WO2001032838A2

WO2001032838A2 - Methods for improving viral vector titers and reducing cell death in cell cultures

Info

Publication number: WO2001032838A2
Application number: PCT/US2000/029549
Authority: WO
Inventors: Manish Singh
Original assignee: Chiron Corporation
Priority date: 1999-10-29
Filing date: 2000-10-27
Publication date: 2001-05-10
Also published as: CA2389057A1; EP1224260A2; WO2001032838A3; AU1346501A; JP2003513623A

Abstract

Disclosed are compositions and methods for the increasing the production of a recombinant viral vector in a cell culture, comprising culturing a recombinant viral vector producing cell line in a basal media which further comprises (a) greater than 1.5 g/L amino acids (or, a selected amino acids in a quantity and amount greater than present in a basal media such as DMEM), and (b) between 0.5% and 4% serum, such that the recombinant viral vectors are produced.

Description

METHODS FOR IMPROVING VIRAL VECTOR TITERS AND REDUCING CELL DEATH IN CELL CULTURES

TECHNICAL FIELD

The present invention relates generally to methods and compositions for improving the production of viral vectors in cell culture.

BACKGROUND OF INVENTION

The manufacture of products using animal cells is currently a major component of the biotechnology industry. Such cells are currently being used for the production of viruses, vaccines, monoclonal antibodies, proteins, and recombinant viral vectors. Optimization of product expression and reduction of cost is important for economic and commercial feasibility of these products.

Unfortunately, the ability to express high amounts of products for long periods of time is limited due to death of cells in culture. Prevention of cell death during culture is a high priority for the production of biopharmaceuticals, however, very little progress has been made in this direction especially for commercial product manufacturing.

One of the major limitations of present commercial production strategies is their limited ability to delay apoptosis, particularly for cultures that do not grow well in suspension (i.e., are anchorage-dependent). This problem is particularly apparent for a large number of vaccine and viral vector applications where the cell lines which are utilized are anchorage dependent cultures.

One of the major components of most culture media, which significantly increases the cost of manufacturing, is serum. However, removal or reduction of serum from a standard of 5-10% usually results in a decrease of product formation. A number of media have been developed for serum-free propagation, however, only a few cell types such CHO and 293 cells have been reliably grown under serum-free/reduced serum conditions. Most commercial production of biological products still require use of serum, and production of viruses and viral vectors routinely require use of higher serum concentrations. Reduction in serum concentrations without sacrificing product yields or product titers is a challenge that has not been completely addressed to date.

The present invention discloses compositions and methods for which increase vector titer and decrease cell death, and further provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methods for the increasing the production of a recombinant viral vector in a cell culture, comprising culturing a recombinant viral vector producing cell line in a basal media which further comprises (a) greater than 1.5 g/L amino acids (or, a selected amino acids in a quantity and amount greater than present in a basal media such as DMEM), and (b) between 0.5% and 4% serum, such that the recombinant viral vectors are produced. Such methods can be utilized to increase the production of viral vector (or viral titer) by the cell line, and/or to decrease cell death during production. Moreover, the effect of such culturing conditions can be utilized to produce an anti-apoptotic effect on cells within the cell culture.

Representative examples of suitable basal media include RPMI or DMEM. To such media is added amino acids (to a final concentration of greater than 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, or, 2.5 g/L) and serum (between about 0.5% and 4%, preferably between about 1 or 2% and 3%. Within certain embodiments, the quantity and type of amino acids added to the basal media is based upon the utilization profile of the cell line to be cultured. For example, rather than adding 1.5, 2, 2.5 or greater g/L of amino acids to a cell culture, selected amino acids can be added in order to supplement a given cell line's utilization profile. Similarly, various types of serum can be used within the context of the present invention, including for example, fetal calf serum and human serum. Further ingredients can also be added to the media, including for example metals such as selenium and zinc, and salts such as magnesium chloride. The methods described herein may be utilized for a wide variety of recombinant viral vector producing cell lines, including for example, cell lines which have been constructed to produce recombinant retroviral vectors, adenoviral vectors, adeno-associated viral vectors and alphavirus vectors. Such vectors may be utilized for a variety of purpose, including for example, for the in vivo or ex vivo production of proteins, or vaccine purposes. Within certain embodiments, the recombinant viral vector producing cell line is an anchorage-dependent cell line. Within further embodiments, the cell line is cultured in a batch mode, or in a perfusion culture system. The method according to the present invention may further comprise a first metal salt comprising a metal selected from the group consisting of Cobalt (Co), Copper (Cu), Manganese (Mn), Molybdenum (Mo), and Selenium (Se) and a second metal salt comprising a metal selected from the group consisting of Iron (Fe), Calcium (Ca), Magnesium (Mg), and Zinc (Zn). Within certain embodiments, the first metal salt may be selected from the group consisting of CoCl .6H₂O, CuCl₂.2H₂O, CuSO , MnCl₂.4H₂O, (NH₄)₆ Mo₇O₂.4H₂O, and Na₂SeO₃. Additionally or alternatively, the second metal salt may be selected from the group consisting of FeCl₃, FeSO , CaCl₂, CaNO₃, MgCl₂, MgSO₄, ZnCl₂, ZnSO₄, FeNO₃.9H₂O.

The present invention also provides compositions for increasing the production of a recombinant viral vector and/or decreasing cell death in a cell culture. Exemplary compositions comprise a basal media; greater than 1.5 g/L amino acids; between 0.5% and 4% serum; a first metal salt wherein said first metal salt comprises a metal selected from the group consisting of Cobalt (Co), Copper (Cu), Manganese (Mn), Molybdenum (Mo), and Selenium (Se); and a second metal salt wherein said second metal salt comprises a metal selected from the group consisting of Iron (Fe), Calcium (Ca), Magnesium (Mg), and Zinc (Zn).

Further provided are compositions wherein the basal media is selected from the group consisting of BME, MEM, DMEM, DMEM-F-12, IMDM, McCoy's 5 A, Media 199, Ham's F-10, Ham's F-12, MS- 162, MS- 174, and RPMI. Within certain embodiments, the basal media is selected from the group consisting of DMEM, MS- 162, and MS- 174. Alternative embodiments of the present invention provide compositions comprising greater than 1.7 g/L amino acids, greater than 2.0 g/L amino acids, or greater than 2.5 g/L amino acids. By certain embodiments, one of the amino acids may be L-Cystine which may be present at a concentration of between 75 and 300 mg/L or, more preferably, at a concentration of between 100 and 200 mg/L. By still further embodiments, one of said amino acids is L-Serine which may be present at a concentration of between 75 and 1000 mg/L or, more preferably, at a concentration of between 200 and 700 mg/L. Other embodiments provide that one of the amino acids may be L-Methionine at a concentration of between 75 and 300 mg/L or, more preferably, at a concentration of between 100 and 200 mg/L.

Still further embodiments of the present invention provide compositions comprising between 1% and 3% serum or, alternatively, between 2% and 3% serum. By certain embodiments, the serum is fetal calf serum or human serum.

By certain embodiments, compositions according to the present invention may comprise a first metal salt selected from the group consisting of CoCl₂.6H₂O, CuCl₂.2H₂O, CuSO₄, MnCl₂.4H₂O, (NH₄)₆ Mo₇O₂.4H2O, and Na₂SeO₃. By preferred embodiments, the first metal salt may be present at a concentration of between 0.003 and 1.0 mg/L or, more preferably, at a concentration of between 0.01 and 0.1 mg/L. Further embodiments provide compositions that may comprise a second metal salt is selected from the group consisting of FeCl₃, FeSO , CaCl₂, CaNO₃, MgCl₂, MgSO₄, ZnCl , ZnSO₄, FeNO₃.9H O. By preferred embodiments, the second metal salt may be MgCl₂ at a concentration of between 50 and 500 mg/L or, more preferably, at a concentration of between 125 and 175 mg/L. Alternatively, the second metal salt may be ZnCl at a concentration of between 1 and 5 mg/L or, more preferably, at a concentration of between 2 and 4 mg/L.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a bar graph which shows vector product produced from DMEM 10% (FBS) vs. MS-162 10% (FBS) for 7 and 8 day cultures.

Figure 2 is a bar graph which shows cell death in cultures of DMEM 10% (FBS) vs. MS-162 10% (FBS) for 7 and 8 day cultures.

Figure 3 is a graph which compares the titer of cells grown in DMEM 11% (FBS), DMEM 2% (FBS), MS-174 2% (FBS), and MS-174 11% (FBS). Figure 4 is a graph which compares the vector production rate of cells grown in DMEM 11% (FBS), DMEM 2% (FBS), MS-174 2% (FBS), and MS-174 11% (FBS).

Figure 5 is a graph which compares LDH production per day of cells grown in DMEM 11% (FBS), DMEM 2% (FBS), MS-174 2% (FBS), and MS-174 11% (FBS).

Figure 6 is a graph which compares total vector production (on a manufacturing scale) for cells grown in DMEM 2% (FBS) and MS-174 2% (FBS).

Figure 7 is a graph which compares cell death (on a manufacturing scale) for cells grown in DMEM 2% (FBS) and MS-174 2% (FBS). Figure 8 is a bar graph which shows vector titer from cells grown on

DMEM 10% (FBS) vs. MS-162 10% (FBS).

Figure 9 is a bar graph which shows vector production from cells grown on DMEM 10% (FBS) vs. MS-162 10% (FBS).

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

Prior to setting forth the invention, it may be helpful to an understanding thereof to first set forth definitions of certain terms that will be used hereinafter.

"Recombinant viral vector" refers to a construct which is capable of delivering, and, within preferred embodiments expressing, one or more gene(s) or sequence(s) of interest in a host cell. Such recombinant viral vectors may be constructed from or derived from a variety of viruses, such as, for example, retroviruses, adenoviruses, adeno-associated viruses, alphaviruses and the like.

"Recombinant adeno-associated virus vector'Or "rAAV vector" refers to a gene delivery vector based upon an adeno-associated virus. The rAAV vectors, should contain 5' and 3' adeno-associated virus inverted terminal repeats (ITRs), and a transgene or gene of interest operatively linked to sequences which regulate its expression in a target cell. Within certain embodiments, the transgene may be operably linked to a heterologous promoter (such as CMV), or, an inducible promoter such as (tet). In addition, the rAAV vector may have a polyadenylation sequence.

"Recombinant Viral Vector Producing Cell Line" refers to a cell line which is used to produce recombinant viral particles, representative examples of such cell lines include retroviral packaging or producer cell lines, and alphavirus packaging or producer cell lines.

"Basal media" refers to a minimal media that, when supplemented with serum, is sufficient to support the growth and/or proliferation of a desired cell line. Representative examples of such media include BME, MEM, DMEM, DMEM-F-12, IMDM, McCoy's 5A, Media 199, Ham's F-10, Ham's F-12, and RPMI. Such media typically provide a mixture of sugar (e.g., glucose), vitamins (e.g., B12, etc.), and one or more salts or buffers, and may be obtained from a variety of commercial sources, including for example, Hyclone, Inc. (Logan, UT), Irvine Scientific (Irvine, CA), Biowhittaker (Walkersville, MD), Gibco-LTI (Gaithersburg, MD), and Sigma Chemical Co. (St. Louis, MO).

As noted above, the present invention provides compositions and methods for the production of a recombinant viral vector in a cell culture, comprising the step of culturing a recombinant viral vector producing cell line in a basal media which further comprises (a) greater than 1.5 g/L amino acids and (b) between 0.5% and

4% serum, such that the recombinant viral vectors are produced.

Such methods can be utilized to increase the production of viral vector (or viral titer) by the cell line, and / or to decrease cell death during production, and result in a variety of advantages. For example, increasing cell viability and product titer can result in (a) a decrease in the resultant cost of recombinant viral particles due to a decreased need for serum; (b) an increase in purity (due to less contaminating dead-cell products); (c) ease in use of anchorage dependent cell lines; and (d) an increase in cell density, as compared to other standard media, thereby resulting in a higher product concentration and/or higher specific activity.

In order to further the understanding of the invention, described in more detail below is: (A) Construction of Recombinant Viral Vector Producing Cell Lines, including methods for generating recombinant viral vector producing cell lines; and (B) Methods for Culturing Recombinant Viral Vector Producing Cell Lines, including methods for selecting appropriate media.

A. Recombinant Viral Vector Producing Cell Lines

1. Construction of retroviral vector producing cell lines

Within one aspect of the present invention, retroviral vectors are provided which are constructed to carry or express a selected gene(s) or sequence(s) of interest. Briefly, retroviral gene delivery vehicles of the present invention may be readily constructed from a wide variety of retroviruses, including for example, B, C, and D type retroviruses as well as spumaviruses and lentiviruses (see RNA Tumor Viruses, 2d ed., Cold Spring Harbor Laboratory, 1985). Such retroviruses may be readily obtained from depositories or collections such as the American Type Culture Collection ("ATCC"; Rockville, Maryland), or isolated from known sources using commonly available techniques.

Any of the above retroviruses may be readily utilized in order to assemble or construct retroviral gene delivery vehicles given the disclosure provided herein, and standard recombinant techniques (e.g., Sambrook et al, Molecular Cloning:

A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Kunkle,

PNAS S2:488, 1985). In addition, within certain embodiments of the invention, portions of the retroviral gene delivery vehicles may be derived from different retroviruses. For example, within one embodiment of the invention, retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous

Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.

Within one aspect of the present invention, retroviral vectors are provided comprising a 5' LTR, a tRNA binding site, a packaging signal, one or more heterologous sequences, an origin of second strand DNA synthesis and a 3' LTR, wherein the vector construct lacks gag/pol or env coding sequences.

Other retroviral vectors may likewise be utilized within the context of the present invention, including for example EP 0,415,731; WO 90/07936; WO

91/0285, WO 9403622; WO 9325698; WO 9325234; U.S. Patent No. 5,219,740; WO 9311230; WO 9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and

Hart, Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993;

Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al, J. Neurosurg. 79:129-

735, 1993 (U.S. Patent No. 4,777,127, GB 2,200,651, EP 0,345,242 and WO91/02805). Packaging cell lines suitable for use with the above described retroviral vectors may be readily prepared (see U.S. Serial No. 08/240,030, filed May 9, 1994; see also U.S. Serial No. 07/800,921, filed November 27, 1991), and utilized to create producer cell lines (also termed vector cell lines or "VCLs") for the production of recombinant vector particles.

Two particularly preferred packaging cell lines, HA-II and HA-LB, were developed based upon the expression of MLV gag/pol and amphotropic env sequences, in the HT-1080 (human fibrosarcoma) cell line. These packaging cell lines were developed using the principle of splitting the retroviral genome, removing the long term repeat (LTR) sequences and replacing them with cytomegalovirus (CMV) immediate early promoter to reduce the potential of generating replication competent retrovirus (RCR). HA-II and HA-LB differed in the number of overlapping sequences. Vector producing cell lines were generated by transduction of VSV-G pseudotyped provector encoding appropriate gene into the appropriate packaging cell line.

2. Recombinant Adeno-Associated Virus Vectors As noted above, a variety of rAAV vectors may be utilized to direct the expression of one or more desired sequence of interests. Briefly, the rAAV should be comprised of, in order, a 5' adeno-associated virus inverted terminal repeat, a transgene or gene of interest operatively linked to a sequence which regulates its expression in a target cell, and a 3' adeno-associated virus inverted terminal repeat. In addition, the rAAV vector may preferably have a polyadenylation sequence.

Generally, rAAV vectors should have one copy of the AAV ITR at each end of the transgene or gene of interest, in order to allow replication, packaging, and efficient integration into cell chromosomes. The ITR consists of nucleotides 1 to 145 at the 5' end of the AAV DNA genome, and nucleotides 4681 to 4536 (i.e., the same sequence) at the 3' end of the AAV DNA genome. Preferably, the rAVV vector may also include at least 10 nucleotides following the end of the ITR (i.e., a portion of the "D region").

Within preferred embodiments of the invention, the transgene sequence will be of about 2 to 5 kb in length (or alternatively, the transgene may additionally contain a "stuffer" or "filler" sequence to bring the total size of the nucleic acid sequence between the two ITRs to between 2 and 5 kb). Alternatively, the transgene may be composed of same heterologous sequence several times (e.g., two nucleic acid molecules which encode FGF-2 separated by a ribosome readthrough, or alternatively, by an Internal Ribosome Entry Site or "IRES"), or several different heterologous sequences (e.g., FGF-2 and FGF-5, separated by a ribosome readthrough or an IRES). Recombinant AW vectors of the present invention may be generated from a variety of adeno-associated viruses, including for example, serotypes 1 through

6. For example, ITRs from any AAV serotype are expected to have similar structures and functions with regard to replication, integration, excision and transcriptional mechanisms.

Within certain embodiments of the invention, expression of the transgene may be accomplished by a separate promoter (e.g., a viral promoter). Representative examples of suitable promoters in this regard include a CMV promoter, RSV promoter, SV40 promoter, or MoMLV promoter. Other promoters that may similarly be utilized within the context of the present invention include cell or tissue specific promoters (e.g., a rod, cone, or ganglia derived promoter), or inducible promoters. Representative examples of suitable inducible promoters include tetracycline-response promoters ("Tet", see, e.g., Gossen and Bujard, Proc. Nαtl. Acαd. Sci. USA. 59:5547-5551, 1992; Gossen et al., Science 268:1166-1169, 1995; Baron et al., Nucl. Acids Res. 25:2723-2729, 1997; Blau and Rossi, Proc. Nαtl. Acαd. Sci. USA. 96:191-199, 1999; Bohl et al., Blood 92:1512-1517, 1998; and Haberman et al., Gene Therapy 5.T 604-1611, 1998), the ecdysone system (see, e.g., No et al., Proc. Natl. Acad. Sci. USA. 93:3346-3351, 1996), and other regulated promoters or promoter systems (see, e.g., Rivera et al., Nat. Med. 2:1028-1032, 1996;). The rAVV vector may also contain additional sequences, for example from an adenovirus, which assist in effecting a desired function for the vector. Such sequences include, for example, those which assist in packaging the rAVV vector in adenovirus particles.

Packaging cell lines suitable for producing adeno-associated viral vectors may be readily prepared given readily available techniques (see, e.g., U.S. Patent No. 5,872,005).

3. Recombinant Alphavirus vectors

The present invention also provides a variety of Alphavirus vectors which may function as gene delivery vehicles. For example, the Sindbis virus is the prototype member of the alphavirus genus of the togavirus family. The unsegmented genomic RNA (49S RNA) of Sindbis virus is approximately 11,703 nucleotides in length, contains a 5' cap and a 3' poly-adenylated tail, and displays positive polarity. Infectious enveloped Sindbis virus is produced by assembly of the viral nucleocapsid proteins onto the viral genomic RNA in the cytoplasm and budding through the cell membrane embedded with viral encoded glycoproteins. Entry of virus into cells is by endocytosis through clatharin coated pits, fusion of the viral membrane with the endosome, release of the nucleocapsid, and uncoating of the viral genome. During viral replication the genomic 49S RNA serves as template for synthesis of the complementary negative strand. This negative strand in turn serves as template for genomic RNA and an internally initiated 26S subgenomic RNA. The Sindbis viral nonstructural proteins are translated from the genomic RNA while structural proteins are translated from the subgenomic 26S RNA. All viral genes are expressed as a polyprotein and processed into individual proteins by post translational proteolytic cleavage. The packaging sequence resides within the nonstructural coding region, therefore only the genomic 49S RNA is packaged into virions.

Several different alphavirus vector systems and packaging cell lines can be readily generated given the disclosure provided herein. Representative examples of such systems include those described within U.S. Patent Nos. 5,843,723, and 5,789,245, and PCT Publication No. WO 95/07994.

4. Other viral gene delivery vectors

In addition to retroviral vectors and alphavirus vectors, numerous other viral vectors systems may also be utilized as a gene delivery vehicle. Representative examples of such gene delivery vehicles include viruses such as pox viruses, such as canary pox virus or vaccinia virus (Fisher-Hoch et al., PNAS 5(5:317-321, 1989; Flexner et al, Ann. N Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 5:17-21, 1990; U.S. Patent Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89/01973); SV40 (Mulligan et al., Nature 277:108-114, 1979); influenza virus (Luytjes et al., Cell 59:1107-1113, 1989; McMicheal et al., N. Eng. J. Med. 309:13-17, 1983; and Yap et al., Nature 273:238-239, 1978); herpes (Kit, Adv. Exp. Med. Biol. 215:219-236, 1989; U.S. Patent No. 5,288,641); HIV (Poznansky, J. Virol. f55:532-536, 1991); measles (EP 0 440,219); Semliki Forest Virus, and coronavirus, as well as other viral systems (e.g., EP 0,440,219; WO 92/06693; U.S. Patent No. 5,166,057). In addition, viral carriers may be homologous, non-pathogenic (defective), replication competent virus (e.g., Overbaugh et al., Science 239:906-910,1988), and nevertheless induce cellular immune responses, including CTL.

B. Methods for Culturing Recombinant Viral Vector Producing Cell Lines

In order to increase the production of viral vector (or viral titer) by the cell line, and / or to decrease cell death during production, the viral vector producing cell line is cultured or incubated in a basal media to which is added (a) greater than 1.5 g/L amino acids (or a specific amount of selected amino acids) and (b) between 0.5% and 4% serum.

Briefly, as noted above basal media refers to a minimal media which, when supplemented with serum, is sufficient to support the growth and/or proliferation of a desired cell line. Basal media typically contains a mixture of sugar (e.g., glucose), vitamins (e.g., B12, etc.), and one or more salts or buffers. Representative examples of such media include BME, MEM, DMEM, DMEM-F-12, IMDM, McCoy's 5 A, Media 199, Ham's F-10, Ham's F-12, and RPMI, and may be obtained from a variety of commercial sources, including for example, Hyclone, Inc. (Logan, UT), Irvine Scientific (Irvine, CA), Biowhittaker (Walkersville, MD), Gibco-LTI (Gaithersburg, MD), and Sigma Chemical Co. (St. Louis, MO).

To such basal media is added a selected quantity of preferably monomeric amino acids. Within certain aspects of the invention, particular amino acids may be added to the basal media a higher proportion than other amino acids, and indeed, only certain amino acids may be specifically added to the basal media. In order to assess which amino acids are preferred, the desired viral-vector producing cell line may be analyzed based upon its amino acid utilization, and a quantity of amino acids added to the basal media to meet this need. As a representative example, producer cell lines (HA-LB/CF8 or HA- II/CF8) are grown in DMEM +10 % FBS at 37°C in 10% CO2 incubators until confluence in a T-flask and culture media is replaced by fresh media (DMEM +10% FBS) on day 6. Media is replaced on a daily basis by fresh media, and spent media is stored at -80°C for further amino acid analysis. Cells are also harvested from these flasks on day 6, 7 and 8 using trypsin-EDTA for a cell counting using trypan blue exclusion. As a baseline, DMEM+10% FBS (unused) sample is also stored for amino acid analysis.

Amino acid analysis may be carried out by a variety of commercial organizations, including for example, the Scientific Research Consortium, Inc. (St. Paul, MN). Briefly, amino acid analysis can be performed on Beckman Instruments, Inc., Models 6300 and 7300 dedicated amino acid analyzers, which incorporate a 10 cm cation-ion exchange column, four sequential lithium-based eluents, and lithium hydroxide for column regeneration. Absorbence is measured at 440 and 570 nm following post-column color development by Ninhydrin reagent at 131°C. Data acquisition and management is accomplished with a computer running Beckman System Gold 8.10 chromatography software. Beckman reference solutions fulfills standardization requirements. (S)-2-Aminoethyl-l-cysteine (S2AEC) or Glucosamine acid is added to the sample as the preferred internal standard. Based on the amino acid analysis, consumption rates for each amino acid is a calculated by subtracting the amount present in used media from the amount present in unused media and divided by the time spent between refeeds. The specific consumption rate is calculated by dividing amino acid consumption rate by the viable cell number. A positive value indicates a net consumption and a negative value indicates a net accumulation of the specific amino acid. Based on intended media usage, amino acid concentration in media is calculated by multiplying desired cell concentration and specific amino acid consumption rates. All the amino acids that are produced by the culture, can be deleted from the media composition.

Utilizing this method, amino acid concentrations based on nutrient selection method combined with a typical basal media compositions of vitamins, buffer, metal salts and other salts is used to design a high density culture media. As an example, a comparison of MS-162 and MS-174 is provided below, versus the amino acid profile of the basal media DMEM ( mg/L). The resultant media MS-162 and MS- 174 had approximately 2.9 or 3 times increase in total amino acid content as compared to DMEM, and did not contain certain amino acids such as L-Alanine or L-Glutamate.

MS-162 MS-174 DMEM

Amino Acid

L-Alanine 0 0 0

L-Arginine, free base 302 400 84

L-Asparagine 39 100 0

L-Aspartic acid 29 88 0

L-Cystine 260 141 62.6

L-Glycine 40 0 30

L-Glutamate 0 0 0

L-Histidine, free base 85 49 42

L-Isoleucine 253 283 105

L-Leucine 353 429 105

L-Lysine.HCl 218 203 146

L-Methionine 143 137 30

L-Phenylalanine 116 112 66

L-Proline 39 0 0

L-Serine 461 458 42

L-Threonine 131 150 95

L-Tryptophan 80 59 16

L-Tyrosine 134 95 103.79

L-Valine 185 217 94

Sum of AA 2867 2921 1021

In addition, certain metal salts may also be added to the culture media in order to enhance vector titer production and decrease cell death. Representative examples of such metal salts include: CoCl₂.6H₂O, CuCl₂.2H₂O, CuSO₄, MnCl₂.4H₂O, (NH₄)₆ Mo₇O₂.4H2O, and Na₂SeO₃.

Metal Salts (mε/LΪ MS-162 MS-174 DMEM

CoC12.6H2O 237.95 0.003 0.03 0

CuC12.2H2O 135.03 0.003 0.03 0

CuSO4 0 0 0

MnC12.4H2O 197.9 0.003 0.03 0

(NH4)6 Mo7 O24.4H2O 1235.95 0.003 0.03 0

Na2SeO3 172.95 0.01 0.01 0

Other metals may also be included such as, e.g., Iron (Fe), Calcium (Ca), Magnesium (Mg), and Zinc (Zn). Exemplary metal salts are presented in the following table:

Metal Salts (mε/L MS-162 MS-174 DMEM

FeC13 162.22 0 0

FeSO4 0 0

CaC12 110.99 265 200 265

CaNO3 0 0

MgC12 95.23 155 155

MgSO4 0 0 97.6

ZnC12 136.29 3 3

ZnSO4 0 0

FeNO3.9H20 0.1 0.1 0.1

MS-162 media was designed to support 1.7 e7 cells/ml of HA-II/CF8 vector producing cell lines and MS-174 was designed to support 1.7 e7 cells/ml of - LB/CF8 VPCL.

MS-174 media was also designed based on rational media development principles. Amino acid consumption rates for HA-LB/CF8 cell lines were calculated and a media was designed to support HA-LB/CF8 cell lines at 1.7 x 10⁷ cells/ml. This cell concentration is 10 times higher than the cell concentration that DMEM can support without exhausting any of its nutrients. In addition to higher amino acids, this media contains metal salts to support growth at low serum, and MgCl₂ to enhance retroviral vector stability.

Utilizing such media, viral vector producing cell lines can be cultured under a variety of conditions. For example, HA-LB/CF8 cells were inoculated in CellCubes with 21,500 cm² - 85,000 cm² of surface area. Inoculation density was maintained between 2 -6 x 10⁴ cells/cm² to provide growth in an exponential phase. Appropriate basal media supplemented with 11% FBS and 2-4 mM L-Glutamine was used for inoculation. Perfusion was initiated on day 1 to remove waste metabolites, collect viral product and supplement cell culture with fresh nutrients. Perfusion rates were gradually increased to 5-7 volume exchanges per day after 7-8 days. Perfusion media was usually changed to appropriate media with low serum on day 4. Levels of dissolved oxygen and pH of the culture were controlled by using appropriate mixture of air, oxygen and CO . DO was usually maintained at 50-80% of air saturation, and pH was controlled at physiological levels. Gradients of DO and pH and their oscillations were minimized by increasing recirculation rate in the CellCube. Samples were collected daily for analysis of glucose, lactate, ammonia and lactate dehyrodogenase using Kodak Ektachem machine. Samples were collected daily for titer analysis to measure retroviral activity.

The titer of vector producing cell lines can be readily determined by a variety of methods. For example, a titer assay can be performed utilizing the transduction of expression (TOE) principle. Briefly, HT-1080 cells were plated on day 1 in 6 well plates and transduced with retroviral vector on day 2. Transduction was conducted using several dilutions of retroviral vector samples. A standard with known amounts of retroviral vector titers was used to generate a standard curve. Factor VIII activity in the supernatant fluid was measured on day 5 using a Factor VIII assay kit from Chromogenix using the manufacturer's supplied instructions. Factor VIII levels measured in the supernatant is directly correlated to retroviral vector titers using a standard curve.

The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES

EXAMPLE 1

INCREASE IN VECTOR TITER AND DECREASE IN CELL DEATH BY USING MS-162 MEDIA

Production of retroviral vector was conducted in T-flasks. In particular, HA-II or HA-LB based vector producing cell lines were inoculated in T-75 flasks at 2-4 x 10⁴ cells/cm² in 15 ml of DMEM supplemented with 10% FBS and 4 mM L- Glutamine. On day 4 or 5 when cells became confluent, media was replaced with appropriate media supplemented with various serum amounts. Media was replaced on a daily basis by fresh media, and spent media was analyzed for retroviral vector concentration.

Cells were grown in 10% DMEM until day 6 and media was switched to MS-162 +10% FBS on day 6. For a controlled comparison, DMEM +10% FBS was also used. Figure 1 shows retroviral vector titers on day 7 and 8 in these two different conditions. Titers in MS-162 + 10% FBS were 35-65% higher than DMEM +10% FBS media. Cell death was also analyzed by comparing LDH levels in the supernatant. LDH is one of the enzymes that are released when cells lyse, and it has been traditionally used in the bioprocess development environment to correlate to the cell death. Figure 2 shows significantly reduced LDH levels for MS-162 media compared to DMEM. This indicates that MS-162 media can prevent apoptosis of cells to provide a sustained level of viable cells for a prolonged time period.

EXAMPLE 2

INCREASE IN VECTOR TITER IN A CONTINUOUS PERFUSION CULTURE BY USING MS-174 MEDIA COMBINED WITH REDUCED SERUM Production of retroviral vector using HA-LB/CF8 VPCL was carried out in 21,250 cm² CellCubes (Pilot Scales) and average of a number of runs are compared for vector titers. DMEM media with 11% serum usually results in a plateau by day 7-8 followed by a decline in the vector titers (Figure 3). Vector titers for DMEM supplemented with 11% or 2% serum is around 0.5-1.5 x 10 eq.cfu/ml for the entire production phase (day 4-13). MS-174 supplemented with 2% or 11% serum results in vector levels in the range of 1-3 x 10⁸ eq.cfu/ml. MS-174 +11% plateaued on day 10 and then dropped similar to DMEM +11% FBS levels. MS-174 +2% FBS, in contrast, increased vector production further during last three days. This demonstrates that MS- 174 + 2% FBS has superior titer capabilities compared to DMEM +2%, DMEM +11% and MS-174 +11% FBS.

EXAMPLE 3

INCREASE IN TOTAL VECTOR PRODUCTION IN A CONTINUOUS PERFUSION CULTURE BY USING MS-174 MEDIA COMBINED WITH REDUCED SERUM

Production of retroviral vectors using HA-LB/CF8 VPCL was carried out in 21,250 cm CellCubes and average of a number of runs are compared for total vector production per day. This measurement is a true indication of the yields and performance of various culture systems. Figure 4 shows total vector production per day for various culture systems. The total vector production is also set forth in Table 1 below:

TABLE 1

Total Vector

Produced (Area Total Vector in

Under the Curve) Harvest

DMEM +2% FBS 6.25E+12 4.45E+12 DMEM + 11% FBS 1.19E+13 5.26E+12

MS-174 +2% FBS 1.66E+13 1.30E+13 MS-174 +11% FBS 1.33E+13 1.10E+13

Briefly, DMEM media with 11% serum showed a peak on day 9 at 2.0 x

10¹² eq.cfu/day followed by a three fold decline to level close to 7 x 10¹¹ eq. cfu/day by day 12. DMEM supplemented with 2% FBS resulted in reduced levels of vector production in the range 5-8 x 10¹¹ eq.cfu day. MS-174 + 2% or 11% FBS resulted in a 2-3 fold higher total vector production with levels in the range of 1.0 - 2.0 x 10 eq.cfu day during days 5-10 of culture. This is a significant improvement over DMEM +2% orl l% FBS cultures. During last three days, MS-174 +11% shows a 3 fold drop in vector production levels, while MS- 174+2% shows further increases in vector production rates. This was an unexpected result since based on DMEM results we would have expected MS-174+11% to perform superior to MS-174+2% FBS.

EXAMPLE 4

COMBINING MS-174 MEDIA WITH REDUCED SERUM CONDITIONS CAN REDUCE APOPTOSIS

Growth of HA-LB/CF8 cells were carried out in 21,250 cm² CellCubes and cell death is compared by plating LDH produced per day in these cultures. When cell lyses, it releases LDH in the surrounding media, which is directly proportional to the number of dead cells. Higher amount of cell death is very undesirable as it increases impurity levels and decreases duration of manufacturing runs. Figure 5 shows LDH/day, which is an indication of the cell death/day during CellCube cultures. All data points represent averages of multiple runs. Standard media conditions (DMEM + 11% FBS) indicates a 4-6 fold higher cell death compared to MS-174 + 2% FBS. This graph also shows MS-174 +2% FBS has 2-3 fold lower cell death compared to DMEM +2% FBS cultures. More importantly, cell death levels during entire MS-174 +2% FBS culture is lower than day 9 of DMEM culture supplemented with either 2% or 11% FBS, indicating that cell death has been delayed significantly. This demonstrates that using this method it is possible to significantly increase duration of vector production phase.

This graph also shows limited effect of rational media design on delaying apoptosis. A comparison of DMEM and MS-174 cultures, both supplemented with 11% FBS, indicate a 3-day delay in cell apoptosis in MS-174 media culture. However, the number of cells dying during last two days of culture is similar in both cultures. Reducing serum levels in these cultures provides a much superior culture conditions showing a delay of 7 days using MS-174+2% FBS culture. EXAMPLE 5 HIGHER TITER AT MANUFACTURING SCALE USING MS-174 MEDIA COMBINED WITH REDUCED SERUM Growth of HA-LB/CF8 cells was carried out in 85,000 cm² CellCubes and two manufacturing scale runs are compared with respect to vector titers. Results are shown in Figures 6 and 7. Briefly, these graphs show 3-10 fold higher titers in MS- 174 +2% FBS media compared to DMEM + 2% FBS media. Titers in DMEM are in the range of 3-6 x 10⁷ eq.cfu/ml, while MS-174 shows titers in the range of 1.5-4.0 x 10⁷ eq.cfu/ml. This provides a significant improvement in vector quality and reduces manufacturing costs considerably.

EXAMPLE 6 HIGHER TOTAL VECTOR PRODUCTION RATE AT MANUFACTURING SCALE USING MS-174 MEDIA COMBINED WITH REDUCED SERUM Growth of HA-LB/CF8 cells was carried out in 85,000 cm2 CellCubes and two manufacturing scale runs are compared for vector production rate. This graph also shows a 2-3 times higher vector production levels in MS-174 media initially, and 5-10 fold higher levels after day 10. This represents a very significant improvement in product yields while considerably reducing cost of manufacturing.

EXAMPLE 7 REDUCTION IN CELL APOPTOSIS IN MANUFACTURING SCALE CULTURES

USING MS-174 MEDIA WITH REDUCED SERUM Growth of HA-LB/CF8 cells was carried out in 85,000 cm² CellCubes and two manufacturing scale runs are compared for cell death. Results are shown in Figure 8, briefly, this graph shows similar cell death levels during 11% FBS cultures (day 1-4), followed by a significant delay in apoptosis for MS-174 media supplemented with 2% FBS. The levels of cell death in MS-174 media during entire production phase (day 5-13) are lower than DMEM. This demonstrates that it is possible to delay apoptosis by changing cell environmental conditions at bioproduction scales. EXAMPLE 8 INCREASE IN VECTOR PRODUCTION FOR HA-LB/EPO VECTOR IN A BATCH CULTURE

HA-LB/EPO producer cell line for retroviral vector was grown as indicated in the Materials and Methods section. Figure 9 shows average titer values for multiple DMEM and MS-162 runs in T-flasks, both supplemented with 10% FBS. MS- 162 media with 10% FBS results in a 60% increase in viral vector titers in the culture supernatant.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

1. A method for increasing the production of a recombinant viral vector in a cell culture, comprising culturing a recombinant viral vector producing cell line in a basal media which comprises (a) greater than 1.5 g/L amino acids and (b) between 0.5% and 4% serum, such that said recombinant viral vectors are produced.

2. A method for decreasing cell death during the production of a recombinant viral vector in a cell culture, comprising culturing a recombinant viral vector producing cell line in a basal media which comprises (a) greater than 1.5 g/L amino acids and (b) between 0.5% and 4% serum, such that said recombinant viral vectors are produced.

3. The method according to claims 1 or 2 wherein said basal media is RPMI or DMEM.

4. The method according to claims 1 or 2 wherein said basal media contains greater than 1.2 g/L of amino acids.

5. The method according to claims 1 or 2 wherein said basal media contains greater than 2 g/L of amino acids.

6. The method according to claims 1 or 2 wherein said basal media contains between about 2% and 3% serum.

7. The method according to claims 1 or 2 wherein said serum is fetal bovine serum.

8. The method according to claims 1 or 2 wherein said viral vector is a recombinant retroviral vector.

9. The method according to claims 1 or 2 wherein said viral vector is a recombinant adenoviral vector.

10. The method according to claims 1 or 2 wherein said viral vector is a recombinant alphavirus vector.

11. The method according to claims 1 or 2 wherein said viral vector is a recombinant adeno-associated virus vector.

12. The method according to claims 1 or 2 wherein said recombinant viral vector producing cell line is an anchorage-dependent cell line.

13. The method according to claims 1 or 2 wherein said recombinant viral vector producing cell line is cultured under conditions of continuous perfusion.

14. The method according to claims 1 or 2 further comprising a first metal salt comprising a metal selected from the group consisting of Cobalt (Co), Copper (Cu), Manganese (Mn), Molybdenum (Mo), and Selenium (Se) and a second metal salt comprising a metal selected from the group consisting of Iron (Fe), Calcium (Ca), Magnesium (Mg), and Zinc (Zn).

15. The method of claim 14 wherein said first metal salt is selected from the group consisting of CoCl₂.6H₂O, CuCl₂.2H₂O, CuSO₄, MnCl₂.4H₂O, (NH₄)₆ Mo₇O₂.4H₂O, and Na₂SeO₃.

16. The method according to claim 14 wherein said second metal salt is selected from the group consisting of FeCl₃, FeSO₄, CaCl , CaNO₃, MgCl , MgSO₄, ZnCl₂, ZnSO₄, FeNO₃.9H₂O.

17. A composition for increasing the production of a recombinant viral vector and/or decreasing cell death in a cell culture, said composition comprising:

(a) a basal media;

(b) greater than 1.5 g/L amino acids;

(c) between 0.5% and 4% serum;

(d) a first metal salt wherein said first metal salt comprises a metal selected from the group consisting of Cobalt (Co), Copper (Cu), Manganese (Mn), Molybdenum (Mo), and Selenium (Se); and

(e) a second metal salt wherein said second metal salt comprises a metal selected from the group consisting of Iron (Fe), Calcium (Ca), Magnesium (Mg), and Zinc (Zn).

18. The composition of claim 17 wherein said basal media is selected from the group consisting of BME, MEM, DMEM, DMEM-F-12, IMDM, McCoy's

5 A, Media 199, Ham's F-10, Ham's F-12, MS-162, MS-174, and RPMI.

19. The composition of claim 17 wherein said basal media is selected from the group consisting of DMEM, MS-162, and MS-174.

20. The composition of claim 17 comprising greater than 1.7 g/L amino acids.

21. The composition of claim 17 comprising greater than 2.0 g/L amino acids.

22. The composition of claim 17 comprising greater than 2.5 g/L amino acids.

23. The composition of claim 17 wherein one of said amino acids is L-Cystine and wherein said L-Cystine is present at a concentration of between 75 and 300 mg/L.

24. The composition of claim 17 wherein one of said amino acids is L-Cystine and wherein said L-Cystine is present at a concentration of between 100 and 200 mg/L.

25. The composition of claim 17 wherein one of said amino acids is L-Serine and wherein said L-Serine is present at a concentration of between 75 and 1000 mg/L.

26. The composition of claim 17 wherein one of said amino acids is L-Serine and wherein said L-Serine is present at a concentration of between 200 and 700 mg/L.

27. The composition of claim 17 wherein one of said amino acids is L-Methionine wherein said L-Methionine is present at a concentration of between 75 and 300 mg/L.

28. The composition of claim 17 wherein one of said amino acids is L-Methionine wherein said L-Methionine is present at a concentration of between 100 and 200 mg/L.

29. The composition of claim 17 comprising between 1% and 3% serum.

30. The composition of claim 17 comprising between 2% and 3% serum.

31. The composition of claim 17 wherein said serum is fetal calf serum.

32. The composition of claim 17 wherein said serum is human serum.

33. The composition of claim 17 wherein said first metal salt is selected from the group consisting of CoCl₂.6H₂O, CuCl₂.2H₂O, CuSO₄, MnCl₂.4H₂O, (NH₄)₆ Mo₇O₂.4H2O, and Na₂SeO₃.

34. The composition of claim 17 wherein said first metal salt is present at a concentration of between 0.003 and l.Omg/L.

35. The composition of claim 17 wherein said first metal salt is present at a concentration of between 0.01 and 0.1 mg/L.

36. The composition of claim 17 wherein said second metal salt is selected from the group consisting of FeCl₃, FeSO , CaCl₂, CaNO₃, MgCl₂, MgSO₄, ZnCl₂, ZnSO₄, FeNO₃.9H₂O.

37. The composition of claim 36 wherein said second metal salt is MgCl₂ and wherein said MgCl₂ is present at a concentration of between 50 and 500 mg/L.

38. The composition of claim 36 wherein said second metal salt is MgCl₂ and wherein said MgCl is present at a concentration of between 125 and 175 mg/L.

39. The composition of claim 36 wherein said metal is ZnCl₂ and wherein said ZnCl₂ is present at a concentration of between 1 and 5 mg/L.

40. The composition of claim 36 wherein said metal is ZnCl and wherein said ZnCl is present at a concentration of between 2 and 4 mg/L.