liter. The basal cell culture medium may also contain Vitamin B1 (thiamine), nicotine amide, Vitamin B5 (D calcium pentothenate), folic acid, and/or i-inositol, which are preferably present in an amount of about 0.2-8.0 mg/liter.

Basal cell culture medium will also typically include a lipid agent, which provides a source of lipids or contributes to lipid formation. Suitable lipid agents which can be used in the basal cell culture medium include, but are not limited to, Human Ex-Cyte^® (Bayer),

ethanolamine (or a salt thereof), sitosterol (a plant steroid), rice hydrolysate (a mixture of proteins and lipids), LTI Defined Lipid Mixture, a mixture of arachidonic acid, cholesterol, DL- alpha-tocopherol -acetate, ethyl alcohol, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitric acid, palmitic acid, Pluronic^® F-68, stearic acid, Tween^® 80, choline chloride, phosphatidylcholine and methyl lineoleate. Preferably, ethanolamine is used in basal cell culture medium. Suitable concentrations of a lipid agent ingredient can be determined by one of ordinary skill in the art. Lipid agents will generally be present in an amount of about 0.05-10 mg/liter.

The components of DMEM/F12 media are provided in Table 1. Table 1 : Components of DMEM/F12

The components of FBS are provided in Table 2. Due to the nature of the purification from animal sources, there can be a significant variance between different samples. Table 2: Composition of 100% FBS

* from Lindl, T. (2002): "Zell- und Gewebekultur". 5th ed. Spektrum Akademischer Verlag, Heidelberg

Glucocorticoid compounds include, but are not limited to, betamethasone, dexamethasone, fludrocortisone acetate hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone. The compounds are readily available from commercial sources such as Sigma-Aldrich, St. Louis, MO. In certain embodiments of the invention, the

glucocorticoid compound is selected from betamethasone, dexamethasone, hydrocortisone, and prednisolone. In one embodiment, the glucocorticoid compound is dexamethasone.

The glucocorticoid compound can be added to a concentration of about 1 nM-1 mM. Preferably glucocorticoid compound is added to a concentration of about 0.1 mM to about 100 mM, about 0.5 mM to about 10 pM, about 1 pM to about 5 mM.

While the glucocorticoid compound can be added to the culture medium prior to addition of cells, i.e., is a component of the cell culture medium, without departing from the spirit of the invention, the glucocorticoid can be added at inoculation or at any time after virus infection, e.g., the glucocorticoid compound can be added within one day of the virus infection or any time later. In this scenario, the glucocorticoid compound may be added to the cell culture one time, two times, three times, or any number of times during the specified time period. One or more glucocorticoid compounds may be used in conjunction. That is, any given single addition of a glucocorticoid compound may include the addition of one or more other

glucocorticoid compounds. Similarly, if there is more than one addition of a glucocorticoid compound, different glucocorticoid compounds may be added at the different additions.

Additional compounds and substances, including glucocorticoid compounds, may be added to the culture before, with or after the addition of glucocorticoid compound— either during or not during the specified time period.

The glucocorticoid compound may be added to the cell culture by any means.

Means of adding glucocorticoid compound include, but are not limited to, dissolved in DMSO, dissolved in organic solvent such as ethanol, dissolved in water, dissolved in culture medium, dissolved in feed medium, dissolved in a suitable medium, in the form in which it is obtained or any combination thereof.

The growth factor can be selected from an EGF, a platelet derived growth factor (PDGF), and a fibroblast growth factor (FGF). The growth factor may be any animal or mammalian growth factor, such as human, bovine, equine, murine, porcine, Chinese hamster, chicken or rat. Preferably, the growth factor is a human growth factor.

In one embodiment, the growth factor is an EGF. The EGF family includes, for example, EGF, TGF-a (transforming growth factor-a), amphiregulin, HB-EGF (heparin-binding EGF-like growth factor), epiregulin, and neuregulin. In one aspect of this embodiment, the EGF family member is EGF. The EGF used in the presently claimed medium is preferably of synthetic or recombinant origin. EGF is available from various commercial sources, including Sigma-Aldrich.

The growth factor can be used in concentration of about 0.5 ng/ml to about 400 ng/ml, about 2 ng/ml to about 200 ng/ml, or about 5 ng/ml to about 40 ng/ml.

Accordingly, in certain embodiments, the cell culture medium contains a basal cell culture medium suitable for culturing primary human cells, which basal cell culture medium is preferably enriched and additionally contains: about 0.5 to about 200 ng/ml of EGF and about about 0.5 mM to about about 10 pM of dexamethasone. In certain embodiments, the cell culture medium contains about 2.0 to about 50 ng/ml of EGF and about 1 pM to about 10 pM of dexamethasone.

The culture media will also contain a mixture of amino acids sufficient to support growth of the cells. The amino acids can be in the basal cell culture media and supplemented as necessary to obtain the concentrations in Table 3.

Table 3 : Amino acid concentrations

Desired final concentration

Amino Acid

(mg/liter)

L- Alanine 5-50

L- Arginine (HC1) 50-100

L- Asparagine (H2O) 5-50

L-Aspartic Acid 5-50

L-Cy stine (di sodium salt) 50-500

L-Glutamic acid 5-50

L-Glutamine 100-600

Glycine 20-200

L-Histidine (HCI.H2O) 50-500

L-Isoleucine 50-500

L-Leucine 50-500

L-Lysine (HC1) 100-500

L-Methionine 20-200

L-Phenylalanine 50-250

L-Proline 10-100

L-Serine 20-200

L-Threonine 50-500

L-Tryptophan 10-100

L-Tyrosine (di sodium salt) 50-500

L- Valine 50-500

The amino acids included in the medium are preferably of synthetic origin. The amounts which are usually included vary for each amino acid but are generally in the range about 10-500 mg/ml. However, L-glutamine is generally present at much higher concentration preferably in the range about 100-600 mg/ml.

In certain embodiments, a commercially available amino acids mixture is added to the basal culture media. Representative amino acid mixtures are available as essential amino acids (e.g., MEM (Minimal Essential Medium) Amino Acids Solution (50X), Coming Inc., Coming, NY, MilliporeSigma, or ThermoFisher Scientific).

Nucleosides are included in the cell culture medium of the present invention.

Examples of nucleosides include adenosine, cytidine, guanosine, thymidine, and uridine. Each of these nucleosides can be found in the commercially available glutamine synthetase expression medium supplement (e.g., GSEM Supplement (50X), Millipore Sigma, St. Louis, MO). Typically, essential amino acid solutions do not contain L-glutamine. In one embodiment, the basal culture media is supplemented with both MEM essential amino acids and GSEM.

The composition of MEM and GSEM are provided in Tables 4 and 5, respectively.

Table 4: IX MEM Essential Amino Acids

The concentrations refer to the amino acids being added to the basal cell culture medium. In cases where a 50X solution is diluted 50-fold to obtain a IX solution, the above concentrations do not take into account any amino acids already in the culture media. If MEM is used as the basal culture medium, addition of IX MEM Essential Amino Acids to the MEM medium will result in a 2X concentration of the essential amino acids.

Table 5: lx GSEM Supplements

The concentrations refer to the supplements being added to the basal cell culture medium. In cases where a 50X solution is diluted 50-fold to obtain a IX solution, the above concentrations do not take into account any of the listed amino acids or nucleosides already in the basal culture media. GSEM Supplement is described in Doyle et al ., Cell and Tissue Culture: Laboratory Procedures 27D, 3-3, (1999)

A basal culture medium will also be supplemented with various trace elements, which will be present in a cell culture medium in only trace amounts. Trace elements for supplementation include, for example, Se⁴⁺, Ag⁺, Al⁺ , Ba²⁺, Cd²⁺, Co²⁺, Cr⁺, Ge⁴⁺ , Br, G, Mn²⁺, F , Si⁴⁺ , V⁵⁺, Mo⁶⁺, Ni²⁺, Rb⁺, Sn²⁺ and Zr⁴⁴ and salts thereof. Particularly common trace elements include non-ferous metal ions of magnesium, copper and zinc; as well as sodium, potassium and selenium. The ions are generally added to the medium in the form of salts such as chlorides and sulphates. Any salt of a given trace element can be used to supplement the basal culture medium. For example, the sodium salt of selenium oxide (sodium selenite, NaiSeCE) is preferably used to provide selenium. Suitable concentrations of trace element-containing compounds can be determined by one of ordinary skill in the art. For example, in the medium of the invention, the concentration of SeCE² can be about 0.007 to about 0.07 mg/L. In a preferred embodiment of the medium of the invention, the concentration of SeCE² is about 0.01 mg/L.

The amounts are typically similar to those provided in the media disclosed in the examples below, but may be varied. A representative commercially available mix (Coming™ Trace Elements A, 1000X) when diluted to IX adds 1.6 pg/mL CuSCE ATEO, 863 pg/mL

ZnSCE AFEO, 17.3 pg/mL Selenite -2Na, 1155.1 pg/mL ferric citrate. Concentrations of the trace elements in the culture media can be lx - 4x concentrations.

Cell Lines

A suitable cell line includes one that hosts HCMV. Suitable host cells include, but are not limited to, epithelial cells ( e.g . ARPE-19 human retinal pigment epithelial cells), endothelial cells (e.g. TIME, HUVEC), fibroblast cells (e.g. MRC-5 human fetal lung fibroblast cells, WI-38 human fetal lung fibroblasts, HFF, etc.) and kidney cells (e.g., Vero African green monkey kidney and BHK baby hamster kidney). Typically, the cell line is a mammalian cell line, such as a rodent, non-human primate (e.g., monkey), or human cell line. In one embodiment, the host cell line is ARPE-19.

Culture conditions

Cell culture is performed to enable the cell to metabolize, grow, divide and/or produce virus of interest. Cell culture conditions suitable for the methods of the present invention are those that are typically employed and known for batch, fed-batch, or continuous culturing of cells, with attention paid to pH, e.g., about 6.5 to about 7.5; dissolved oxygen (O2), e.g., between about 5-90% of air saturation and carbon dioxide (CO2), agitation and humidity, and temperature.

Cell culture methods may comprise growth adhering to surfaces, growth in suspension, or combinations thereof. Culturing can be done, for instance, in dishes, well plates, T-flasks, shake flasks, stirred vessels, spinner flasks, roller bottles or in bioreactors, using batch, fed-batch, continuous systems, hollow fiber, and the like. High density cell culture systems, such as Coming HyperFlask® and HyperStack® systems can also be used. In one embodiment, a suitable cell culturing vessel for large scale vims production is a bioreactor. Bioreactor processes and systems have been developed to optimize gas exchange, to supply sufficient oxygen to sustain cell growth and productivity, and to remove CO2. Maintaining the efficiency of gas exchange is an important criterion for ensuring successful scale up of cell culture and protein production. Such systems are well-known to the person having skill in the art.

The culture may be ran for any length of time and may be determined by one of skill in the art, based on relevant factors such as the quantity and quality of recoverable vims, and the level of contaminating cellular species (e.g. proteins and DNA) in the supernatant, which will complicate recovery of the vims of interest. Suitable conditions for culturing cells are known. See, e.g., Tissue Culture, (1973), Kruse and Paterson (Eds.), Academic Press, and Freshney,

2000, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Liss Inc.).

Methods of propagating cyto egalovirus, including HCMV, are known in the art. See, e.g., Britt, 2010, Human Cytomegalovirus: Propagation, Quantification, and Storage; in Current Protocols in Microbiology, 18:E: 14E.3 : 14E.3.1-14E.3.17; Chambers et al., 1971, Appl Microbiol 22:914-8; and Schneider-Ohrum et al., 2016, J Virol 90: 10133-10144. In some embodiments, cell cultures are inoculated with cyto egalovirus and cultured for a period of time ( e.g ., 24 hours to 3 weeks) before harvesting the virus. Various methods of harvesting virus from cell culture are known in the art. In some embodiments, the cytomegalovirus is harvested from the cell culture by collecting the medium. In some embodiments, the cytomegalovirus is harvested from the cell culture by lysing the cells (e.g., by mechanical or non-mechanical lysis methods). In some embodiments, the cyto egalovirus is harvested from the cell culture by adding a cell permeation agent to release virus from host cells.

In order to achieve large-scale production of virus through cell culture, it is preferred in the art to have cells capable of growing in suspension or microcarriers. Accordingly, in some embodiments, the cells used to propagate the HCMV are grown on microcarriers. A microcarrier is a support matrix allowing for the growth of adherent cells in spinner flasks or bioreactors (such as stirred bioreactors, rotating wall microgravity bioreactors and fluidized bed bioreactors). Microcarriers are typically 125 - 250 mM spheres with a density that allows them to be maintained in suspension with gentle stirring. Microcarriers can be made from a number of different materials including, but not limited to, DEAE-dextran, glass, polystyrene plastic, acrylamide, and collagen. The microcarriers can have different surface chemistries including, but not limited to, extracellular matrix proteins, recombinant proteins, peptides and charged molecules. In one embodiment, the microcarriers are Cytodex™ microcarriers (GE Healthcare Life Sciences) which are based on cross-linked dextran matrices.

In some embodiments, the processes described herein employ a nuclease to remove contaminating nucleic acids, which are mostly from the host cell. Exemplary nucleases suitable for use in the invention include BENZONASE^®, PULMOZYME^®, or any other DNase and/or RNase commonly used within the art. In preferred embodiments of the invention, the nuclease is BENZONASE^®, which rapidly hydrolyzes nucleic acids by hydrolyzing internal phosphodiester bonds between specific nucleotides. BENZONASE^® is a genetically engineered variant of an endonuclease normally expressed in Serratia marcescens, and can be commercially obtained from Millipore Sigma (Burlington, MA). The concentration at which the nuclease is employed is preferably within the range of 1-100 units/ml.

In certain embodiments, the nuclease is added to the post-harvest cell culture medium. The batch can then be incubated at temperatures ranging from the culturing

temperature (e.g., about 37°C), or at room temperature (around 20°C) or lower (e.g., around 0°C), wherein, in general, longer incubation times are required at lower temperatures to achieve the same result.

In certain embodiments, the nuclease can be employed before the cell culture medium is harvested. It may be added just seconds prior to (or virtually concomitant with) the harvesting step, but preferably, the nuclease is added to the culture at least one minute before the harvesting step and up to several days before the harvesting step. The cell culture with the added nuclease can then be incubated above process temperature, e.g., around 40°C, or at the culturing temperature (e.g., about 37°C), or at room temperature (around 20°C) or lower (e.g., around 0°C), wherein, in general, longer incubation times are required at lower temperatures to achieve the same result.

Cytomegalovirus

The cell culture media of the present invention can be used to produce any cyto egalovirus, including wild-type cyto egalovirus, a recombinant cytomegalovirus, and modified cytomegalovirus (for example, cytomegalovirus containing a transgene). In some embodiments, the cytomegalovirus is a human cytomegalovirus (HHV-5). In some

embodiments, the HCMV is AD169 strain, Toledo strain or Towne strain. In some

embodiments, the cytomegalovirus is a recombinant HCMV, particularly, a conditional replication defective HCMV. In some embodiments, the cyto egalovirus is a recombinant HCMV delivering one or more transgenes. The processes of the invention can also be used to purify both infectious and noninfectious HCMV particles. See Schneider-Ohrum et al., 2016, J Virol. 90: 10133-10144.

In certain embodiments, the HCMV is a recombinant HCMV vaccine. See, e.g., Lilja et al, 2012, Vaccine 30:6980. In certain embodiments, the HCMV is a recombinant vaccine vector expressing a heterologous antigen. The antigen can be from another pathogen or be a tumor antigen. See, e.g., U.S. Pat. No. 9,249427 (pathogen antigen or tumor antigen), U.S. Patent Application Publication No. 20160354461 (HIV, SIV or TB antigens) and International Patent Application Publication No. W01998/010311 Al (HIV or malarial antigens); Marzi et al, 2016, Sci Rep 6:21674 (Ebola virus); and Tierney et al, 2012, Vaccine 30:3047 (tetanus toxin).

In one embodiment, the HCMV is a conditional replication defective HCMV (rdHCMV). As used herein, the term "conditional replication defective virus" refers to virus particles that can replicate in a certain environments but not others. In certain embodiments, a virus is made a conditional replication defective virus by destabilization of one or more proteins essential for viral replication such as IE1/2, UL51, UL52, UL79 and UL84, or a combination thereof, for example, by fusion with a destabilizing polypeptide such as a sequence of FKBP or a derivative thereof. Preferred FKBP derivatives have one or more of the following substitutions at the denoted amino acid positions F15S, V24A, H25R, F36V, E60G, M66T, R71G, D100G, D100N, E102G, K105I and L106P. The FKBP derivative having the F36V and L106P substitutions is particularly preferred. The nucleic acids encoding the wild type, non-destabilized essential proteins are modified in the conditional replication defective virus. In more preferred embodiments, one or more essential proteins are destabilized. Such fusion proteins can be stabilized by the presence of a stabilizing agent such as Shield- 1, commercially available from Cheminpharma LLC (Farmington, CT) or Clontech Laboratories, Inc. (Mountain View, CA).

See, e.g., U.S. Patent Application Publication No. 2009/0215169; and Clackson et al., 1998, Proc Natl Acad Sci USA 95: 10437-42).

In one embodiment, the conditional replication defective HCMV is derived from AD 169 strain that has a restored gH complex expression due to a repair of a mutation in the UL131 gene. As used herein, the terms "pentameric gH complex" or“gH complex” refer to a complex of five viral proteins on the surface of the HCMV virion. The complex is made up of proteins encoded by UL128, UL130, and UL131 assembled onto a gH/gL scaffold. See Wang and Shenk, 2005, Proc Natl Acad Sci USA 102: 1815; and Ryckman et al. , 2008, J. Virol. 82:60.

In one embodiment, the genome of the rdHCMV has a destabilized IE1/2 and UL51 and has a sequence as shown in SEQ ID NO: l . See U.S. Pat. No. 9,546,355.

Manufacture of Conditional Replication Defective HCMV

rdHCMV can be propagated in the presence of a stabilizing agent such as Shield- 1 on permissive cell types, preferably human epithelial cells, and more preferably human retinal pigmented epithelial cells. In additional embodiments, the human retinal pigmented epithelial cells are ARPE-19 cells deposited with the American Type Culture Collection (ATCC) as Accession No. CRL-2302. In some embodiments, Shield-1 is present at a concentration of at least 0.5 mM in the tissue culture media. In preferred embodiments, Shield- 1 is present at a concentration of at least 2.0 pM in the tissue culture media. Shield- 1 can be used to control replication of the rdHCMV in conjunction with FKBP (FK506 binding protein). See, e.g., U.S. Pat. No. 9,546,355.

In certain embodimetns, Shield- 1 is one of the components to be removed during the purification steps described herein. After purification of rdHCMV from cell culture media containing Shield- 1, there may be a small amount of residual Shield- 1 remaining in the rdHCMV preparation. In one embodiment, the level of Shield- 1 in the HCMV composition after purification is at least 10-fold below the level used to sustain replication in tissue culture. In another embodiment, the level of Shield- 1 in the rdHCMV preparation after purification is 0.05 pM or less. In another embodiment, the level of Shield- 1 in the rdHCMV preparation after purification is undetectable. Determination of Shield-1 levels in a composition can be detected using a LC/MS (liquid chromatography-mass spectroscopy) or HPLC/MS (high performance liquid

chromatography-mass spectroscopy) assays. These techniques combine the physical separation capabilities of LC or HPLC with the mass analysis capabilities of and can detect chemicals of interest in complex mixtures.

Compositions Including Media, Cells, and, Optionally, Viruses

The invention also includes compositions that include a medium of the invention, in combination with cells for use in virus production. These compositions can also, optionally, include viruses produced in the cells of the invention. Thus, in a certain embodiment, the invention provides a cell culture media as described herein, in combination with cells, e.g., ARPE-19 cells. In one aspect of the embodiment, the invention provides a cell culture media as described herein, in combination with APRE-19 cells infected with HCMV. The HCMV can include attenuating and/or other beneficial mutations as described herein.

Isolation of HCMV

Following cell culture, HCMV is isolated from the cell culture. Typically, cyto egalovirus is released from mammalian host cells into the culture media without any intervention. However, in some cases, cytomegalovirus can by released from cells by by adding a cell permeation agent to release virus from host cells or by lysing host cells. HCMV obtained from culture media, whether released by cells or through cell lysis, will contain one or more contaminants selected from proteins (e.g., host cell proteins, cell culture serum proteins or exogenously added proteins), and nucleic acids (e.g., host cell DNA). The cyto egalovirus preparations can by purified by at least one filtration, chromatography, clarification,

precipitation, or fractionation step or any combination thereof. A representative purification process is provided in U.S. Provisional Application No. 62/661,928, filed on April 24, 2018.

An exemplary complete purification process involves: (1) Nuclease digestion (with Benzonase^®) prior to harvest in a bioreactor containing cell culture medium and microcarrier beads having cells infected with HCMV; (2) harvest of cell culture medium from microcarrier beads; (3) clarification of harvested cell culture medium by a 1.2-micron glass-fiber filter (e.g. Sartorius Sartopore^® GF Plus); (4) capture of viral particles by anion exchange (AEX) membrane chromatography (e.g., Sartorius Sartobind^® Q); (5) polishing purification by Capto™ Core 700 chromatography (GE Healthcare) operated in a flow through mode; (6) concentration and buffer exchange by 750-kDa hollow fiber TFF (e.g., GE Healthcare or Spectrum). In one aspect, a sterile filtration using a 0.2-micron filter selected from Sartorius Sartobran^® P,

Millipore Durapore™ PVDF filter, or Pall SuporLife^® is performed before or after the TFF step in (6). In an alternative aspect, no sterile filtration is performed and the process is run completely aseptically.

In one embodiment, a 750-kDa hollow fiber TFF (GE Healthcare or Spectrum) step may also be included between the AEX and polishing chromatography.

All references, publications, patent applications, and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference.

Examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention.

EXAMPLES

ANALYTICAL METHODS

Viral particle concentration

A relative size distribution and total concentration of viral particles in the HCMV sample can be obtained by flow cytometry (e.g. Apogee Flow Systems). As the individual particles pass through the instrument’s flow cell, they are illuminated by a 405 nm laser and the scattered light is detected. The obtained distribution is a relative size distribution of light scattering signal on a logarithmic scale. Counts in total particles per milliliter were also determined.

Relative infectivity assay

This analytical procedure is a cell-based relative infectivity assay based on expression of a HCMV protein following infection. ARPE-19 cells were planted and incubated in 96-well micro-titer plates followed by infection with serial dilutions of HCMV samples, controls, and standards. The infected cells were incubated for approximately 1 to 5 days then fixed with a fixative (e.g. formaldehyde). After washing, the plate is incubated with a monoclonal antibody (MAb) reactive with a HCMV protein. After incubation with the anti- HCMV MAb, the plate is washed and incubated with a detection antibody. After this final incubation, the plate is washed and read on a plate reader instrument. The relative infectivity assigned to the test article is based on signal generated with an assay reference standard tested on the same plate.

EXAMPLE 1 : Screening of individual supplements on ARPE-19 cell growth at 96-well plate format

Despite the preference to avoid animal serum for vaccine production, the propagation of HCMV in ARPE-19 cells requires Fetal Bovine Serum (FBS) in order to obtain sufficient quantities. FBS contains various growth factors, fatty acids, proteins, trace metals, amino acids, etc; some of which are crucial to support mammalian cell growth and virus production within the cell. For serum-free media, supplementation of the media with serum components such as growth factors has been shown to be necessary to achieve levels of virus production similar to that obtained with serum-containing media. In an attempt to optimized virus production in serum-containing media, various supplements were investigated in FBS- containing medium for the purpose of boosting ARPE-19 cell growth and/or CMV virus production without increasing the FBS concentration. This investigation of the effect of supplements on ARPE-19 cell growth was performed on 96-well format, T-flask format and microcarrier inside bioreactors.

The supplements evaluated for their effect on ARPE-19 cell growth include: Epidermal Growth Factor (EGF), Hydrocortisone, Dexamethasone, Insulin, Non-Essential Amino Acids [Glycine, L- Alanine, L- Asparagine, L-Aspartic acid, L-Glutamic Acid, L-Proline, and L-Serine], Essential Amino Acids [L- Arginine, L-Cystine, L-Histidine, L-Isoleucine, L- Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L- Threonine, L-Tryptophan, L-Tyrosine, and L-Valine] (Vendors: MilliporeSigma, Therm oFisher)0, Glutathione, and Linoleic Acid-Oleic Acid-Albumin (Sigma-Aldrich Corp., St. Louis, MO). Several concentrations and combinations were tested as indicated in Table 6

Table 6: Supplements used for screening

Cells were seeded at 1E4 vc/cm² (viable cells/cm²) and growth was monitored by staining the cells on day 2, 3, 4, 5, 6, 7, and 8 post plant with cell nuclear stain (Hoechst) and quantifying the nuclear stain on an imager to generate cell growth data for each day. The cell growth data was analyzed statistically to assess the impact of the supplement on cell growth.

Components with p-value of < 0.05 were deemed to have positive effect on cell growth. These include EGF, essential amino acid, linoleic and oleic acid. See Figure 1.

To confirm that these supplements also had an effect on viral infectivity, combinations of multiple supplements at various concentrations were tested in the 96-well plate. ARPE-19 cells were planted at 1E4 viable cell/cm² in DMEM/F12 containing either 10% FBS or 2% FBS with or without supplements. The cells were allowed to grow for 4 days and growth was monitored by staining the cells on day 2, 3, 4, 5, 6, 7, and 8 post plant with cell nuclear stain (Hoechst) and quantifying the nuclear stain on an imager to generate cell growth data for each day. The cultures were then exchanged into production medium containing 2% FBS with or without supplements and infected with HCMV virus at the multiplicity of infection (MOI) of 0.1 pfu/cell. Culture samples were collected 3 and 4 days post infection and analyzed for virus infectivity (% Response).

The viral infective was plotted in Figure 2.

The data shown suggests that Media #16 (DMEM/F12+2%FBS+ 10 ng/mL EGF + lx essential amino acid + 2.5 mcM Dexamethasone) produced the highest virus infectivity compared to other combinations as well as control without supplements.

EXAMPLE 2: Demonstration of the effect of select supplements on ARPE-19 cell growth in T- flasks

An ARPE-19 Working Cell Bank was planted onto T-175 flasks at a plant density of ~le4 vc/cm² using 2 different growth media as listed below. Medium #1 : DMEM/F-12 + 10% FBS + 6 mM L-Glutamine

Medium #2: DMEM/F-12 + 10% FBS + 6 mM L-Glutamine + 10 ng/mL Epidermal Growth Factor (EGF), 2.5 mM Dexamethasone, lx essential amino acid cocktail, lx GSEM (Sigma- Aldrich; L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, adenosine, guanosine, cytidine, uridine, and thymidine), and lx Trace Elements A (Corning; Cupric Sulfate, Ferric Citrate, Sodium Selenite, and Zinc Sulfate).

One flask from each medium was harvested daily for 7 days. Cells were passaged in the medium once they reach the targeted cell density of 8e4 vc/cm² or 6.67e7 cells/mL based on the daily cell counts (instrument used: Cedex cell counter). Cells in medium #1 were passaged 6 times and cells in medium #2 were passaged 8 times. Growth rate and doubling time were calculated. Cells grown in medium #2 (supplemented medium) had higher growth rate and lower doubling time in the first 3 passages compared to medium #1 (non-supplemented medium). See Figure 3.

The supplements added in combination specified above boost ARPE-19 cell growth in the presence of 10%FBS.

Another study was performed to investigate the effect of certain supplements on the growth of ARPE-19 cells with a lower amount of FBS (2%). The study was performed on T- flasks as described above.

ARPE-19 Working Cell Bank was planted onto T-175 flasks at plant density of ~le4 vc/cm2 using 3 different growth media listed below.

Medium #3: DMEM/F-12 + 10% FBS + 6 mM L-Glutamine

Medium #4: DMEM/F-12 + 2% FBS + 6 mM L-Glutamine

Medium #5: DMEM/F-12 + 2% FBS + 6 mM L-Glutamine + 10 ng/mL Epidermal Growth Factor (EGF), 2.5 mM Dexamethasone, lx essential amino acid cocktail, lx Linoleic and Oleic acid mixture (Sigma-Aldrich).

Cells were passaged in the media specified above for 3 passages under the same passaging schedule (7 day-6 day-4 day). The plant density for each passage was 1E4 vc/cm².

The harvest density from each passage was measured by vicell cell counter. The average doubling time was calculated from the harvest density and plant density. The results are shown in Figure 4.

Surprisingly, cells passaged in medium #5 had equivalent doubling time to cells passaged in medium #3 despite medium #5 having lower FBS concentration than in medium #3.

This shows that FBS concentration could be reduced but that adding certain supplements could provide an equivalent or superior effect on cell culture. In passage 36 and 37, the doubling time in medium #5 is also significantly lower than the doubling time in medium #4 which indicated the positive effect of that the supplements had on ARPE-19 cell growth.

These results indicate that the combination of EGF and dexamethasone, with different combinations of amino acids, was able to reduce doubling time of the ARPE-19 cells grown in media containing 2% FBS compared to ARPE-19 cells grown in media containing 10% FBS.

EXAMPLE 3 : Demonstration of Improved ARPE-19 cell growth with addition of select supplements at various sera types

Various supplements were investigated in cell culture medium containing either fetabl bovine serum (FBS), bovine serum (BS) for the purpose of boosting ARPE-19 cell growth concentration. This investigation was performed on 96-well format. Cells were seeded at 1E4 vc/cm² (viable cells/cm²) and growth was monitored by staining the cells on day 2, 3, 4, 5, 6, 7, and 8 post plant with cell nuclear stain (Hoechst) and quantifying the nuclear stain on an imager to generate cell growth data for each day. The cell growth data was analyzed statistically to assess the impact of the supplement on cell growth. The results are shown in Figures 5A-C.

The addition of supplements (EGF, essential amino acid, linoleic/oleic acid, and dexamethasone) to the DMEM/F12 media with 2%FBS resulted in 40% higher growth compared to 10%FBS alone. Similar observations were made when we tested the supplements in the presence of donor bovine serum (DBS) and Heat-Inactivated Bovine serum (BS).

EXAMPLE 4: Demonstration of Improved ARPE-19 Cell Growth in the presence of Select Supplements in a Microcarrier System inside Bioreactor

The effect of certain supplements was also tested in a microcarrier system inside bioreactors. Cells were planted on Cytodex™-l microcarrier inside bioreactors with 2 different growth media. The cell growth in the bioreactors was measured using nucleocounter cell counting method. The average doubling time of the cells in the growth phase was calculated.

Medium #6: DMEM/F-12 + 10% FBS + 6 mM L-Glutamine

Medium #7 : DMEM/F-12 + 10% FBS + 6 mM L-Glutamine+ 2.5 mM Dexamethasone, 10 ng/mL EGF, lx essential amino acid + lx GSEM + lx Trace-Element A (Trace Elements A includes Cupric Sulfate, Ferric Citrate, Sodium Selenite, and Zinc Sulfate). Table 7: ARPE-19 Population Doubling Time in Microcarrier inside a Bioreactor

Based on the data presented above, an improved cell culture media will include include the following supplements to enhance ARPE-19 cell growth in the microcarrier system.

EXAMPLE 5: Demonstration of Improved CMV Production in the presence of Select

Supplements in Microcarrier System inside Bioreactor

The impact of the certain supplements on virus production was also assessed.

Cells were planted on Cytodex™-l microcarrier inside bioreactors with either DMEM/F12 +

10% FBS + 6 mM L-Glutamine growth medium or DMEM/F12 + 10% FBS + L-Glutamine + supplements. These bioreactors were then media-exchanged into infection/production medium containing DMEM/F12 + 1% FBS+ 5 mM Shield-1 or DMEM/F12+ 1% FBS + 5 mM Shield-1 + supplements. The specific components of the various growth media is provided in Table 8. The bioreactors were then infected with sufficient amount of virus seed and virus production was monitored over 15 days using Apogee flow cytometry (total parti cles/mL) and IRVE (infectivity).

Table 8:

The volumetric production of virus as measured by Apogee flow cytometry is shown in Figure 6. Condition #3 yielded 35% higher total particle/mL compared to Condition #1 and approximately 20% higher total particle/mL compared to Condition #2.

Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. A cell culture medium comprising a basal cell culture medium that comprises serum and is further supplemented with between about 0.1 mM and about 10 mM of dexamethasone, between about 0.1 ng/mL and about 100 ng/mL of epidermal growth factor (EGF), IX MEM essential amino acids, 1X-4X glutamine synthetase expression medium (GSEM) supplement and trace elements.

2. The medium of claim 1, wherein the basal cell culture medium is

DMEM/F-12.

3. The medium of claim 1 or 2, wherein said serum is fetal bovine serum

(FBS).

4. The medium of claim 3, wherein the FBS is present at a concentration between about 2% and about 10%, inclusive.

5. The medium of any one of claims 1-4, wherein the dexamethasone is present at a concentration between about 1 and about 10 mM.

6. The medium of any one of claims 1-5, wherein the EGF is present at a concentration between about 1 and about 40 ng/mL.

7. The medium of any one of claims 1 to 6, wherein the IX MEM essential amino acids consist of arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine.

8. The medium of any one of claims 1 to 7, wherein the IX GSEM supplement consists of a mixture of L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L- proline, L-serine, adenosine, guanosine, cytidine, uridine, and thymidine.

9. The medium of any one of claims 1 to 8, wherein the trace elements are selected from cupric sulfate, ferric citrate, sodium selenite, and zinc sulfate.

10. A composition comprising the medium of any one of claims 1 to 9 and cells selected from ARPE-19, Vero, MDCK, MRC-5, BHK, CEM, and LL-I cells.

11. The composition of claim 10, wherein said composition further comprises a virus.

12. The composition of claim 11, wherein said virus is a human herpesvirus.

13. The composition of claim 12, wherein said human herpesvirus is human cyto egalovirus (HCMV).

14. The composition of claim 13, wherein said HCMV is a conditional replication defective HCMV mutant.

15. The composition of claim 14, wherein the conditional replication defective

HCMV mutant has a genomic sequence of SEQ ID NO: 1.

16. A method of increasing virus production in cells, said method comprising culturing the composition of any of claims 10 to 15, wherein said composition provides increased production of the virus by at least 10% relative to the production of virus grown in a composition that does not contain the supplements of claim 1.