[go: up one dir, main page]

WO2005087915A2 - Utilisation d'ethanolamine pour renforcer la croissance cellulaire dans des systemes membranaires - Google Patents

Utilisation d'ethanolamine pour renforcer la croissance cellulaire dans des systemes membranaires Download PDF

Info

Publication number
WO2005087915A2
WO2005087915A2 PCT/US2005/007926 US2005007926W WO2005087915A2 WO 2005087915 A2 WO2005087915 A2 WO 2005087915A2 US 2005007926 W US2005007926 W US 2005007926W WO 2005087915 A2 WO2005087915 A2 WO 2005087915A2
Authority
WO
WIPO (PCT)
Prior art keywords
serum
cell side
ethanolamine
cell
medium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2005/007926
Other languages
English (en)
Other versions
WO2005087915A3 (fr
Inventor
Michael Gramer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biovest International Inc
Original Assignee
Biovest International Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biovest International Inc filed Critical Biovest International Inc
Publication of WO2005087915A2 publication Critical patent/WO2005087915A2/fr
Publication of WO2005087915A3 publication Critical patent/WO2005087915A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/46—Amines, e.g. putrescine

Definitions

  • Culturing mammalian cells is an essential technique for research into cellular processes, production of recombinant therapeutic proteins, and generation of expanded cells for transplantation purposes.
  • Cell culture studies have led to the determination of numerous metabolic processes and the identification of growth factors, hormones and their receptors (Bio Techniques, 5:534-542, 1987).
  • membrane systems such as hollow fiber systems provide a powerful tool for culturing cells for the production of proteins, and, in particular, culturing hybridomas for the production of antibodies.
  • Membrane systems such as hollow fiber systems provide improved cell retention and high density cell growth.
  • Conventional cell culture media comprise basal nutrient media supplemented with serum from various sources, most often fetal bovine serum, horse serum or human serum.
  • serum is undesirable for several reasons. Growth media containing serum may vary in composition, hormone content, and contaminants, thereby introducing extraneous factors and/or infections agents into the culture system (Bio Technology, 11:49-53, 1993; Pharm. Technol, 48:56, 1987).
  • serum is expensive and impractical for large-scale production of therapeutics. Further, variance between serum lots and laboratory protocols is also a problem.
  • Serum-free media provide many important advantages over serum-containing media, including lot-to-lot consistency, biological uniformity, and freedom from adventitious agents.
  • Serum-free media are generally cost effective and low in protein content. All of these factors contribute to a more controlled examination of cellular, molecular and metabolic processes (ATCC Connection, 16(2):5-6, 1996). Serum-free technology is not entirely problem free. Many serum-free media are highly specific to a particular cell type, are for short-term culturing only, or contain components extracted from serum. Very few available products can support primary cell cultures. Ethanolamine has been reported to aid in the growth of hybridomas in serum-free medium in T-flasks (Murakami et al. (1982) PNAS 79:1158-1162). In addition, the use of ethanolamine in serum free medium has been reported (Glassy et al. (1988) Biotechnol.
  • ethanolamine at 20 ⁇ M was added to the basal medium on the non-cell side, while hybridoma cells were quickly weaned off serum on the cell side (Klerx et al. (1988) J Immunol. Meth. Il l : 179-188).
  • Klerx et al. (1988) J Immunol. Meth. Il l : 179-188).
  • cells were maintained at 10% serum concentration of total media components for 7 days and then subsequently weaned to 0% of total media components over the course of the ensuing 8 days.
  • Klerx et al cells were weaned from 10% to 2% serum concentration of total media components over the course of approximately two weeks.
  • DMEM Dulbecco's Modified Eagle's Medium
  • BME Basal Media Eagle
  • MEM Minimum Essential Eagle's Medium
  • Ham's F12 Ham's F12
  • RPMI Iscove's Modified Dulbecco's Medium (1MDM)
  • McCoy's McCoy's, or Waymouth's
  • the present invention provides methods for enhancing cell growth in a membrane system having a cell side and a non-cell side, by culturing cells in the presence of serum at an initial concentration on the cell side and in the absence of serum on the non-cell side, wherein the non-cell side has a medium containing ethanolamine, wherein the serum concentration on the cell side is maintained at greater than 20% (e.g., at least about 25-50%, 50-75% or 75-100%) of the initial concentration, and wherein the cell growth is enhanced compared to growth in the absence of ethanolamine.
  • suitable membrane systems include hollow fiber systems, such as hollow fiber bioreactors.
  • the present invention provides methods for culturing cells in a membrane system having a cell side and a non-cell side, by culturing cells in the presence of serum at an initial concentration on the cell side and in the absence of serum on the non-cell side, wherein the non-cell side comprises a medium containing ethanolamine, and wherein the serum concentration on the cell side is maintained at greater than 20% (e.g., at least about 25-50%, 50-75% or 75-100%) of the initial concentration.
  • the present invention provides methods for enhancing cell viability in a membrane system having a cell side and a non-cell side, by culturing cells in the presence of serum at an initial concentration on the cell side and in the absence of serum on the non-cell side, wherein the non-cell side has a medium containing ethanolamine, wherein the serum concentration on the cell side is maintained at greater than 20% (e.g., at least about 25-50%, 50-75% or 75-100%) of the initial concentration and wherein cell viability is enhanced when cells are cultured in the presence of ethanolamine compared to when cells are cultured in the absence of ethanolamine.
  • the present invention provides methods for enhancing antibody production in a membrane system having a cell side and a non-cell side, by culturing antibody producing cells in the presence of serum on the cell side and in the absence of serum on the non-cell side, wherein the non-cell side comprises a medium containing ethanolamine, and wherein antibody production is enhanced when cells are cultured in the presence of ethanolamine compared to antibody production when cells are cultured in the absence of ethanolamine.
  • the non-cell side medium contains less than about 100 mg/L ethanolamine. In another embodiment, the medium contains less than about 10 mg/L ethanolamine.
  • the non-cell side medium containing ethanolamine also contains basal medium.
  • the basal medium may include Ham's F-12.
  • the basal medium may include Ham's F-12 and a second basal medium selected from the group consisting of Dulbecco's modified Eagle's medium (DMEM), Essential modified Eagle's medium (EMEM), RPMI-1640, and Iscove's modified Dulbecco's Medium (IMDM).
  • DMEM Dulbecco's modified Eagle's medium
  • EMEM Essential modified Eagle's medium
  • RPMI-1640 RPMI-1640
  • Iscove's modified Dulbecco's Medium IMDM
  • the medium containing ethanolamine contains at least one supplement.
  • the supplement may be, but is not limited to, an amino acid, glucose, glutamine, methionine, cystine, cysteine, tryptophan, sodium bicarbonate and/or combinations thereof.
  • the medium contains glucose (e.g., less than about 10 g/L), glutamine (e.g., less than about 5 g/L), methionine (e.g., less than about 100 mg/L), cystine (e.g., less than about 100 mg/L), tryptophan (e.g., less than about 100 mg/L) and sodium bicarbonate (e.g., less than about 10 g/L).
  • the medium also contains fats or insulin.
  • the cells may be cultured in the presence of serum on both the cell side and the non-cell side (e.g., for less than 24 hours), prior to culturing the cells in the presence of serum on the cell side and in the absence of serum on the non-cell side.
  • the serum concentration is maintained between about 2-15% of the total media components on the cell side, more preferably, between about 8-12% of the total media components.
  • the serum concentration is maintained at greater than 2% of the total media components for at least 7 days (e.g., at least 7-14 days, 14-28 days or more, or for the entire duration of the culturing step).
  • the serum concentration is maintained at about 50%, at least about 75%, at least about 90%, or at about 100% (i.e., without weaning) of the initial serum concentration.
  • the serum concentration on the cell side is maintained at at least about 25% of the initial serum concentration for at least about 14- 28 days or more, or for the entire duration of the culturing step.
  • the cultured cells may be hybridoma or heterohybridoma cells (e.g., human, sheep or rodent hybridomas or heterohybridomas, such as rat, murine, rabbit, and hamster hybridomas or heterohybridomas).
  • the cells may be Chinese hamster ovary (CHO), NS/O, SP2/O or murine hybridomas or heterohybridomas .
  • Methods of the present invention serve to enhance cell viability and growth in the absence of serum on the non-cell side of a membrane system.
  • the cultured cells e.g., hybridomas
  • the methods of the invention serve to enhance cell growth and, thus, antibody production when cells are cultured in the presence of ethanolamine compared to when cells are cultured in the absence of ethanolamine.
  • antibody production is enhanced by at least about 20% or by at least about 50%.
  • Figure 1 depicts the effect of bioreactor extracapillary space (EC) medium supplements on cell growth in a micro bioreactor. Rho 1D4 cells were inoculated in
  • fetal bovine serum-supplemented medium at 5xl0 ⁇ /ml in the bioreactor intracapillary space (IC) (0.2 ml culture space), and were harvested three days later.
  • the EC reservoir (non cell side) contained 16 ml basal medium (Basal), or basal medium supplemented with 10% fetal bovine serum (FBS), 10% newborn calf serum (NCS), 10% adult bovine serum (ABS) or a serum-free medium supplement SITE+3 (SITE+3). Data shown are the averages and standard deviations of duplicate bioreactors.
  • Figure 2 depicts the effect of removing components of the serum-free medium supplement. Rho 1D4 cells were inoculated in 10% FBS-supplemented medium at
  • the EC (non-cell side) reservoir contained 16 ml basal medium (Basal), the basal medium plus the complete SITE+3 supplement (All), or the basal medium with the
  • FIG. 1D4 depicts the effect of ethanolamine, bovine serum albumin (BSA), and fatty acid addition to medium.
  • Rho 1D4 cells were inoculated in 10% FBS- supplemented medium at 5xl ⁇ 6/ml in the bioreactor IC (0.2 ml culture space), and were harvested three days later.
  • the EC reservoir (non-cell side medium) contained 16 ml basal medium (Basal), or basal medium supplemented with BSA (B), the BSA/fatty acid complex (BF), ethanolamine (E), BSA with ethanolamine (BE), or the BSA/fatty acid complex with ethanolamine (BFE). Data shown are the averages and standard deviations of duplicate bioreactors. Figure 4 depicts the effect of various supplements in a 5-ml micro-bioreactor.
  • Rho 1D4 cells were inoculated in 10% FBS-supplemented medium at 5xl0 ⁇ /ml in the bioreactor IC (0.2 ml culture space), and were harvested three days later.
  • the EC reservoir (non-cell side medium) contained 5 ml basal medium (Basal), or basal medium supplemented 10% FBS (FBS), ethanolamine (E), or the BSA/fatty acid complex with ethanolamine (BFE). Data shown are the averages and standard deviations of duplicate bioreactors.
  • Figure 5 depicts the T-flask growth curve of rho 1D4 cells. Cells were grown in 10% FBS-supplemented medium with (closed circles) or without (open circles) 4 mg/L ethanolamine. Data shown are the averages and standard deviations of triplicate T- flasks.
  • Figure 6 depicts the effect of different media on cell growth in the ResCu
  • Rho 1D4 cells were inoculated in the EC space of three bioreactors at 5xl ⁇ 6/ml with 10% FBS.
  • the IC medium reservoir contained either basal medium, basal medium supplemented with 10% FBS, or basal medium supplemented with 4 mg/L ethanolamine.
  • GUR is short for glucose uptake rate.
  • Figure 7 depicts the effect of different media on cell growth in the AcuSyst- Maximizer. Rho 1D4 cells were inoculated in the EC space of two bioreactors at
  • FIG. 8 depicts the growth of three other murine hybridomas in the micro- bioreactor.
  • Cells were inoculated in 10% FBS-supplemented medium at 5xl0 ⁇ /ml in the bioreactor IC (0.2 ml culture space), and were harvested three days later.
  • the EC reservoir contained 5 ml basal medium (Basal), basal medium supplemented with 10% FBS (FBS), or basal medium supplemented with 4 mg/L ethanolamine (Basal+E). Data shown are the averages and standard deviations of duplicate bioreactors.
  • the present invention is based, in part, on the discovery that cell growth and cell viability in a membrane system, such as a hollow fiber system, can be significantly enhanced using a cell culture medium containing ethanolamine, even when serum is present on the cell side, hi view of the well known fact that serum alone can fully support cell growth, the discovery that adding ethanolamine significantly increases cell growth and viability was unexpected. h particular, by way of the present invention, it was found that when including ethanolamine in a serum free medium on the non-cell side of a membrane system, with serum on the cell side maintained at greater than 20% of its initial concentration, unexpected improvements in cell growth and viability are achieved.
  • the methods of the present invention provide the advantage of minimizing the use of expensive serum components on both sides of the membrane and reducing the risks, such as exposure to contaminants, extraneous factors and infectious agents, that come with excessive use of serum.
  • Minimizing or, alternatively, eliminating entirely the use of serum on the non-cell side of a membrane system in accordance with the methods disclosed herein is particularly advantageous in that media components on the non-cell side of membrane systems are known to be consumed at a far greater rate than components on the cell side.
  • Ethanolamine is an important element for optimal cell growth. Without wishing to be bound to any particular theory, cell growth requires the synthesis of various cellular components, including, in particular, phospholidids.
  • the phosphate group of a phospholipid is capped with either ethanolamine, choline, serine, glycerol and inositol. Choline, serine and inositol are generally present in standard basal media. In addition, cells readily synthesize sufficient glycerol to support cell growth. However, while cells can synthesize ethanolamine, cells generally cannot synthesize sufficient ethanolamine to support optimal cell growth. Accordingly, the addition of ethanolamine to standard basal media is beneficial for cell growth.
  • cell side refers to the region or side of the membrane system on which cells are cultured.
  • non-cell side refers to the region or side on which cells are not cultured.
  • the cell side will be separated from the non-cell side by a membrane.
  • the cell side and non-cell side regions are defined by the hollow fibers themselves, i such cases, cells are usually cultured in the extracapillary (EC) space outside of the hollow fibers.
  • EC extracapillary
  • the EC space will be considered the cell side while the intracapillary (IC) space will be considered the non-cell side.
  • the IC space when cells are cultured in the IC space, the IC space will be considered the cell side while the EC space will be considered the non-cell side.
  • the term "serum,” as used herein, includes sera derived from both human and animal sources. Suitable sera include, but are not limited to, fetal bovine serum (FBS) (also known as fetal calf serum (FCS)), newborn calf serum (NCS), adult bovine serum (ABS), bovine calf serum (BCS), horse serum (HS), human, chicken, goat, porcine, rabbit, bovine, fetal, calf and sheep sera.
  • FBS fetal bovine serum
  • FCS newborn calf serum
  • ABS adult bovine serum
  • BCS bovine calf serum
  • HS horse serum
  • Serum can be obtained from a variety of commercial sources such as Sigma Aldrich Co. (St. Louis, MO). Serum may be used at any concentration easily determinable by one skilled in the art so as to optimize culturing conditions, hi various embodiments, the concentration of serum may be less than about 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%. In a particular embodiment, the concentration of serum maybe between about 5% and about 25% (for example, at about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25%). In a particular embodiment the concentration of serum is about 10%. The concentrations of serum as used on the cell side and the non-cell side may differ. In addition, the concentrations of serum as used on a particular side may differ over time.
  • basal medium refers to a medium containing several art-recognized (non-serum derived) nutrients for culturing cells and supporting cell growth.
  • the basal medium may support cell growth on its own or, in some instances, may require additional components, including serum or serum-derived components, to support cell growth.
  • the basal medium may be any of a variety of basal media known in the art, including, but not limited to, Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's Modified Eagle's Medium (DMEM), Essential Modified Eagle's Medium (EMEM), Minimal Essential Medium Eagle (MEM), Basal Medium Eagle (BME), Click's Medium, L-15 Medium Leibovitz, McCoy's 5 A Medium, Glasgow Minimum Essential Medium (GMEM), NCTC 109 Medium, Williams' Medium E, RPMI-1640, and Medium 199.
  • IMDM Iscove's Modified Dulbecco's Medium
  • DMEM Dulbecco's Modified Eagle's Medium
  • EMEM Essential Modified Eagle's Medium
  • MEM Minimal Essential Medium Eagle
  • BME Basal Medium Eagle
  • Click's Medium L-15 Medium Leibovitz
  • McCoy's 5 A Medium Glasgow Minimum Essential Medium (GMEM), NCTC 109 Medium, Williams' Medium
  • a medium specifically developed for a particular cell type/line or cell function e.g., Madin-Darby Bovine Kidney Growth Medium, Madin-Darby Bovine Kidney Maintenance Medium, various hybridoma media, Endothelial Basal Medium, Fibroblast Basal Medium, Keratinocyte Basal Medium, and Melanocyte Basal Medium may also be utilized (all available from Sigma Aldrich Co.; St. Louis, MO).
  • Presence of serum refers to the presence (e.g., cell side or non-cell side) of a sufficient number of components derived from serum that together are able to support cell growth and/or cell viability.
  • Presence of serum does not exclude the presence of some serum components (e.g., that are able to traverse the membrane and diffuse from the cell side to the non-cell side). However, these components are not alone sufficient to support cell growth.
  • absence of serum refers to the absence of a sufficient number of components derived from serum that together are able to support cell growth and/ or cell viability.
  • ethanolamine refers to the chemical well-known in the art.
  • Ethanolamine is commonly referred to as 2-aminoethanol, monoethanolamine, beta-aminoethanol, beta-aminoethyl alcohol, ethylolamine, glycinol, olamine, MEA, UASF EK-1597, colamine, beta-hydroxyethylamine, all of which are encompassed by the term "ethanolamine.”
  • Ethanolamine has the molecular formula, C 2 H 7 NO, and the structural formula, H 2 N(CH ) 2 OH. Ethanolamine is available from numerous commercial suppliers, including Moravek Biochemicals (Brea, CA).
  • Ethanolamine as used herein, is also intended to encompass other known forms of ethanolamine including, but not limited to, phosphoethanolamine and phosphatidyl ethanolamine.
  • cell viability refers generally to the lifetime and physical condition of cultured cells.
  • cell growth refers to the division and proliferation of cultured cells.
  • hybrida refers to a B cell obtained from an animal, e.g., a human or rodent, fused to an immortalized cell. Hybridomas are often used for the production of monoclonal antibodies.
  • membrane systems provide an efficient and effective means for culturing cells, often for the subsequent production of proteins.
  • membrane systems are ideal for culturing hybridomas for the production of antibodies.
  • Membrane systems provide improved cell retention and high density cell growth.
  • the term "membrane system,” as used herein, refers to a cell culture system that has a growth region (e.g., compartment) separated, by a membrane, from a medium region (e.g., compartment).
  • the membrane system often has a permeable or semi-permeable membrane (e.g., hollow fibers or capillaries) that allows for the bi-directional diffusion of gases, ions and small molecules for optimal cell culturing conditions.
  • the membranes have a molecular weight cut-off (usually less than 100 kD, and most often in the range of 10 to 30 kD) designed so as to, in part, allow smaller components to pass through, while retaining growth factors and product (e.g., antibodies secreted by the cultured cells).
  • Hollow fiber systems e.g. , hollow fiber bioreactors such as hollow fiber micro- bioreactors or large scale batch hollow fiber bioreactors
  • Perfused tanks and flask systems e.g., CL-1000 from Integra Biosciences, Chur, Switzerland are other examples of membrane systems.
  • hollow fiber systems have a plurality of semi-permeable hollow fibers potted in a housing to create a space inside the fibers (intracapillary or IC space) separate from that outside the fibers (extracapillary or EC space). Communication between the IC and the EC space is entirely through diffusion through fiber pores.
  • Cells are usually placed on the EC side of the fiber, but, in other embodiments, cells maybe placed on the IC side. Cells are retained better when grown in the EC space. Additionally, when cells are cultured for the purpose of collecting a secreted product (e.g., antibodies), growing the cells in EC space is preferred. However, if cells are cultured with the intention of ultimately recovering the cells, growing the cells in IC space is preferred.
  • a secreted product e.g., antibodies
  • the non-cell side medium may be circulated at a fast rate so as to provide sufficient oxygen, which diffuses through the porous fibers into the cell mass. Conversely, carbon dioxide created from cell respiration and acid neutralization diffuses from the cell mass, across the fibers and ultimately is carried away in the non-cell side medium.
  • the non-cell side medium provides the bulk of nutrition for components small enough to pass through the fibers.
  • the non-cell side of the membrane system contains a medium that lacks serum, but includes ethanolamine.
  • Ethanolamine is added or included in the serum-free medium in an amount necessary to support cell growth, as can be determined by a skilled practitioner in light of various factors including, but not limited to, culturing conditions, the nature of the cell being cultured, and the nature of the medium components.
  • the serum-free medium contains less than about 100 mg/L ethanolamine.
  • the medium contains less than about 10 mg/L, less than about 5 mg/L, less than about 2 mg/L, less than about 1 mg/L, or less than about .5 mg/L ethanolamine.
  • the ethanolamine can be added and maintained at these concentrations or added at greater concentrations and allowed to be reduced by consumption down to these concentrations.
  • ethanolamine uniquely maintains and enhances cell growth in a non-cell side environment that lacks sufficient serum components to support cell growth
  • the cells may first be cultured in the presence of serum on both the cell side and non-cell side prior to culturing the cells in the absence of serum on the non-cell side.
  • the serum on the non-cell side can become adsorbed to, for example, the silicone surfaces used in tubing and oxygenation to optimize the initial cell culture conditions.
  • the silicone surfaces often used in membrane systems are hydrophobic and interact with hydrophobic fats (Gramer et al. (1998) Biotechnol. Prog. 14:203-209).
  • the silicone surfaces on the non-cell side attract fats away from the serum on the cell side and deplete the cell side serum of necessary fats.
  • the serum coats the silicone surfaces and blocks the surface with fats.
  • additional fats will not be drawn to the non-cell side and will remain on the cell side, thereby enhancing the culturing conditions.
  • utilizing medium containing serum on the non-cell side for a limited time is often advantageous. Following this initial period, the serum on the cell side is sufficient to support serum feeding. Furthermore, continued use of serum beyond this initial period is undesirable in light of the unpredictability (in terms of its components), risks (potential contaminants), and costs associated with serum use.
  • the cells are cultured in the presence of serum on the non-cell side for less than 120 hours, 96 hours, 72 hours, 48 hours or 24 hours.
  • serum is added to the non-cell side at times less than 120 hours, 96 hours, 72 hours, 48 hours or 24 hours after inoculation.
  • serum is added on the day of inoculation.
  • serum is added only on the day of inoculation.
  • the serum may be added during any of the initial days of culturing (e.g., only during the day of inoculation) and subsequently allowed to gradually dilute out of the non-cell side.
  • the non-cell side medium contains basal medium including, but not limited to, any of the basal media described above, hi a particular embodiment, the combination of Ham's F-12 and DMEM in a 1 :1 ratio (D/F12) is used.
  • D/F12 is commercially available; for example, a modified form of D/F12 containing trace elements and pyruvate is available from Sigma Aldrich Co. as Product No. D0547.
  • trace elements and pyruvate are not present in the basal medium, such components may be added as necessary.
  • Other combinations of basal media may be used in the practice of the present invention.
  • the combinations include Ham's F-12, regardless of the identity of the other basal medium component, preferably in a 1 : 1 ratio.
  • Ham's F-12 contains important components for cell growth not necessarily provided for by the other basal media.
  • particular components of Ham's F-12 can serve as substitutes for particular low molecular weight serum components.
  • the non-cell side medium includes at least one supplement.
  • supply refers to additional components that supplement other media components so as to promote cell growth.
  • Suitable cell culture supplements include, for example, glucose, sodium bicarbonate, and amino acids such as glutamine, methionine, cystine, cysteine and tryptophan. Each or a combination of supplements are added as necessary. As is well known in the art, factors to be considered in determining the nature and the concentration of supplements to be added include culturing conditions, the nature of the cell being cultured, and the nature of the basal medium. For example, a skilled practitioner must account for the various supplements, such as glucose and glutamine, already present in certain commercially available basal media formulations (e.g., D/F12 available from Sigma Aldrich). A balanced medium may require additional supplements.
  • the non-cell side medium includes less than 10 g/L glucose, less than about 5 g/L glutamine, less than about 100 mg/L methionine, less than about 100 mg/L cystine, less than about 100 mg/L tryptophan, and/or less than about 10 g/L sodium bicarbonate.
  • the non-cell side medium is as described below:
  • the non-cell side medium may contain fats or insulin as necessary to support and enhance cell growth.
  • the hydrophobic components of fats and insulin aid in cell growth in a membrane system, in part, by becoming adsorbed to the silicone surface area used in tubing and for oxygenation on the non-cell side.
  • fats will remain on the cell side associated with serum as necessary for optimal cell growth.
  • the non-cell side medium can be supplemented with a variety of growth factors, cytokines, serum, etc., depending on the cells being cultured.
  • suitable growth factors include: basic fibroblast growth factor (bFGF), vascular endothelial growth factor (NEGF), epidermal growth factor (EGF), transforming growth factors (TGF ⁇ and TGF/3), platelet derived growth factors (PDGFs), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), insulin, erythropoietin (EPO), and colony stimulating factor (CSF).
  • suitable hormone additives are estrogen, progesterone, testosterone or glucocorticoids such as dexamethasone.
  • cytokine medium additives are interferons, interleukins or tumor necrosis factor . (T ⁇ F ⁇ ).
  • Salt solutions may also be added to the media, including Alseverr's Solution, Dulbecco's Phosphate Buffered Saline (DPBS), Earle's Balanced Salt Solution, Gey's Balanced Salt Solution (GBSS), Hanks' Balanced Salt Solution (HBSS), Puck's Saline A, and Tyrode's Salt Solution.
  • DPBS Dulbecco's Phosphate Buffered Saline
  • GBSS Gey's Balanced Salt Solution
  • HBSS Hanks' Balanced Salt Solution
  • Puck's Saline A Puck's Saline A
  • Tyrode's Salt Solution may also be added to the media, including Alseverr's Solution, Dulbecco's Phosphate Buffered Saline (DPBS), Earle's Balanced Salt Solution, Gey's Balanced Salt Solution (GBSS), Hanks' Balanced Salt Solution (HBSS), Puck's Saline A, and Tyrode's Salt Solution.
  • CELL SIDE MEDIUM According to the present invention, cells are typically cultured on the cell side in the presence of serum.
  • Any suitable animal derived serum may be used, as is well known in the art. Suitable sera include, but are not limited to, fetal bovine serum (FBS) (also known as fetal calf serum (FCS)), newborn calf serum ( ⁇ CS), adult bovine serum (ABS), bovine calf serum (BCS), horse serum (HS), human, chicken, goat, porcine, rabbit and sheep sera.
  • FBS fetal bovine serum
  • ⁇ CS newborn calf serum
  • ABS adult bovine serum
  • BCS bovine calf serum
  • HS horse serum
  • human chicken, goat, porcine, rabbit and sheep sera.
  • Serum replacements may also be used, such as controlled process serum replacement-type (CPSR; 1 or 3) or bovine embryonic fluid. Specific purified growth factors or cocktails of multiple growth factors can also be added.
  • CPSR controlled process serum replacement-type
  • the cell side also contains ethanolamine.
  • the cell side also contains basal medium.
  • Other medium components including, but not limited to, those listed above (e.g., fats, insulin, supplements, etc.) may be added as necessary.
  • the selection of the cell side media and culture conditions varies depending, in part, on cell type and may be empirically determined by one skilled in the art.
  • the serum level is maintained at more than about 20% of its initial concentration.
  • the terms "maintain" and “maintained” refer to sustaining serum concentration above or at a particular concentration.
  • the serum level is maintained at about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% of the initial concentration. In other embodiments, the serum is maintained at greater than 2% of the total media components, for example, between about 2-15%, preferably between about 8-12%, of the total media components.
  • the serum level is preferably maintained at these levels for at least 7-14 days (e.g., 15, 16, 17, 18, 19, 20, 21, 22 or 28 days) or the entire duration of the culturing step.
  • the methods of the invention are particularly suited for culturing a wide variety of cells including, in particular, cells that produce antibodies (e.g., monoclonal antibodies).
  • Membrane systems such as hollow fiber bioreactors, are particularly suited for the mass production of antibodies by culturing hybridomas or heterohybridomas.
  • Suitable hybridomas include, but are not limited to, rodent hybridomas (such as murine, rat, hamster and rabbit), sheep hybridomas and human hybridomas.
  • the hybridoma cells are Chinese hamster ovary (CHO) hybridoma cells, NS/O hybridomas, SP2/O hybridomas, or murine hybridoma cells.
  • the culturing techniques of the present invention serve to enhance antibody (e.g., idiotype) production.
  • antibody production is enhanced by at least about 200%, 150%, 100%, 75%, 50%, 40%, 30%, 20%, 10% or 5%.
  • Example 1 Use of Ethanolamine in Culturing Murine Hybridomas in Hollow Fiber Bioreactors hi this study, a 10 kD MWCO hollow-fiber micro bioreactor was used to determine if a less expensive form of serum other than 10% fetal bovine serum (FBS) or a more defined supplement could be added to the medium reservoir (non-cell side) of the bioreactor to optimize growth of a murine hybridoma. Neither 10% newborn calf serum nor 10% adult bovine serum served as a replacement for 10% FBS in the medium reservoir.
  • FBS fetal bovine serum
  • Optimal growth was attained when 10% fetal bovine serum (FBS) was used in both the IC and EC compartments.
  • FBS fetal bovine serum
  • serum is usually added only to the cell side of the bioreactor which reduces serum usage by a factor of about 100.
  • the hollow-fiber micro bioreactor was used to determine if a less expensive form of serum or a more defined supplement could be added to the non-cell side of the bioreactor. The results are generalized by examining a total of four different murine hybridoma cell lines.
  • MATERIALS AND METHODS Media Basal medium (Sigma Chemical Company) consisted of DME/F12 with trace elements and 2.5 mM L-glutamine, and was additionally supplemented with 1.5 M L- glutamine, 2.44 g/L sodium bicarbonate, 0.066 g/L penicillin G (Irvine Scientific), and 0.144 g/L streptomycin sulfate (Irvine Scientific). Serum-supplemented media were prepared by adding fetal bovine serum (FBS), newborn calf serum (NCS) or adult bovine serum (ABS) (all from Sigma) to basal medium.
  • FBS fetal bovine serum
  • NCS newborn calf serum
  • ABS adult bovine serum
  • the serum-free medium supplement SITE+3 from Sigma was used as a 1:50 dilution to give final concentrations of 10 ⁇ g/L selenium, 20 mg/L insulin (bovine pancreas), 11 mg/L transferrin (human), 4 mg/L ethanolamine, 1 g/L bovine serum albumin (BSA), 9.4 mg/L linoleic acid, and 9.4 mg/L oleic acid.
  • BSA bovine serum albumin
  • 9.4 mg/L linoleic acid 9.4 mg/L oleic acid.
  • the bioreactor fibers were made of regenerated cellulose (cuprophane) with a 10 kD MWCO.
  • the bioreactors were flushed before use to remove the wetting agent in the fibers and residuals from the sterilization process.
  • the IC (0.2 ml) was then primed with 10% FBS-supplemented medium, and the EC (5 or 16 ml depending on the diameter of silicone tubing used) was rinsed and filled with basal medium plus other components as described in the Results section.
  • T-Flask Growth Curve Cells (rho 1D4) were cultured for one week in 10% FBS-supplemented basal medium which contained an additional 1 g/L glucose and 1 g/L sodium bicarbonate with or without 4 mg/L ethanolamine. Cells at about 5xl ⁇ 5/ml and 95% viability were pelleted at 200xg and resuspended in the same fresh medium at about 5xl ⁇ 4/ml. Cells (5 ml) in each of the two media were inoculated into thirty T-25 flasks. Three flasks were harvested each day for cell counts and antibody determination until the viability dropped below 25%.
  • Basal medium supplemented with 4 mg/L ethanolamine was circulated from the IC medium reservoir, through the oxygenator, through the fiber IC compartment, and was returned to the bottle.
  • the EC medium was changed 3 times per week by adding 20 ml of 10% FBS-supplemented medium through the bottom port while harvesting 20 ml through the top port.
  • the IC medium bottles were changed daily to keep the glucose concentration above 1.5 g/L.
  • the IC reservoir initially contained 200 ml of medium, and this volume was increased up to 2 L to support the glucose demand, after which the medium was supplemented with an additional 1 g/L glucose and 1 g/L sodium bicarbonate.
  • the results from control bioreactors (basal medium or 10% FBS-supplemented medium in the IC) were generated previously (Gramer, 1998).
  • MWCO MWCO.
  • the total surface area based on fiber ID was 1.1 m ⁇ and the EC volume was about 100 ml.
  • Cells (5x10 ⁇ ) were inoculated simultaneously into the EC compartment of two instruments in 10% FBS-supplemented medium.
  • Basal medium was circulated in the IC which contained an additional 1 g/L glucose and 1 g/L sodium bicarbonate; in one instrument, this medium also contained 4 mg/L ethanolamine.
  • the pH was controlled at pH 7.2 and the temperature was controlled at 37°C.
  • Fresh basal medium was continually added to and removed from the IC medium reservoir. The rate of IC medium addition was increased daily from 25 ml/hr up to 400 ml/hr based on off-line glucose analyses to keep the glucose above 2 g/L.
  • the rho 1D4 murine hybridoma cell line was inoculated at 5xl0 6 /ml with 10% fetal bovine serum (FBS) into the IC compartment (0.2 ml volume) of a series of micro bioreactors.
  • the EC reservoir contained either 16 ml of basal medium or 16 ml of basal medium supplemented with 10% FBS, 10% newborn calf serum (NCS), 10% adult bovine serum (ABS), or a serum-free medium supplement SITE+3. Cells were harvested and counted after three days of growth in an incubator. As expected, growth was poor in the bioreactor containing only basal medium in the EC reservoir, while growth was very good in the bioreactor with 10% FBS in the EC reservoir (Fig. 1).
  • the finding of a defined supplement to replace FBS in the reservoir of the bioreactor was fortunate, but the supplement cost was still quite high.
  • the SITE+3 supplement contains insulin, transferrin, selenium, ethanolamine, and BSA with oleic and linoleic acid. Of these components, selenium (in the trace elements) and a small amount of linoleic acid are already found in the basal medium. Selenium, ethanolamine, insulin, and the fatty acids (the equilibrium portions not bound by BSA) are small enough to pass through the 10 kD hollow-fiber membrane.
  • Transferrin and BSA cannot pass through the membrane, but these proteins are buffers of potentially toxic metals and hydrophobic substances, respectively (Glassy et al, 1988).
  • each component was selectively removed from the EC supplement, and the micro bioreactor experiment was repeated.
  • cell growth was poor in the basal EC medium control bioreactor and very good in the bioreactor containing the complete SITE+3 medium supplement in the EC reservoir (Fig. 2).
  • Cell growth was not reduced by removing insulin, transferrin, or selenium from the SITE+3 supplement.
  • cell growth was greatly suppressed by the removal of ethanolamine or the BSA/fatty acid complex.
  • Ethanolamine in T-Flasks Cells were grown in 10% FBS-supplemented medium in a T-flask with or without 4 mg/L ethanolamine. There was very little difference in the viable cell density (Fig. 5), total cell density (not shown), and the antibody concentration (Fig. 5) over the 6-day growth period. The discrepancy between the T-flask and hollow-fiber bioreactor data suggests that T-flasks should not be used to optimize the medium formulation for cells grown in a hollow-fiber system.
  • the reduction of the EC medium reservoir also results in a reduction of the silicone surface area.
  • these data suggest that the ratios of cell side volume to reservoir volume and silicone surface may be important scale-up parameters.
  • Two types of production-scale hollow-fiber systems were used to test the preceding hypothesis. The first was a simple hollow-fiber system (ResCu-Primer HF) placed in an incubator that is operated through batch changes of IC and EC media. The second was an automated system (AcuSyst-Maximizer) that provides automatic temperature and pH control with continuous IC and EC media addition and removal.
  • the ratios of initial non cell side volumes and silicone surface areas to cell side volumes are compared in Table 1 to the same ratios for the micro bioreactors (note that the cells are placed in the IC in micro bioreactors and in the EC in the large hollow-fiber bioreactors). Based on the results in this paper, the two larger production systems should not require the BSA/lipid supplement on the non-cell side of the membrane since the ratio of silicone to cell side volume is smaller than that for the 5-ml bioreactor. However, the two production scale bioreactors could still potentially benefit from the ethanolamine supplement. Results for three simple batch operated production systems are shown in Figure 6.
  • the cell side of the bioreactors contained basal medium supplemented with 10% FBS
  • the reservoir side contained either basal medium, basal medium supplemented with ethanolamine, or basal medium supplemented with 10% FBS.
  • cell growth was very poor with only basal medium in the reservoir.
  • the addition of either ethanolamine or FBS to the medium reservoir resulted in good cell growth (as estimated by the glucose uptake rate), and growth with the two supplements was essentially identical.
  • the metabolic activity of cells grown in the automated systems was more extensively characterized (Fig 7). Cells were inoculated on the EC side in basal medium supplemented with 10% FBS.
  • the IC (non-cell) side contained basal medium with or without 4 mg/L ethanolamine.
  • the cells grew fairly well without the addition of ethanolamine to the IC medium reservoir.
  • the metabolic activity increased at a much faster rate when ethanolamine was added to the basal medium.
  • the two cultures consumed medium at the same rate and displayed similar glucose uptake rates and lactate production rates.
  • the oxygen uptake rate and antibody production rate were about 70% and 50% higher respectively for the bioreactor with ethanolamine relative to the bioreactor without ethanolamine.
  • rho 1D4 murine hybridoma cell line Optimal growth of the rho 1D4 murine hybridoma cell line in a hollow-fiber system required the addition of ethanolamine or FBS to the basal medium reservoir.
  • Three other murine hybridoma cell lines were tested in 5-ml micro bioreactors to determine if this is might be a general requirement for this cell type. As shown in Figure 8, the results for these three additional cell lines paralleled those of the rho 1D4 cell line; cell growth was greatly enhanced by the addition of ethanolamine or FBS, and growth with these two supplements was comparable.
  • the rho 1D4 cell line was created with the NS-1 fusion partner while the three cell lines in Fig. 8 were created using the Sp2/0 fusion partner.
  • DISCUSSION A novel hollow-fiber micro bioreactor was used as a screening tool to look for less expensive alternatives to FBS supplementation on the non-cell side of the membrane. Less expensive forms of serum such as newborn calf serum and adult bovine serum did not substitute for FBS (Fig 1).
  • FBS could be replaced by the addition of just ethanolamine to the basal medium (Fig. 4).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention est fondée en partie sur la découverte que la croissance cellulaire, la viabilité cellulaire et la production d'anticorps dans un système membranaire tel qu'un système de fibres creuses peuvent être renforcées par la culture de cellules en présence de sérum du côté cellulaire et dans un milieu contenant de l'éthanolamine en l'absence de sérum du côté non cellulaire. Par conséquent, cette invention concerne des techniques permettant d'améliorer la croissance de cellules tout en minimisant l'utilisation de sérum.
PCT/US2005/007926 2004-03-08 2005-03-08 Utilisation d'ethanolamine pour renforcer la croissance cellulaire dans des systemes membranaires Ceased WO2005087915A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US55111904P 2004-03-08 2004-03-08
US60/551,119 2004-03-08

Publications (2)

Publication Number Publication Date
WO2005087915A2 true WO2005087915A2 (fr) 2005-09-22
WO2005087915A3 WO2005087915A3 (fr) 2006-06-01

Family

ID=34976297

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2005/007926 Ceased WO2005087915A2 (fr) 2004-03-08 2005-03-08 Utilisation d'ethanolamine pour renforcer la croissance cellulaire dans des systemes membranaires

Country Status (1)

Country Link
WO (1) WO2005087915A2 (fr)

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8309347B2 (en) 2007-03-05 2012-11-13 Terumo Bct, Inc. Cell expansion system and methods of use
US8691565B2 (en) 2008-03-05 2014-04-08 Terumo Bct, Inc. Method of reseeding adherent cells grown in a hollow fiber bioreactor system
US8906688B2 (en) 2007-04-13 2014-12-09 Terumo Bct, Inc. Cell expansion system and methods of use
KR20150110807A (ko) * 2013-01-31 2015-10-02 아지노모토 가부시키가이샤 다능성 줄기 세포의 안정한 미분화 유지 증식을 실시하기 위한 배양 방법
US9441195B2 (en) 2006-05-22 2016-09-13 Biovest International, Inc. Method and system for the production of cells and cell products and applications thereof
US9617506B2 (en) 2013-11-16 2017-04-11 Terumo Bct, Inc. Expanding cells in a bioreactor
US9677042B2 (en) 2010-10-08 2017-06-13 Terumo Bct, Inc. Customizable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US9725768B2 (en) 2012-08-31 2017-08-08 Biovest International, Inc. Methods for producing high-fidelity autologous idiotype vaccines
US9732313B2 (en) 2011-06-10 2017-08-15 Biovest International, Inc. Method and apparatus for virus and vaccine production
US9902928B2 (en) 2012-08-28 2018-02-27 Biovest International, Inc. Biomanufacturing suite and methods for large-scale production of cells, viruses, and biomolecules
US10077421B2 (en) 2014-04-24 2018-09-18 Terumo Bct, Inc. Measuring flow rate
US10093956B2 (en) 2011-06-10 2018-10-09 Biovest International, Inc. Method and apparatus for antibody production and purification
US10119118B2 (en) 2006-09-13 2018-11-06 Abbvie Inc. Modified serum-free cell culture medium
CN109897880A (zh) * 2019-03-18 2019-06-18 郑州伊美诺生物技术有限公司 体外培养制备afp单克隆抗体的方法及其应用
US10577576B2 (en) 2012-08-20 2020-03-03 Terumo Bct, Inc. System for expanding cells
US11008547B2 (en) 2014-03-25 2021-05-18 Terumo Bct, Inc. Passive replacement of media
US11104874B2 (en) 2016-06-07 2021-08-31 Terumo Bct, Inc. Coating a bioreactor
US11608486B2 (en) 2015-07-02 2023-03-21 Terumo Bct, Inc. Cell growth with mechanical stimuli
US11624046B2 (en) 2017-03-31 2023-04-11 Terumo Bct, Inc. Cell expansion
US11629332B2 (en) 2017-03-31 2023-04-18 Terumo Bct, Inc. Cell expansion
US11667881B2 (en) 2014-09-26 2023-06-06 Terumo Bct, Inc. Scheduled feed
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GRAMER MICHAEL J ET AL: "Optimal NSO cell growth in a hollow fiber bioreactor requires increased serum concentration or a cholesterol supplement on the cell side of the fiber." BIOTECHNOLOGY PROGRESS, vol. 19, no. 6, 2003, pages 1762-1766, XP002366086 ISSN: 8756-7938 *
INOUE YUICHI ET AL: "Production of recombinant human monoclonal antibody using ras-amplified BHK-21 cells in a protein-free medium" BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, vol. 60, no. 5, 1996, pages 811-817, XP002366085 ISSN: 0916-8451 *
KLERX J P A M ET AL: "IN-VITRO PRODUCTION OF MONOCLONAL ANTIBODIES UNDER SERUM-FREE CONDITIONS USING A COMPACT AND INEXPENSIVE HOLLOW FIBER CELL CULTURE UNIT" JOURNAL OF IMMUNOLOGICAL METHODS, vol. 111, no. 2, 1988, pages 179-188, XP002366082 ISSN: 0022-1759 cited in the application *
MICHAEL J. GRAMER ET AL.: "Screening tool for hollow-fiber bioreactor process development" BIOTECHNOL. PROG., vol. 14, 1998, pages 203-209, XP002366087 *
TAKAZAWA Y ET AL: "HIGH-DENSITY CULTURE OF MOUSE-HUMAN HYBRIDOMA IN SERUM-FREE DEFINED MEDIUM" BIOTECHNOLOGY AND BIOENGINEERING, vol. 31, no. 2, 1988, pages 168-172, XP002366084 ISSN: 0006-3592 *
VAN ERP R ET AL: "MONITORING OF THE PRODUCTION OF MONOCLONAL ANTIBODIES BY HYBRIDOMAS PART I. LONG-TERM CULTIVATION IN HOLLOW FIBER BIOREACTORS USING SERUM-FREE MEDIUM" JOURNAL OF BIOTECHNOLOGY, vol. 20, no. 3, 1991, pages 235-248, XP002366083 ISSN: 0168-1656 cited in the application *

Cited By (49)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11345882B2 (en) 2006-05-22 2022-05-31 Biovest International, Inc. Extra-capillary fluid cycling system and method for a cell culture device
US9441195B2 (en) 2006-05-22 2016-09-13 Biovest International, Inc. Method and system for the production of cells and cell products and applications thereof
US9534198B2 (en) 2006-05-22 2017-01-03 Biovest International, Inc. Extra-capillary fluid cycling system and method for a cell culture device
US10723993B2 (en) 2006-05-22 2020-07-28 Biovest International, Inc. Method and system for the production of cells and cell products and applications thereof
US10119118B2 (en) 2006-09-13 2018-11-06 Abbvie Inc. Modified serum-free cell culture medium
US8785181B2 (en) 2007-03-05 2014-07-22 Terumo Bct, Inc. Cell expansion system and methods of use
US9260698B2 (en) 2007-03-05 2016-02-16 Terumo Bct, Inc. Cell expansion system and methods of use
US8309347B2 (en) 2007-03-05 2012-11-13 Terumo Bct, Inc. Cell expansion system and methods of use
US8906688B2 (en) 2007-04-13 2014-12-09 Terumo Bct, Inc. Cell expansion system and methods of use
US8691565B2 (en) 2008-03-05 2014-04-08 Terumo Bct, Inc. Method of reseeding adherent cells grown in a hollow fiber bioreactor system
US10577582B2 (en) 2008-03-05 2020-03-03 Terumo Bct, Inc. Method of reseeding adherent cells grown in a hollow fiber bioreactor system
US9428729B2 (en) 2008-03-05 2016-08-30 Terumo Bct, Inc. Method of reseeding adherent cells grown in a hollow fiber bioreactor system
US9677042B2 (en) 2010-10-08 2017-06-13 Terumo Bct, Inc. Customizable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US9725689B2 (en) 2010-10-08 2017-08-08 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US10870827B2 (en) 2010-10-08 2020-12-22 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11613727B2 (en) 2010-10-08 2023-03-28 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11746319B2 (en) 2010-10-08 2023-09-05 Terumo Bct, Inc. Customizable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US10669519B2 (en) 2010-10-08 2020-06-02 Terumo Bct, Inc. Customizable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11773363B2 (en) 2010-10-08 2023-10-03 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US9732313B2 (en) 2011-06-10 2017-08-15 Biovest International, Inc. Method and apparatus for virus and vaccine production
US10093956B2 (en) 2011-06-10 2018-10-09 Biovest International, Inc. Method and apparatus for antibody production and purification
US10570434B2 (en) 2011-06-10 2020-02-25 Biovest International, Inc. Method and apparatus for antibody production and purification
US10577576B2 (en) 2012-08-20 2020-03-03 Terumo Bct, Inc. System for expanding cells
US10662401B2 (en) 2012-08-28 2020-05-26 Biovest International, Inc. Biomanufacturing suite and methods for large-scale production of cells, viruses, and biomolecules
US9902928B2 (en) 2012-08-28 2018-02-27 Biovest International, Inc. Biomanufacturing suite and methods for large-scale production of cells, viruses, and biomolecules
US9725768B2 (en) 2012-08-31 2017-08-08 Biovest International, Inc. Methods for producing high-fidelity autologous idiotype vaccines
KR102211266B1 (ko) * 2013-01-31 2021-02-04 아지노모토 가부시키가이샤 다능성 줄기 세포의 안정한 미분화 유지 증식을 실시하기 위한 배양 방법
US10745669B2 (en) 2013-01-31 2020-08-18 Ajinomoto Co., Ltd. Culture method for stable proliferation of pluripotent stem cell while maintaining undifferentiated state
KR20150110807A (ko) * 2013-01-31 2015-10-02 아지노모토 가부시키가이샤 다능성 줄기 세포의 안정한 미분화 유지 증식을 실시하기 위한 배양 방법
US10662411B2 (en) 2013-01-31 2020-05-26 Ajinomoto Co., Inc. Culture method for stable proliferation of pluripotent stem cell while maintaining undifferentiated state
US9617506B2 (en) 2013-11-16 2017-04-11 Terumo Bct, Inc. Expanding cells in a bioreactor
US11708554B2 (en) 2013-11-16 2023-07-25 Terumo Bct, Inc. Expanding cells in a bioreactor
US10557112B2 (en) 2013-11-16 2020-02-11 Terumo Bct, Inc. Expanding cells in a bioreactor
US10633625B2 (en) 2013-11-16 2020-04-28 Terumo Bct, Inc. Expanding cells in a bioreactor
US11667876B2 (en) 2013-11-16 2023-06-06 Terumo Bct, Inc. Expanding cells in a bioreactor
US11008547B2 (en) 2014-03-25 2021-05-18 Terumo Bct, Inc. Passive replacement of media
US11795432B2 (en) 2014-03-25 2023-10-24 Terumo Bct, Inc. Passive replacement of media
US10077421B2 (en) 2014-04-24 2018-09-18 Terumo Bct, Inc. Measuring flow rate
US11667881B2 (en) 2014-09-26 2023-06-06 Terumo Bct, Inc. Scheduled feed
US11608486B2 (en) 2015-07-02 2023-03-21 Terumo Bct, Inc. Cell growth with mechanical stimuli
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion
US11634677B2 (en) 2016-06-07 2023-04-25 Terumo Bct, Inc. Coating a bioreactor in a cell expansion system
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11104874B2 (en) 2016-06-07 2021-08-31 Terumo Bct, Inc. Coating a bioreactor
US11999929B2 (en) 2016-06-07 2024-06-04 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11624046B2 (en) 2017-03-31 2023-04-11 Terumo Bct, Inc. Cell expansion
US11702634B2 (en) 2017-03-31 2023-07-18 Terumo Bct, Inc. Expanding cells in a bioreactor
US11629332B2 (en) 2017-03-31 2023-04-18 Terumo Bct, Inc. Cell expansion
CN109897880A (zh) * 2019-03-18 2019-06-18 郑州伊美诺生物技术有限公司 体外培养制备afp单克隆抗体的方法及其应用

Also Published As

Publication number Publication date
WO2005087915A3 (fr) 2006-06-01

Similar Documents

Publication Publication Date Title
WO2005087915A2 (fr) Utilisation d'ethanolamine pour renforcer la croissance cellulaire dans des systemes membranaires
EP0435911B1 (fr) Milieu de culture de cellules pour l'amelioration de la croissance des cellules, de la longivite de la culture et de l'expression du produit
Ham et al. [5] Media and growth requirements
Yao et al. Animal‐cell culture media: History, characteristics, and current issues
JP3221681B2 (ja) 動物細胞培養
US5232848A (en) Basal nutrient medium for cell culture
US6306644B1 (en) Device for cultivating different mammal cells at the same time
US20140335616A1 (en) Defined systems for epithelial cell culture and use thereof
EA022219B1 (ru) Способ получения полипептида или вируса, представляющих интерес, в непрерывной клеточной культуре
Heath et al. Methods for increasing monoclonal antibody production in suspension and entrapped cell cultures: biochemical and flow cytometric analysis as a function of medium serum content
US7390651B2 (en) C3A serum-free clonal cell line and methods of use
EP3207121B1 (fr) Procedes de replication de culture cellulaire de production d'eculizumab a grande echelle
CN114317428B (zh) 一种含中药小分子的干细胞无血清培养基及其制备方法
Sato et al. Media for culture of mammalian cells
Honegger Overview of cell and tissue culture techniques
CN102405279B (zh) 改善单细胞克隆的方法
JP2016508376A (ja) 組換えタンパク質産生の増加のための製剤及び方法
CN103484426A (zh) 一种无动物源的低蛋白培养基
Custer Physiological studies of hybridoma cultivation in hollow fiber bioreactors.
Keenan et al. Serum-free media
Pechan et al. Cell specific nutrient feeding in perfusion mode enhances hMSC growth in stirred tank bioreactor process
RU2786997C2 (ru) Процессы культивирования в системе посевных ферментеров и способы их применения
KR20250076588A (ko) 무혈청 배지
Sharfstein Metabolic studies of mammalian cells grown in culture
KR20230072208A (ko) 저혈청 배지 첨가제 및 이의 용도

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase in:

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase