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WO2023079058A1 - Culture cellulaire avec production réduite de lactate - Google Patents

Culture cellulaire avec production réduite de lactate Download PDF

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Publication number
WO2023079058A1
WO2023079058A1 PCT/EP2022/080777 EP2022080777W WO2023079058A1 WO 2023079058 A1 WO2023079058 A1 WO 2023079058A1 EP 2022080777 W EP2022080777 W EP 2022080777W WO 2023079058 A1 WO2023079058 A1 WO 2023079058A1
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Prior art keywords
polynucleotide
cell population
cell
aspartate
production
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PCT/EP2022/080777
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English (en)
Inventor
Shilpa NARGUND
Klaus Mauch
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Yokogawa Insilico Biotechnology Gmbh
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Publication of WO2023079058A1 publication Critical patent/WO2023079058A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present invention relates to cell culture methods and compositions thereof that in one aspect improve the yield and quality of a biologic.
  • the cell culture methods include expressing a polynucleotide of interest to improve the yield and quality of a biologic produced in a mammalian cell culture.
  • the biopharmaceutical industry widely uses mammalian cell cultures to produce biologies such as proteins, vaccines, genes, cells or tissues that will ultimately be used for therapeutic purposes.
  • the process used for growing mammalian cells in in vitro cultures must meet the nutritional needs of the cells and provide a conducive environment with respect to aeration, temperature, pH and osmolality.
  • the development of a robust and efficient process for culturing mammalian cells is critical to obtaining high yields and consistent quality of the biologic.
  • lactate is one of the main waste product that is secreted in the cell culture vessel, and it has been shown to inhibit cell growth and protein production.
  • the production of excessive lactate by mammalian cell cultures has been a long-standing problem that not only affects the yield but also the quality of biologies or biopharmaceutical products.
  • the excessive lactate produced leads to a decrease in pH and a concomitant addition of an alkali in pH-controlled processes.
  • the addition of an alkali leads to an increase in osmolarity of the cell culture medium.
  • the invention seeks to address one or more of the above-mentioned limitations or needs and in one aspect provides a method for reducing the production of lactate in a cell population, the method comprising:
  • polynucleotide described herein in a cell population
  • the polynucleotide is selected from the group consisting of a polynucleotide encoding a transporter of aspartate across the cell membrane, a polynucleotide encoding a protein involved in biosynthesis of aspartate, or any combination thereof, wherein expressing the polynucleotide results in an increased amount of aspartate in the cell when compared to a cell population that does not express the polynucleotide, wherein the increased amount of aspartate reduces the production of lactate in the cell population.
  • method for reducing the production of lactate of cells in culture comprising: expressing a polynucleotide described herein in one or more cells in culture, wherein the polynucleotide is selected from the group consisting of a polynucleotide encoding a transporter of aspartate across the cell membrane, a polynucleotide encoding a protein involved in biosynthesis of aspartate, or any combination thereof, wherein expressing the polynucleotide results in an increased amount of aspartate in the cell when compared to a cell that does not express the polynucleotide, wherein the increased amount of aspartate reduces the production of lactate of the cells in culture.
  • a method for reducing the production of lactate in a cell population comprising:
  • the polynucleotide is selected from the group consisting of a polynucleotide encoding a transporter of aspartate across the cell membrane, a polynucleotide encoding a protein involved in biosynthesis of aspartate, or any combination thereof, wherein expressing the polynucleotide results in an increased amount of aspartate in the cell when compared to a cell population that does not express the polynucleotide, wherein the increased amount of aspartate reduces the production of lactate in the cell population.
  • the invention also provides a method for producing a cell population with a reduced production of lactate, the method comprising introducing a polynucleotide described herein into a cell population and culturing the cell population under conditions suitable for expression of the polynucleotide, wherein the polynucleotide is selected from the group consisting of a polynucleotide encoding a transporter of aspartate across the cell membrane, a polynucleotide encoding a protein involved in biosynthesis of aspartate, or any combination thereof, wherein expressing the polynucleotide results in an increased amount of aspartate in the cell when compared to a cell population that does not express the polynucleotide, wherein the increased amount of aspartate reduces the production of lactate in the cell population, thereby producing a cell population with a reduced production of lactate.
  • a method for preparing a cell population for the production of a biologic of interest comprising:
  • the polynucleotide is selected from the group consisting of a polynucleotide encoding a transporter of aspartate across the cell membrane, a polynucleotide encoding a protein involved in biosynthesis of aspartate, or any combination thereof, wherein expressing the polynucleotide results in an increased amount of aspartate in the cell when compared to a cell population that does not express the polynucleotide, wherein the increased amount of aspartate reduces the production of lactate in the cell population, thereby preparing a cell population for the production of a biologic of interest.
  • a method for the production of a biologic of interest in a cell population comprising: - providing a cell population expressing a polynucleotide or vector described herein;
  • the polynucleotide is selected from the group consisting of a polynucleotide encoding a transporter of aspartate across the cell membrane, a polynucleotide encoding a protein involved in biosynthesis of aspartate, or any combination thereof, wherein expressing the polynucleotide results in an increased amount of aspartate in the cell when compared to a cell population that does not express the polynucleotide, wherein the increased amount of aspartate reduces the production of lactate in the cell population, thereby providing a method for the production of a biologic of interest in the cell population.
  • a method for reducing the production of lactate in a cell population comprising:
  • the polynucleotide is selected from the group consisting of a polynucleotide encoding a transporter of aspartate across the cell membrane, a polynucleotide encoding a protein involved in biosynthesis of aspartate, or any combination thereof, wherein expressing the polynucleotide results in an increased amount of aspartate in the cell when compared to a cell population that does not express the polynucleotide, wherein the increased amount of aspartate reduces the production of lactate in the cell population.
  • a method for reducing the production of lactate in a cell population comprising:
  • the polynucleotide is selected from the group consisting of a polynucleotide encoding a transporter of aspartate across the cell membrane, a polynucleotide encoding a protein involved in biosynthesis of aspartate, or any combination thereof, wherein expressing the polynucleotide results in an increased amount of aspartate in the cell when compared to a cell population that does not express the polynucleotide.
  • the reduction of the production of lactate in the cell population, or cells in culture results in a reduced secretion of lactate into the cell medium that contains the cell population.
  • the method further comprises introducing a further polynucleotide into the cell population, wherein the further polynucleotide encodes a biologic of interest.
  • the method may comprise a step of isolating the biologic of interest from the cell population or cell culture medium.
  • the amount of aspartate is increased by expressing a polynucleotide that increases uptake of aspartate into the cell. In another embodiment, the amount of aspartate is increased by expressing a polynucleotide that increases the synthesis of aspartate in the cell. In any embodiment of the invention, the amount of aspartate is increased by increasing the expression of (a) an endogenous gene encoding a transporter of aspartate across the cell membrane, (b) an endogenous gene encoding a protein involved in biosynthesis of aspartate, or (c) a combination of (a) and (b). In this embodiment, the invention provides a method for reducing the production of lactate in a cell population, the method comprising:
  • polynucleotide is selected from the group consisting of a polynucleotide encoding a transporter of aspartate across the cell membrane, a polynucleotide encoding a protein involved in biosynthesis of aspartate, or any combination thereof, wherein increasing the expression of the polynucleotide results in an increased amount of aspartate in the cell, wherein the increased amount of aspartate reduces the production of lactate in the cell population.
  • expressing a polynucleotide described herein may be increasing the expression of (a) an endogenous gene encoding a transporter of aspartate across the cell membrane, (b) an endogenous gene encoding a protein involved in biosynthesis of aspartate, or (c) a combination of (a) and (b).
  • the endogenous promoter of one or more of the genes in (a), (b) or (c) may be modified to increase expression.
  • the method may comprise producing a biologic of interest in the cell population or providing conditions suitable for the production thereof.
  • the biologic of interest may be a protein or polypeptide, vaccine, gene, cell, tissue, or blood component(s).
  • the biologic may be an antibody, a receptor, a growth factor or signalling molecule, a G-protein coupled receptor (GPCR), a chimeric antigen receptor (CAR).
  • GPCR G-protein coupled receptor
  • CAR chimeric antigen receptor
  • the yield or quantity of the biologic may be obtained as a secreted product present in the cell culture medium or it may be contained within the cell or cell population and isolated thereafter.
  • the yield of the biologic of interest is increased by at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, or at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74% at least 76%, at least 78% or at least 80% or more when compared to a cell population that does not express the polynucleotide.
  • the biologic of interest is characterised by having consistent quality profiles compared to a biologic of interest obtained from a cell population that does not express the polynucleotide.
  • the pH of the cell culture medium containing the cell population which express any polynucleotide described herein is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8 or about 7.9.
  • the pH is between about 6.8 and 7.3.
  • the osmolality of the cell culture medium containing the cell population which express any polynucleotide described herein is about 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, , 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445 or 450 mOSM/kg.
  • the osmolality is between about 270 to 350 mOSM/kg.
  • the polynucleotide for use in any method described herein may be selected from the group consisting of: - a polynucleotide encoding a a transporter of aspartate across the cell membrane including SLC1A1 , SLC1A2, SLC1A3, SLC1A6, SLC1A7, SLC17A1 , SLC17A2, SLC17A3, SLC17A4, SLC17A6, SLC17A7, SLC17A8, SLC17A9, SLC38A7, SLC38A10, SLC7A13, preferably SLC1 A3;
  • polynucleotide encoding a protein (preferably enzyme) involved in biosynthesis of aspartate including Asparaginase (ASPG), Glutamate Oxaloacetate Transaminase 1 (GOT1 ) and Glutamate Oxaloacetate Transaminase 2 (GOT2); and
  • a polynucleotide encoding a protein involved in the synthesis of aspartate may comprise, consist essentially of or consist of one or more of the nucleotide sequences shown in SEQ ID NOs: 2 or 3.
  • the cell population is a mammalian cell population.
  • the mammalian cell population may be any one of, but not limited to, Chinese Hamster Ovarian (CHO) cells such as CHO-K1 , CHO-S, CHO-DG44, CHO-MK; Human Embryonic Kidney (HEK) cells such as HEK 293; Baby Hamster Kidney (BHK) cells such as BHK 21 ; PER.C6; murine myeloma cells such as NSO, SP2/0; HUVEC or derivatives of any of these cells.
  • CHO Chinese Hamster Ovarian
  • HEK Human Embryonic Kidney
  • BHK Baby Hamster Kidney
  • PER.C6 murine myeloma cells
  • NSO NSO
  • SP2/0 HUVEC or derivatives of any of these cells.
  • the cell population is a CHO cell population.
  • the cell population is cultured in a batch, fed-batch or continuous process.
  • the fed-batch process is a high-density culture, constantly-fed batch or an exponential-fed-batch process.
  • the continuous process is a perfusion process wherein fresh medium is continuously fed, spent media is continuously removed while the cells are retained inside the culture vessel.
  • the perfusion process is such that the biologic of interest is harvested from the cell culture vessel continuously or at intervals.
  • the polynucleotide may be transiently or stably expressed in the cell population. In another embodiment, the polynucleotide may be transiently or stably expressed in the cell population before the biologic of interest is expressed in the cell population. In yet another embodiment, the polynucleotide may be transiently or stably expressed in the cell population after the biologic of interest is expressed in the cell population. In another embodiment, the polynucleotide may be transiently or stably expressed in the cell population at substantially the same time as the biologic of interest is expressed in the cell population.
  • the amount of aspartate in the cell population is increased by at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68% or at least 70% or more when compared to a cell population that does not express the polynucleotide.
  • an increase in the amount of aspartate is to be understood to be an increase in the cellular uptake of aspartate from the cell culture medium. In another embodiment, an increase in the amount of aspartate is to be understood to be an increase in the intracellular synthesis of aspartate.
  • the production of lactate in the cell population is decreased by at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, or at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68% or at least 70% or more when compared to a cell population that does not express the polynucleotide.
  • a decrease in the production of lactate is to be understood to be a decrease in the secretion of lactate into the cell culture medium in which the cell population is contained.
  • a decrease in the production of lactate is to be understood to be a decrease in the intracellular synthesis of lactate.
  • a decrease in the production of lactate is to be understood as an increase in uptake of lactate from the cell culture medium in which the cell population is contained.
  • the increase in uptake from the cell culture medium may reflect a cessation of production of lactate by the cell population and instead the cell population relying upon the lactate in the cell culture medium as a carbon source.
  • the production of ammonia in the cell population is decreased by at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, or at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68% or at least 70% or more when compared to a cell population that does not express the polynucleotide.
  • a decrease in the production of ammonia is to be understood to be
  • the growth rate of the cell population is increased by at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, or at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74% at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%
  • the yield of the biologic produced by the cell population is increased by at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, or at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74% at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%,
  • the density of the cell population when any polynucleotide described herein is introduced in to the cell population is about 10 3 cells/ml, about 10 4 cells/ml, about 10 4 cells/ml, about 10 5 cells/ml or about 10 6 cells/ml or more.
  • a cell population comprising a vector, polynucleotide or polypeptide described herein.
  • the polynucleotide is contained within a vector and/or is operably linked to one or more regulatory elements to facilitate expression.
  • the polynucleotide may be operably linked to a promoter suitable for use in the cell of interest.
  • the polynucleotide comprises a sequence that functions as a reporter.
  • the present invention also relates to a composition of cells when prepared by any method of the invention, wherein greater than 20% of the cells express a polynucleotide described herein.
  • the composition includes greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98 or 99% of cells that express the polynucleotide described herein.
  • any polynucleotide described herein for use in any method described herein for: reducing the production of lactate in a cell population; producing a cell population with a reduced production of lactate; preparing a cell population for the production of a biologic of interest; production of a biologic of interest in a cell population.
  • the present invention provides a biologic obtained or obtainable by a method of the invention as described herein.
  • Figure 1 shows the relevant biochemical pathways and enzymes involved in uptake of aspartate and de novo synthesis of aspartate in mammalian cells.
  • Lactate is one of the main waste products accumulated during mammalian cell culture, and it has been shown to inhibit cell growth and protein production.
  • the production of excessive lactate by mammalian cell cultures has been a long-standing problem that not only affects the yield but also the quality of biologies or biopharmaceutical products.
  • the excessive lactate production causes a decrease in pH as lactate is acidic and leads to the addition of alkali in pH-controlled cell culture processes.
  • the addition of an alkali in turn leads to an increase in osmolarity of the cell culture medium.
  • Fluctuations in pH and increases in osmolarity are both known to adversely affect the yield and quality of secreted products. For instance, increases to osmolality can lead to cell growth inhibition and decreased antibody yield.
  • the inventors herein sought to generate improved cell culture methods that can be used to produce improved quality and yield of biologies such as proteins, vaccines, genes, cells or tissues. This has been achieved by increasing levels of intracellular aspartate which surprisingly results in reduced production of lactate by the cells. As a result of reduced production of lactate, pH fluctuations are reduced and the subsequent increase in osmolarity of the cell culture medium is avoided. Thus the adverse affects of pH fluctuations and high osmolality on the yield and quality a biologic that is intended to be produced by the cell culture method are avoided. The inventors therefore provide robust and efficient mammalian cell culture process that can generate high yields of any biologic of interest.
  • biological refers to any therapeutic product that is obtained from living organisms including the cell population described herein and includes proteins, vaccines, genes, cells, tissues, blood or blood components and sub-categories thereof known to a skilled person in the art.
  • cell population refers to a biological cell derived from any living organism which is maintained or grown under an artificial, controlled environment.
  • mammalian cell culture refers to a biological cell derived from any mammal which is maintained or grown under an artificial, controlled environment.
  • derivative of mammalian cell lines refers to a cell line obtained from the original cell line by knockout, addition, deletion or other modification of one or more native or non-native genes.
  • protein shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex).
  • the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond.
  • non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.
  • polypeptide or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.
  • antibody includes a protein capable of specifically binding to one or a few closely related antigens by virtue of an antigen binding domain contained within a Fv.
  • This term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, de-immunized antibodies, synhumanized antibodies, half-antibodies, bispecific antibodies).
  • An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallizable (Fc).
  • Exemplary forms of antibodies comprise a four-chain structure as their basic unit.
  • Full-length antibodies comprise two heavy chains ( ⁇ 50 to 70 kD) covalently linked and two light chains ( ⁇ 23 kDa each).
  • a light chain generally comprises a variable region (if present) and a constant domain and in mammals is either a K light chain or a A light chain.
  • a heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s).
  • Heavy chains of mammals are of one of the following types a, 6, £, y, or p.
  • Each light chain is also covalently linked to one of the heavy chains.
  • the two heavy chains and the heavy and light chains are held together by inter-chain disulfide bonds and by non-covalent interactions.
  • the number of inter-chain disulfide bonds can vary among different types of antibodies.
  • Each chain has an N-terminal variable region (VH or VL wherein each are -110 amino acids in length) and one or more constant domains at the C- terminus.
  • the constant domain of the light chain (CL which is -110 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain (CH1 which is 330 to 440 amino acids in length).
  • the light chain variable region is aligned with the variable region of the heavy chain.
  • the antibody heavy chain can comprise 2 or more additional CH domains (such as, CH2, CH3 and the like) and can comprise a hinge region between the CH1 and CH2 constant domains.
  • Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass.
  • batch process generally refers to a method of culturing cells whereby nutrients are fed in a base medium to cells at the beginning of the culture in the culture vessel.
  • fed-batch process generally refers to a method of culturing cells whereby one or more nutrients are fed (supplied) to the culture vessel during culturing.
  • a culture supported by a base medium may be fed a medium that is added to prevent nutrient depletion.
  • high-density culture generally refers to a process whereby a high cell density is reached in the culture vessel.
  • the controlled addition of nutrients or changes in physical conditions such as temperature can be implemented to affect the growth rate of the culture.
  • constantly-fed batch process generally refers to a method of culturing cells whereby the feed rate of a growth-limiting substrate is constant, i.e. the feed rate is invariant during the culture.
  • exponential-fed-batch process generally refers to a method of culturing cells whereby the required feed rate (volumetric or mass) must be increased exponentially with time.
  • continuous process generally refers to a method of culturing cells whereby the cells and/or the cell culture medium are continuously supplied to and removed from the culture vessel.
  • perfusion process generally refers to a continuous process whereby fresh medium is continuously fed, spent media is continuously removed while the cells are retained inside the culture vessel. Further, the biologic of interest may be harvested continuously or at intervals from the cell culture vessel.
  • growth rate of a cell population generally refers to increases in cell biomass.
  • the invention provides for methods that increase the levels of aspartate within the cell which the inventors have surprisingly found has an effect on reducing production of lactate by the cell.
  • the invention contemplates any means for increasing aspartate levels within a cell, which may include means to increase aspartate uptake into a cell and to increase intracellular synthesis of aspartate.
  • Aspartate is an amino acid that is required for protein synthesis, nucleotide synthesis and the malate-aspartate shuttle.
  • a common route of biosynthesis is through the uptake of glutamine and conversion to aspartate via the tricarboxylic acid (TCA) cycle in the mitochondria. Subsequently, the aspartate is transported to the cytosol for use in synthesis of proteins and nucleotides.
  • TCA tricarboxylic acid
  • Another route of synthesis is via the uptake and conversion of asparagine to aspartate in the cytosol.
  • a third route is by reversing the flux through the aspartate-malate shuttle to produce aspartate from malate or oxaloacetate sourced from the mitochondria (Figure. 1 ).
  • Non-limiting means for increasing aspartate are described here and may include expression of one or more of the transporters involved in transporting aspartate across the cell membrane and expression of one or more of the enzymes involved in biosynthesis of aspartate such as Asparaginase (ASPG), Glutamate Oxaloacetate Transaminase 1 (GOT1) and Glutamate Oxaloacetate Transaminase 2 (GOT2).
  • Asparaginase is an enzyme that catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia.
  • Glutamic-oxaloacetic transaminase is a pyridoxal phosphatedependent enzyme which exists in cytoplasmic and mitochondrial forms, GOT1 and GOT2, respectively as outlined above. GOT plays a role in amino acid metabolism and the urea and tricarboxylic acid cycles.
  • increased aspartate may be achieved by increasing expression of: one or more transporters involved in aspartate transport across the cell membrane including SLC1A1 , SLC1A2, SLC1A3, SLC1A6, SLC1A7, SLC17A1 , SLC17A2, SLC17A3, SLC17A4, SLC17A6, SLC17A7, SLC17A8, SLC17A9, SLC38A7, SLC38A10 and SLC7A13; one or more of the enzymes involved in biosynthesis of aspartate such as Asparaginase (ASPG), Glutamate Oxaloacetate Transaminase 1 (GOT1 ) and Glutamate Oxaloacetate Transaminase 2 (GOT2); a combination of two or more of the above.
  • Asparaginase Asparaginase
  • GAT1 Glutamate Oxaloacetate Transaminase 1
  • Glutamate Oxaloacetate Transaminase 2 Glutamate Oxaloacetate
  • the preferred method for increasing the amount of intracellular aspartate is to increase the uptake of aspartate by the cells from the culture medium.
  • transporters that facilitate the uptake of aspartate from extracellular medium.
  • Most of the transporters are excitatory amino acid transporters (EAATs) that can transport excitatory amino acids including aspartate and glutamate.
  • EAATs are sodium dependent transporters that are driven by sodium electrochemical gradient across the cell membrane.
  • Another major family of transporters that may play a role in transporting aspartate are the vesicular glutamate transporters.
  • the invention contemplates expressing one or more genes that encode transporters of aspartate across the cell membrane.
  • Non limiting examples include SLC1A1 (NCBI Gene ID: 100760895), SLC1A2 (NCBI Gene ID: 100752285), SLC1A3 (NCBI Gene ID: 100689464; SEQ ID NO:1), SLC1A6 (NCBI Gene ID: 100770833) and SLC1A7 (NCBI Gene ID: 100767052)
  • SEQ ID NO: 1 nucleotide sequence of SLC1A3:
  • SLC17A1 NCBI Gene ID: 100755850
  • SLC17A2 NCBI Gene ID: 100755263
  • SLC17A3 (NCBI Gene ID: 100755554)
  • SLC17A4 (NCBI Gene ID: 100756142)
  • SLC17A6 NCBI Gene ID: 100773912
  • SLC17A7 NCBI Gene ID: 100756172
  • SLC17A8 (NCBI Gene ID: 100763319), SLC17A9 (NCBI Gene ID: 100758178), SLC38A7 (NCBI Gene ID: 100762550), SLC38A10 (NCBI Gene ID: 100763275), SLC7A13 (NCBI Gene ID: 100758319).
  • SLC1 A1 refers to a gene also known as Solute Carrier Family 1 Member 1 , Sodium-Dependent Glutamate/Aspartate Transporter 3, Excitatory Amino Acid Transporter 3, EAAT3, Excitatory Amino Acid Carrier 1 or EAAC1 .
  • SLC1 A2 refers to a gene also known as Solute Carrier Family 1 Member 2, Sodium-Dependent Glutamate/Aspartate Transporter 2, Excitatory Amino Acid Transporter 2, Glutamate/Aspartate Transporter II or EAAT2.
  • SLC1A3 refers to a gene also known as solute carrier family 1 member 3, Sodium-Dependent Glutamate/Aspartate Transporter 1 , Excitatory Amino Acid Transporter 1 , EAAT1 , GLAST, GLAST-1 or GLAST1 .
  • SLC1 A6 refers to a gene also known as Solute Carrier Family 1 Member 6, Sodium-Dependent Glutamate/Aspartate Transporter, Excitatory Amino Acid Transporter 4 or EAAT4.
  • SLC1A7 refers to a gene also known as Solute Carrier Family 1 Member 7, Excitatory Amino Acid Transporter 5, EAAT5 or AAAT.
  • SLC17A1 refers to a gene also known as Solute Carrier Family 17 Member 1.
  • SLC17A2 refers to a gene also known as Solute Carrier Family 17 Member 2.
  • SLC17A3 refers to a gene also known as Solute Carrier Family 17 Member 3.
  • SLC17A4 refers to a gene also known as Solute Carrier Family 17 Member 4.
  • SLC17A6 refers to a gene also known as Solute Carrier Family 17 Member 6.
  • SLC17A7 refers to a gene also known as Solute Carrier Family 17 Member 7.
  • SLC17A8 refers to a gene also known as Solute Carrier Family 17 Member 8.
  • SLC17A9 refers to a gene also known as Solute Carrier Family 17 Member 9.
  • SLC38A7 refers to a gene also known as Solute Carrier Family 38 Member 7 or SNAT7.
  • SLC38A10 refers to a gene also known as Solute Carrier Family 38 Member 10.
  • SLC7A13 refers to a gene also known as Solute Carrier Family 7 Member 13, AGT-1 or XAT2.
  • the invention includes expressing one or more genes involved in the synthesis of aspartate within a cell.
  • Non limiting examples include ASPG asparaginase according to Gene ID: 100750655, GOT1 according to Gene ID: 14718 (SEQ ID NO: 2) and GOT2 according to Gene ID: 2806 (SEQ ID NO: 3).
  • SEQ ID NO: 2 nucleotide sequence of GOT 1 :
  • ASPG refers to a gene also known as Asparaginase, 60 KDa Lysophospholipase or C14orf76.
  • GOT1 refers to a gene also known as Glutamic-Oxaloacetic Transaminase 1 , Glutamate Oxaloacetate Transaminase 1 , Aspartate Aminotransferase Cytoplasmic, Aspartate Aminotransferase 1 , Aspartate Transaminase 1 or Transaminase A.
  • G0T2 refers to a gene also known as Glutamic-Oxaloacetic Transaminase 2, Glutamate Oxaloacetate Transaminase 2, Glutamic-Oxaloacetic Transaminase 2 Mitochondrial, Aspartate Aminotransferase Mitochondrial, Aspartate Aminotransferase 2, Aspartate Transaminase 2.
  • polynucleotides or nucleic acids or fragments thereof provide for increased amount of intracellular aspartate.
  • the polynucleotide may comprise a sequence of DNA or RNA, including one having an open reading frame that encodes a polypeptide and is capable, under appropriate conditions, of being expressed as a polypeptide for the purpose of increasing the amount of aspartate in a cell population. Accordingly, expressing a polynucleotide described herein in a cell population refers to expressing the protein or polypeptide encoded by the polynucleotide, which ultimately leads to an increase in the amount of intracellular aspartate.
  • nucleic acid also encompasses genomic DNA, cDNA, mRNA, splice variants, antisense RNA, RNAi, DNA comprising one or more single-nucleotide polymorphisms (SNPs), and vectors comprising the subject nucleic acid sequences. Also encompassed in this term are nucleic acids that are homologous or substantially similar or identical to the nucleic acids encoding a given protein. Thus, the subject invention provides genes encoding a subject protein, and homologs thereof.
  • Polynucleotides or nucleic acids described herein refer to polymeric forms of nucleotides of any length.
  • the polynucleotides can contain deoxyribonucleotides, ribonucleotides, and/or their analogs or derivatives.
  • nucleic acids can be naturally occurring DNA or RNA, or can be synthetic analogs, as known in the art.
  • Polynucleotides also encompass genomic DNA, genes, gene fragments, exons, introns, regulatory sequences, or regulatory elements, such as promoters, enhancers, initiation and termination regions, other control regions, expression regulatory factors, and expression controls; DNA comprising one or more single-nucleotide polymorphisms (SNPs), allelic variants, isolated DNA of any sequence, and cDNA; mRNA, tRNA, rRNA, ribozymes, splice variants, antisense RNA, antisense conjugates, RNAi, and isolated RNA of any sequence; recombinant polynucleotides, heterologous polynucleotides, branched polynucleotides, labelled polynucleotides, hybrid DNA/RNA, polynucleotide constructs, vectors comprising the subject nucleic acids, nucleic acid probes, primers, and primer pairs.
  • SNPs single-nucleotide polymorphisms
  • Polynucleotides encompass modified nucleic acid molecules, with alterations in the backbone, sugars, or heterocyclic bases, such as methylated nucleic acid molecules, peptide nucleic acids, and nucleic acid molecule analogs, which may be suitable as, for example, probes if they demonstrate superior stability and/or binding affinity under assay conditions. They also encompass single-stranded, double-stranded, and triple helical molecules that are either DNA, RNA, or hybrid DNA/RNA and that may encode a full- length gene or a biologically active fragment thereof.
  • Polynucleotides may include single nucleotide polymorphisms. Single nucleotide polymorphisms (SNPs) occur frequently in eukaryotic genomes. The nucleotide sequence determined from one individual of a species may differ from other allelic forms present within the population. Thus, in some embodiments, a subject polynucleotide encodes variant polypeptides that include insertions, deletions, or substitutions compared with the polypeptides described herein. Conservative amino acid substitutions include serine/threonine, valine/leucine/isoleucine, asparagine/histidine/glutamine, glutamic acid/aspartic acid, etc.
  • Nucleic acids encoding the proteins and polypeptides of the subject invention may be cDNA or genomic DNA or a fragment thereof.
  • the term "gene” shall be intended to mean the open reading frame encoding specific proteins and polypeptides of the subject invention, and introns, as well as adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression, up to about 20 kb beyond the coding region, but possibly further in either direction.
  • the gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into a host genome.
  • the subject polynucleotides are isolated and obtained in substantial purity, generally as other than an intact chromosome.
  • the DNA will be obtained substantially free of other nucleic acid sequences that do not include a sequence or fragment thereof of the subject genes, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant," i.e., flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.
  • a polynucleotide encoding a polypeptide is operably linked to a heterologous nucleic acid molecule, which can be a functional nucleic acid molecule or can act as a tag to identify the presence of the polynucleotide.
  • a heterologous nucleic acid molecule can comprise or encode a transcriptional regulatory element (e.g., a promoter, an enhancer, or a silencer), a translational regulatory element (e.g., a Kozak consensus sequence, a start codon, a ribosome binding sequence, a stop codon, or a poly-adenylation signal), or a combination of transcriptional and/or translational regulatory elements.
  • a heterologous nucleic acid molecule also can encode a peptide, which can function as a tag and/or a detectable label, or can function as a cellular localization domain, which facilitates transport of the expressed polypeptide into or out of a particular cellular compartment.
  • any polynucleotide disclosed herein can be contained in a vector, which can be a cloning vector or an expression vector, Suitable vectors may include prokaryotic vectors, eukaryotic vectors, or shuttle vectors (e.g., a vector that can be passaged in both prokaryotic and eukaryotic cells, or in different types of eukaryotic cells).
  • the vector is an expression vector, which contains one or more regulatory elements that facilitate expression of the polypeptide in the cell population.
  • host cells which contain a polynucleotide encoding a polypeptide of the invention, wherein the polynucleotide can, but need not, be contained in a vector (e.g., an expression vector).
  • the polynucleotide encoding the polypeptide is stably integrated into the genome of the host cell.
  • An expression vector (or the polynucleotide) generally contains or encodes a promoter sequence, which can provide constitutive or, if desired, inducible or tissue specific or developmental stage specific expression of the encoding polynucleotide, a poly-A recognition sequence, and a ribosome recognition site or internal ribosome entry site, or other regulatory elements such as an enhancer, which can be cell specific.
  • the vector may also contain elements required for replication in a prokaryotic (e.g., bacterial) or eukaryotic (e.g., insect, yeast (e.g., Pichia) and/or mammalian) host or both, as desired.
  • Such vectors which include plasmid vectors and viral vectors such as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, semliki forest virus and adeno-associated virus vectors, are well known and can be purchased from a commercial source (Promega, Madison Wis.; Stratagene, La Jolla Calif.; GIBCO/BRL, Gaithersburg Md.; Invitrogen Corp., Carlsbad Calif.) or can be constructed by one skilled in the art (see, for example, Meth. Enzymol., Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990); Jolly, Cane. Gene Ther.
  • An inducible promoter such as the tetracycline (tet) promoter can be particularly useful for driving expression of a polynucleotide encoding any polypeptide described herein.
  • tetracycline or a tetracycline analogue
  • expression of the encoded polypeptide is induced.
  • Viral expression vectors can be particularly useful for introducing a polynucleotide into a cell.
  • Viral vectors provide the advantage that they can infect host cells with relatively high efficiency and can infect specific cell types.
  • a polynucleotide encoding a polypeptide of the invention can be cloned into a baculovirus vector, which then can be used to infect an insect host cell, thereby providing a means to produce large amounts of the encoded polypeptide.
  • the viral vector also can be derived from a virus that infects cells of an organism of interest, for example, vertebrate host cells such as mammalian, host cells.
  • Viral vectors can be particularly useful for introducing a polynucleotide useful in performing a method of the invention into a target cell.
  • Viral vectors have been developed for use in particular host systems, particularly mammalian systems and include, for example, retroviral vectors, lentivirus vectors, adenovirus vectors, adeno- associated virus vectors, herpesvirus vectors, vaccinia virus vectors, and the like (see Miller and Rosman, BioTechniques 7:980-990, 1992; Anderson et al., Nature 392:25-30 Suppl., 1998; Verma and Somia, Nature 389:239-242, 1997; Wilson, New Engl. J. Med.
  • a polynucleotide encoding a polypeptide, which can be contained in a vector, can be introduced into a cell by any of a variety of methods known in the art (Sambrook et al., “Molecular Cloning: A laboratory manual” (Cold Spring Harbor Laboratory Press 1989); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1987, and supplements through 1995), each of which is incorporated herein by reference).
  • Such methods include, for example, transfection, lipofection, microinjection, electroporation and, with viral vectors, infection; and can include the use of liposomes, microemulsions or the like, which can facilitate introduction of the polynucleotide into the cell and can protect the polynucleotide from degradation prior to its introduction into the cell.
  • liposomes, microemulsions or the like which can facilitate introduction of the polynucleotide into the cell and can protect the polynucleotide from degradation prior to its introduction into the cell.
  • the selection of a particular method will depend, for example, on the cell into which the polynucleotide is to be introduced.
  • sequences can be modified to generate functional derivative so long as the polypeptide retains its ability to increase the amount of aspartate within a cell.
  • Other sequences can readily be obtained by one of skill in the art, for example, in from the publicly available GenBank database.
  • Functional derivatives to be used in accordance with the invention may have at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74% at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or is 100% identical to any polynucleotide described herein.
  • the invention contemplates use of a polynucleotide that is at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or is 100% identical to the polynucleotide according to SEQ ID NO: 1 , 2 or 3.
  • Such functional derivatives retain the ability to increase the amount of aspartate within a cell.
  • genetic control elements may be employed to regulate gene expression of the polypeptide or protein. Such genetic control elements should be selected to be active in the relevant host cell. Control elements may be constitutively active or may be inducible under defined circumstances. Inducible control elements are particularly useful when the expressed protein is toxic or has otherwise deleterious effects on cell growth and/or viability. In such instances, regulating expression of the polypeptide or protein through inducible control elements may improve cell viability, cell density, and /or total yield of the expressed polypeptide or protein. A large number of control elements useful in the practice of the present invention are known and available in the art.
  • constitutive mammalian promoters that may be used in accordance with the present invention include, but are not limited to, the hypoxanthine phosphoribosyl transferase (HPTR) promoter, the adenosine deaminase promoter, the pyruvate kinase promoter, the beta-actin promoter as well as other constitutive promoters known to those of ordinary skill in the art.
  • HPTR hypoxanthine phosphoribosyl transferase
  • adenosine deaminase promoter the pyruvate kinase promoter
  • beta-actin promoter as well as other constitutive promoters known to those of ordinary skill in the art.
  • viral promoters that have been shown to drive constitutive expression of coding sequences in eukaryotic cells include, for example, simian virus promoters, herpes simplex virus promoters, papilloma virus promoters, adenovirus promoters, human immunodeficiency virus (HIV) promoters, Rous sarcoma virus promoters, cytomegalovirus (CMV) promoters, the long terminal repeats (LTRs) of Moloney murine leukemia virus and other retroviruses, the thymidine kinase promoter of herpes simplex virus as well as other viral promoters known to those of ordinary skill in the art.
  • simian virus promoters herpes simplex virus promoters
  • papilloma virus promoters papilloma virus promoters
  • adenovirus promoters include human immunodeficiency virus (HIV) promoters, Rous sarcoma virus promoters,
  • Inducible promoters drive expression of operably linked coding sequences in the presence of an inducing agent and may also be used in accordance with the present invention.
  • the metallothionein promoter induces transcription of downstream coding sequences in the presence of certain metal ions.
  • Other inducible promoters will be recognized by and/or known to those of ordinary skill in the art.
  • the gene expression sequence will also include 5' non-transcribing and 5' non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • Enhancer elements can optionally be used to increase expression levels of the polypeptides or proteins to be expressed. Examples of enhancer elements that have been shown to function in mammalian cells include the SV40 early gene enhancer, as described in Dijkema et al., EMBO J. (1985) 4: 761 and the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus (RSV), as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and human cytomegalovirus, as described in Boshart et al., Cell (1985) 41 :521.
  • LTR long terminal repeat
  • RSV Rous Sarcoma Virus
  • nucleic acids suitable for introducing into host cells nucleic acids sufficient to achieve expression of the polypeptides or proteins of interest are well known in the art. See, for example, Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281 :40-46 (1979); Levinson et al.; EP 117,060; and EP 117,058, all incorporated herein by reference.
  • mammalian cells preferred methods of transformation include the calcium phosphate precipitation method of Graham and van der Erb, Virology, 52:456-457 (1978) or the lipofectamineTM (Gibco BRL) Method of Hawley-Nelson, Focus 15:73 (1193).
  • General aspects of mammalian cell host system transformations have been described by Axel in U.S. Pat. No. 4,399,216 issued Aug. 16, 1983.
  • Keown et al. Methods in Enzymology (1989)
  • Keown et al. Methods in Enzymology, 185:527-537 (1990)
  • Mansour et al. Nature, 336:348-352 (1988).
  • Non-limiting representative examples of suitable vectors for expression of polypeptides or proteins in mammalian cells include pCDNA 1 ; pCD, see Okayama, et al. (1985) Mol. Cell Biol. 5:1136-1142; pMCIneo Poly-A, see Thomas, et al. (1987) Cell 51 :503-512; and a baculovirus vector such as pAC 373 or pAC 610.
  • the polynucleotide is stably transfected into the host cell.
  • the present invention can be used with either transiently or stably transfected mammalian cells.
  • the biologies of interest that may be produced according to the methods described in accordance with the invention include proteins or polypeptides vaccines, genes, cells, or tissues.
  • the biologic may be an antibody, a receptor, a growth factor or signalling molecule, a G-protein coupled receptor (GPCR), a chimeric antigen receptor (CAR).
  • GPCR G-protein coupled receptor
  • CAR chimeric antigen receptor
  • the cell population may have a further polynucleotide introduced that encodes a biologic of interest as described herein.
  • polypeptide that is expressible in a host cell may be produced in accordance with the present invention.
  • the polypeptide may be expressed from a gene that is endogenous to the host cell, or from a gene that is introduced into the host cell through genetic engineering.
  • the polypeptide may be one that occurs in nature or may alternatively have a sequence that was engineered or selected by the hand of man.
  • An engineered polypeptide may be assembled from other polypeptide segments that individually occur in nature or may include one or more segments that are not naturally occurring.
  • Polypeptides that may desirably be expressed in accordance with the present invention will often be selected on the basis of an interesting biological or chemical activity.
  • the present invention may be employed to express any pharmaceutically or commercially relevant enzyme, receptor, antibody, hormone, regulatory factor, antigen, binding agent, etc.
  • Antibodies are proteins that have the ability to specifically bind a particular antigen. Any antibody that can be expressed in a host cell may be used in accordance with the present invention.
  • the antibody to be expressed is a monoclonal antibody.
  • the monoclonal antibody is a chimeric antibody.
  • a chimeric antibody contains amino acid fragments that are derived from more than one organism.
  • Chimeric antibody molecules can include, for example, an antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions.
  • a variety of approaches for making chimeric antibodies have been described. See e.g., Morrison et al., Proc. Natl. Acad Sci. U.S.A. 81 , 6851 (1985); Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S. Patent No.
  • the monoclonal antibody is a human antibody derived, e.g., through the use of ribosome-display or phage-display libraries (see, e.g., Winter et al., U.S. Patent No. 6,291 ,159 and Kawasaki, U.S. Patent No. 5,658,754) or the use of xenographic species in which the native antibody genes are inactivated and functionally replaced with human antibody genes, while leaving intact the other components of the native immune system (see, e.g., Kucherlapati et al., U.S. Patent No. 6,657,103).
  • the monoclonal antibody is a humanized antibody.
  • a humanized antibody is a chimeric antibody wherein the large majority of the amino acid residues are derived from human antibodies, thus minimizing any potential immune reaction when delivered to a human subject.
  • amino acid residues in the complementarity determining regions are replaced, at least in part, with residues from a non-human species that confer a desired antigen specificity or affinity.
  • Such altered immunoglobulin molecules can be made by any of several techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad Sci.
  • Humanized antibodies can be commercially produced by, for example, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain. For further reference, see Jones et al Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), all of which are incorporated herein by reference.
  • the monoclonal, chimeric, or humanized antibodies described above may contain amino acid residues that do not naturally occur in any antibody in any species in nature. These foreign residues can be utilized, for example, to confer novel or modified specificity, affinity or effector function on the monoclonal, chimeric or humanized antibody.
  • the antibodies described above may be conjugated to drugs for systemic pharmacotherapy, such as toxins, low-molecular-weight cytotoxic drugs biological response modifiers, and radionuclides.
  • antibodies include, but are not limited to, those that recognize any one or a combination of proteins including, but not limited to, the above-mentioned proteins and/or the following antigens: CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL- 1a, IL- 1 p, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-10, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-13 receptor, IL-18 receptor subunits, FGL2, PDGF-0 and analogs thereof (see US Patent Nos.
  • VEGF vascular endothelial growth factor
  • TGF TGF-p2, TGF-01
  • EGF receptor see US Patent No. 6,235,883
  • VEGF receptor hepatocyte growth factor
  • osteoprotegerin ligand interferon gamma
  • B lymphocyte stimulator BlyS, also known as BAFF, THANK, TALL-1 , and zTNF4; see Do and Chen-Kiang (2002), Cytokine Growth Factor Rev.
  • Receptors are typically trans-membrane glycoproteins that function by recognizing an extracellular signalling ligand. Receptors typically have a protein kinase domain in addition to the ligand recognizing domain, which initiates a signalling pathway by phosphorylating target intracellular molecules upon binding the ligand, leading to developmental or metabolic changes within the cell.
  • the receptors of interest are modified so as to remove the transmembrane and/or intracellular domain(s), in place of which there may optionally be attached an Ig domain.
  • receptors to be produced in accordance with the present invention are receptor tyrosine kinases (RTKs).
  • RTKs receptor tyrosine kinases
  • the RTK family includes receptors that are crucial for a variety of functions numerous cell types.
  • Non-limiting examples of RTKs include members of the fibroblast growth factor (FGF) receptor family, members of the epidermal growth factor receptor (EGF) family, platelet derived growth factor (PDGF) receptor, tyrosine kinase with immunoglobulin and EGF homology domains-l (TIE-1) and TIE-2 receptors and c-Met receptor.
  • FGF fibroblast growth factor
  • EGF epidermal growth factor receptor
  • PDGF platelet derived growth factor
  • TIE-1 tyrosine kinase with immunoglobulin and EGF homology domains-l
  • TIE-2 receptors c-Met receptor
  • RTK's include fetal liver kinase 1 (FLK-1) or vascular endothelial cell growth factor receptor 2 (VEGFR-2)), fins-like tyrosine kinase-l (Fit- 1 ), sometimes referred to as vascular endothelial cell growth factor receptor 1 (VEGFR-1), neuropilin-1 , endoglin, endosialin, Axl and tumor necrosis factor alpha and beta receptors Those of ordinary skill in the art will be aware of other receptors that can be produced in accordance with the present invention.
  • the invention can also be used to harvest recombinant fusion proteins comprising, for example, any of the above-mentioned proteins.
  • recombinant fusion proteins comprising one of the above-mentioned proteins plus a multimerization domain, such as a leucine zipper, a coiled coil, an Fc portion of an immunoglobulin, or a substantially similar protein, can be produced using the methods of the invention. See e.g. W094/10308; Lovejoy et al. (1993), Science 259: 1288-1293; Harbury et al. (1993), Science 262: 1401-05; Harbury et al. (1994), Nature 371 :80-83; Hakansson et al.
  • receptor fusions whereby only part of the endogenous receptor is produced (e.g. only all or part of the extracellular region) fused or linked to another protein (e.g. Fc of an antibody).
  • Fc a protein in which a portion of a receptor is fused to an Fc portion of an antibody
  • etanercept a p75 TNFR:Fc
  • CTLA4:Fc belatacept
  • growth factors are typically glycoproteins that are secreted by cells and bind to and activate receptors on other cells, initiating a metabolic or developmental change in the receptor cell.
  • Non-limiting examples of mammalian growth factors and other signalling molecules include cytokines; epidermal growth factor (EGF); platelet-derived growth factor (PDGF); fibroblast growth factors (FGFs) such as aFGF and bFGF; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta, including TGF-beta 1 , TGF- beta 2, TGF-beta 3, TGF-beta 4, or TGF-beta 5; insulin-like growth factor-l and -II (IGF-I and IGF-II); des(l-3) -IGF-I (brain IGF-1), insulin like growth factor binding proteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferonalpha, -beta, and - gamma; colony stimulating factors (CSFs
  • G-protein coupled receptors are proteins that have seven transmembrane domains. Upon binding of a ligand to a GPCR, a signal is transduced within the cell which results in a change in a biological or physiological property of the cell.
  • GPCRs are the components of a modular signalling system that connects the state of intracellular second messengers to extracellular inputs. These genes and gene-products are potential causative agents of disease.
  • G protein Following ligand binding to the GPCR, a conformational change is transmitted to the G protein, which causes the alpha-subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the Py-subunits.
  • the GTP bound form of the o- subunit typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cyclic AMP (e.g., by activation of adenylate cyclase), diacylglycerol or inositol phosphates.
  • second messengers such as cyclic AMP (e.g., by activation of adenylate cyclase), diacylglycerol or inositol phosphates.
  • cyclic AMP e.g., by activation of adenylate cyclase
  • diacylglycerol diacylglycerol
  • inositol phosphates inosi
  • GPCRs are a major target for drug action and development. A skilled person will understand that any component of the GPCR pathway, including those mentioned above, may be targeted to produce a biologic on interest.
  • the methods used to measure the yield of a biologic are specific to the type of biologic.
  • the titer of a monoclonal antibody secreted by CHO cells can be measured using enzyme-linked immunosorbent assay (ELISA).
  • Affinity chromatography such as the Protein A chromatography column or reversed phase-high performance liquid chromatography (RP-HPLC) are also commonly used to measure the titer of monoclonal antibodies.
  • the yield of the therapeutic protein is often called the cell specific productivity and is expressed as grams therapeutic/cell/day. If the cells themselves are the biologies, the yield maybe measured as cells/mL or g biomass/mL.
  • the quality attributes that are important for a biologic are specific to the biologic.
  • the quality attributes are chosen such they confirm the safety, purity, identity and potency of the biologic.
  • the commonly measured quality attributes of a monoclonal antibody secreted by CHO cells are glycosylation, charge variants, sequence variants, aggregates and low molecular weight species.
  • the methods used to isolate and purify the biologic depend on the biologic and the quality attributes of interest. If the biologic is secreted by the cells into the cell culture broth or medium, the biologic is separated from the broth or medium after harvest using methods such as filtration . The filtrate is usually further purified to obtain the biologic. Chromatographic techniques are widely used for isolation and purification of the biologic. The chromatographic techniques may purify the biologic based on differences in size, mass, charge, affinity or hydrophobicity/hydrophilicity. Two or more orthogonal chromatography techniques may be used to obtain the biologic of the required quality.
  • any mammalian cell or cell type susceptible to cell culture may be utilized in accordance with the present invention.
  • mammalian cells that may be used in accordance with the present invention include BALB/c mouse myeloma line (NSO/I, ECACC No: 85110503); human retinoblasts (PER-C6 (CruCell, Leiden, The Netherlands)); monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (eg 293 or 293T); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (CHO); mouse sertoli cells (TM4); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
  • hybridoma cell lines any number of commercially and non-commercially available hybridoma cell lines may be utilized in accordance with the present invention.
  • hybridoma cell lines might have different nutrition requirements and/or might require different culture conditions for optimal growth and polypeptide or protein expression and will be able to modify conditions as needed.
  • cells will be selected or engineered to produce high levels of protein or polypeptide.
  • cells are genetically engineered to produce high levels of protein, for example by introduction of a gene encoding the protein or polypeptide of interest and/or by introduction of control elements that regulate expression of the gene (whether endogenous or introduced) encoding the polypeptide of interest.
  • Certain polypeptides may have detrimental effects on cell growth, cell viability or some other characteristic of the cells that ultimately limits production of the polypeptide or protein of interest in some way. Even amongst a population of cells of one particular type engineered to express a specific polypeptide, variability within the cellular population exists such that certain individual cells will grow better and/or produce more polypeptide of interest.
  • the cell line is empirically selected for robust growth under the particular conditions chosen for culturing the cells.
  • individual cells engineered to express a particular polypeptide are chosen for large-scale production based on cell growth, final cell density, percent cell viability, titer of the expressed polypeptide, quality attributes of the express protein or any combination of these or any other conditions deemed important by a skilled person.
  • cell population and “cell culture” are used interchangeably.
  • Cell culture process Typical procedures for culturing cells include batch processes, fed-batch processes and continuous processes.
  • Batch culture processes traditionally comprise inoculating a culture vessel with a seed culture of a particular cell density, growing the cells under conditions conducive to cell growth and viability, harvesting the culture when the cells reach a specified cell density, and purifying the expressed polypeptide.
  • Fed- batch culture processes include an additional step or steps of supplementing the batch culture with nutrients and other components that are consumed during the growth of the cells as bolus or continuous feeds.
  • Continuous processes comprise continuous addition and removal of cells and/or culture medium to the culture vessel. In certain preferred embodiments, cells are retained in the culture vessel while fresh culture medium is continuously added and spent medium is continuously removed from the culture vessel.
  • a persistent and unsolved problem with culturing cells by any of the processes mentioned above is the production of metabolic waste products, which have detrimental effects on cell growth, viability, and production and quality of expressed polypeptides.
  • lactate and ammonium Two metabolic waste products that have particularly detrimental effects are lactate and ammonium, which are produced as a result of glucose and glutamine metabolism, respectively.
  • ammonium also accumulates in cell cultures as a result of non-metabolic degradation over time.
  • the present invention can be employed in any system in which cells are cultured including, but not limited to, batch, fed-batch and continuous processes. In certain preferred embodiments of the present invention, the cells are grown in fed-batch.
  • Traditional media formulations including commercially available media such as Ham's FIO (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) may be used in accordance with the methods described herein.
  • Proprietary media formulations that are used inhouse or commercially available may also be used in accordance with the methods described herein.
  • Non-limiting examples of proprietary commercial media include HyClone CCM5 CHO media (Cytiva), EX-CELL® CD CHO (Sigma), CD OptiCHOTM medium (ThermoFisher), CELLiSTTM media (Ajinomoto Inc.).
  • serum-free, animal-derived component-free, chemically defined media are used.
  • any of these media formulations disclosed in the present invention may optionally be supplemented as necessary with hormones and/or other growth factors, particular ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), amino acids, lipids, protein hydrolysates, or glucose or other energy source.
  • ions such as sodium, chloride, calcium, magnesium, and phosphate
  • buffers such as sodium, chloride, calcium, magnesium, and phosphate
  • vitamins nucleosides or nucleotides
  • trace elements inorganic compounds usually present at very low final concentrations
  • amino acids amino acids
  • lipids protein hydrolysates
  • glucose or other energy source glucose or other energy source.
  • chemical inductants such as hexamethylene- bis(acetamide) (“HMBA") and sodium butyrate (“NaB”
  • HMBA hexamethylene- bis(acetamide)
  • a nucleic acid sufficient to achieve expression may be introduced into the host cell line by any number of well-known techniques.
  • cells are screened to determine which of the host cells have actually taken up the vector and express the polypeptide or protein of interest.
  • the cell is propagated in culture by any of the variety of methods well known to one of ordinary skill in the art.
  • the cell expressing the polypeptide or protein of interest is typically propagated by growing it at a temperature and in a medium that is conducive to the survival, growth and viability of the cell.
  • the initial culture vessel can be of any size, but is often smaller than the culture vessel of the production bioreactor used in the final production of the polypeptide or protein of interest, and frequently cells are passaged several times in bioreactors of increasing volume prior to seeding the production bioreactor.
  • the cell culture can be agitated or shaken to increase oxygenation of the medium and dispersion of nutrients to the cells. Alternatively or additionally, special sparging devices that are well known in the art can be used to increase and control oxygenation of the culture.
  • the starting cell density in the production bioreactor can be chosen by one of ordinary skill in the art. In accordance with the present invention, the starting cell density in the production bioreactor can be as low as a single cell per culture volume. In preferred embodiments of the present invention, starting cell densities in the production bioreactor can range from about 2 x 10 2 viable cells per mL to about 2 x 10 3 , 2 x 10 4 , 2 x 10 5 , 2 x 10 6 , 5 x 10 6 , 1 x 10 7 or 1 x 10 8 viable cells per mL and higher.
  • Initial and intermediate cell cultures may be grown to any desired density before seeding the next intermediate or final production bioreactor. It is preferred that most of the cells remain alive prior to seeding, although total or near total viability is not required.
  • the cells may be removed from the supernatant, for example, by low-speed centrifugation. It may also be desirable to wash the removed cells with a medium before seeding the next bioreactor to remove any unwanted metabolic waste products or medium components.
  • the medium may be the medium in which the cells were previously grown or it may be a different medium or a washing solution selected by the practitioner of the present invention.
  • the cells may then be diluted to an appropriate density for seeding the production bioreactor.
  • the cells are diluted into the same medium that will be used in the production bioreactor.
  • the cells can be diluted into another medium or solution, depending on the needs and desires of the practitioner of the present invention or to accommodate particular requirements of the cells themselves, for example, if they are to be stored for a short period of time prior to seeding the production bioreactor.
  • the cell culture is maintained in the initial growth phase under conditions conducive to the survival, growth and viability of the cell culture.
  • the precise conditions will vary depending on the cell type, the organism from which the cell was derived, and the nature and character of the expressed polypeptide or protein.
  • the production bioreactor can be any volume that is appropriate for large-scale production of the biologic of interest.
  • the volume of the production bioreactor is at least 500 liters.
  • the volume of the production bioreactor is 1000, 2500, 5000, 8000, 10,000, 12,000 liters or more, or any volume in between.
  • the production bioreactor may be constructed of any material that is conducive to cell growth and viability that does not interfere with expression or stability of the produced polypeptide or protein.
  • the temperature of the cell culture in the initial growth phase will be selected based primarily on the range of temperatures at which the cell culture remains viable.
  • CHO cells grow well at 37°C.
  • most mammalian cells grow well within a range of about 25°C to 42°C.
  • mammalian cells grow well within the range of about 35°C to 40°C.
  • Those of ordinary skill in the art will be able to select appropriate temperature or temperatures in which to grow cells, depending on the needs of the cells and the production requirements of the practitioner.
  • the temperature of the initial growth phase is maintained at a single, constant temperature. In another embodiment, the temperature of the initial growth phase is maintained within a range of temperatures. For example, the temperature may be steadily increased or decreased during the initial growth phase. Alternatively, the temperature may be increased or decreased by discrete amounts at various times during the initial growth phase.
  • One of ordinary skill in the art will be able to determine whether a single or multiple temperatures should be used, and whether the temperature should be adjusted steadily or by discrete amounts.
  • the cells may be grown during the initial growth phase for a greater or lesser amount of time, depending on the needs and the requirement of the cells themselves.
  • the cells are grown for a period of time sufficient to achieve a viable cell density that is a given percentage of the maximal viable cell density that the cells would eventually reach if allowed to grow undisturbed.
  • the cells may be grown for a period of time sufficient to achieve a desired viable cell density of 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of maximal viable cell density.
  • the cells are allowed to grow for a defined period of time. For example, depending on the starting concentration of the cell culture, the temperature at which the cells are grown, and the intrinsic growth rate of the cells, the cells may be grown for O, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days. In some cases, the cells may be allowed to grow for a month or more. The practitioner will be able to choose the duration of the initial growth phase depending on biologic production requirements and the needs of the cells themselves.
  • the cell culture may be agitated or shaken during the initial culture phase in order to increase oxygenation and dispersion of nutrients to the cells.
  • it can be beneficial to control or regulate certain internal conditions of the bioreactor during the initial growth phase, including but not limited to pH, temperature, oxygenation, etc.
  • pH can be controlled by supplying an appropriate amount of acid or base and oxygenation can be controlled with sparging devices that are well known in the art.
  • the cells may be maintained in the subsequent production phase until a desired cell density or production titer is reached.
  • the cells are maintained in the subsequent production phase until the titer to the recombinant polypeptide or protein reaches a maximum.
  • the culture may be harvested prior to this point, depending on the production requirement of the practitioner or the needs of the cells themselves.
  • the cells may be maintained for a period of time sufficient to achieve a viable cell density of 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of maximal viable cell density.
  • the viable cell density may be desirable to allow the viable cell density to reach a maximum, and then allow the viable cell density to decline to some level before harvesting the culture. In an extreme example, it may be desirable to allow the viable cell density to approach or reach zero before harvesting the culture.
  • the cell culture is maintained for a subsequent production phase under a second set of culture conditions conducive to the survival and viability of the cell culture and appropriate for expression of the desired polypeptide or protein at commercially adequate levels.
  • the cells are allowed to grow for a defined period of time during the subsequent production phase.
  • the cells may be grown for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days.
  • the cells may be allowed to grow for a month or more.
  • the practitioner of the present invention will be able to choose the duration of the subsequent production phase depending on polypeptide or protein production requirements and the needs of the cells themselves.
  • nutrients or other medium components observed to have been depleted during monitoring of the cell culture.
  • hormones and/or other growth factors particular ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), amino acids, lipids, or glucose or other energy source.
  • supplementary components may all be added to the cell culture at one time, or they may be provided to the cell culture in a series of additions.
  • the supplementary components are provided to the cell culture at multiple times in proportional amounts.
  • the cell culture is fed continually with these supplementary components.
  • the cell culture may be agitated or shaken during the subsequent production phase in order to increase oxygenation and dispersion of nutrients to the cells.
  • pH can be controlled by supplying an appropriate amount of acid or base and oxygenation can be controlled with sparging devices that are well known in the art.
  • the biologic of interest In general, it will typically be desirable to isolate and/or purify the biologic of interest according to the present invention.
  • the biologic of interest is secreted into the medium and thus cells and other solids may be removed, as by centrifugation or filtering for example, as a first step in the purification process.
  • This embodiment is particularly useful when used in accordance with the present invention, since the methods and compositions described herein result in increased cell viability. As a result, fewer cells die during the culture process, and fewer proteolytic enzymes are released into the medium which can potentially decrease the yield of the biologic of interest.
  • the biologic is removed continuously from the bioreactor while the cells are retained in the bioreactor till the end of the culture. In other embodiments, the biologic is harvested along with the cells at the end of the culture.
  • the biologic of interest is bound to the surface of the host cell.
  • the media is removed and the host cells expressing the biologic are lysed as a first step in the purification process. Lysis of mammalian host cells can be achieved by any number of means well known to those of ordinary skill in the art, including physical disruption by glass beads and exposure to high pH conditions.
  • Biologies such as polypeptides or proteins may be isolated and purified by standard methods including, but not limited to, chromatography (e.g., protein A chromatography, ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or differential solubility, ethanol precipitation or by any other available technique for the purification of proteins (See, e.g., Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, S.J. and Hames, B.D. (eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, M.P., Simon, M.L, Abelson, J.N.
  • chromatography e.g., protein A chromatography, ion exchange, affinity, size exclusion, and hydroxyapatite chromatography
  • gel filtration e.g., gel filtration, centrifugation, or differential solubility,
  • the protein may be isolated by binding it to an affinity column comprising antibodies that were raised against that protein and were affixed to a stationary support.
  • affinity tags such as an influenza coat sequence, poly-histidine, or glutathione-S-transferase can be attached to the protein by standard recombinant techniques to allow for easy purification by passage over the appropriate affinity column.
  • Protease inhibitors such as phenyl methyl sulfonyl fluoride (PMSF), leupeptin, pepstatin or aprotinin may be added at any or all stages in order to reduce or eliminate degradation of the polypeptide or protein during the purification process. Protease inhibitors are particularly desired when cells must be lysed in order to isolate and purify the expressed polypeptide or protein.
  • PMSF phenyl methyl sulfonyl fluoride
  • leupeptin leupeptin
  • pepstatin or aprotinin
  • aprotinin may be added at any or all stages in order to reduce or eliminate degradation of the polypeptide or protein during the purification process.
  • Protease inhibitors are particularly desired when cells must be lysed in order to isolate and purify the expressed polypeptide or protein.
  • purification technique will vary depending on the character of the polypeptide or protein to be purified, the character of the cells from which the polypeptide or protein is expressed, and the composition
  • a CHO-K1 cell line stably expressing a recombinant IgG protein is transfected with an expression vector encoding the SLC1A3 gene (NCBI Gene ID: 100689464).
  • the green fluorescent protein (GFP) reporter gene is then co-expressed with SLC1A3 that includes an internal ribosome entry site (IRES) element under the control of the human cytomegalovirus (CMV) promoter.
  • the transfection is to be carried out using lipofectamine.
  • the IgG producing CHO-K1 cells are similarly transfected with expression vectors encoding the GFP reporter gene alone.
  • the transfected pools of cells are enriched by flow cytometry and further validated by Western blotting.
  • the SLC1A3 overexpressing and control CHO-K1 cells are cultured in fed-batch mode for 10 days in 1 L shake flasks (Corning, NY, USA) with CD FortiCHOTM medium (Thermo Fisher Scientific, Waltham, MA, USA) as the basal medium and CD EfficientFeedTM C AGTTM Nutrient Supplement (Thermo Fisher Scientific, Waltham, MA, USA) as the feed medium.
  • the feed medium is added to the shake flasks at 3% v/v on alternate days starting from day 3 of the culture.
  • the shake flasks are maintained in a humidified incubator at 37°C and 5% CO2 on orbital shakers at 160 rpm. The cultures are sampled every day.
  • the titer of the IgG is measured using Protein A HPLC.
  • the cell count, viability and cell diameter are measured using ViCell XR (Beckman Coulter, Brea, CA, USA).
  • the Cedex Bio Analyzer (Roche, Basel, Switzerland) is used to measure concentrations of glucose, glutamine, glutamate, lactate and ammonia in the cell culture media.
  • the IgG secreted by the cells is first separated from the cells and cell debris through filtration.
  • the IgG in the filtrate is captured using a Protein A chromatography column which binds the constant region of the monoclonal antibody.
  • an anion exchange chromatography step is used to bind host cell proteins, DNA and aggregates while the IgG flows through.
  • the last polishing step employs a cation exchange chromatography column to separate the charge variants of the IgG.
  • RM is the rate of uptake or secretion of the metabolite M
  • ACM is the change in concentration of metabolite M in the spent media over the time interval At
  • Xav is the average of the viable cell biomass over the time interval At.
  • the cell culture performance of the CHO-K1 cell lines overexpressing SLC1A3 is to be compared to the control.
  • Table 1 shows expected specific rates of growth, uptake or secretion by the cells in the exponential growth phase i.e. between day 4 to day 7.
  • the specific rates of the SLC1A3 overexpressing cells are normalized to those of the control cells. For example, if the specific uptake rate of aspartate by control cells is 2 mmol/cell/day and 2.4 mmol/cell/day by the SLC1 A3 overexpressing cells, the normalized specific rate is 1.2. In other words, the SLC1A3 overexpressing cells uptake 20% more aspartate than the control.
  • the specific aspartate uptake rate is expected to be 20% ⁇ 5% higher than the control.
  • the specific lactate secretion by SLC1A3 overexpressing cells is expected to be 50% ⁇ 5% lower than the control despite having a higher specific growth rate.
  • the peak concentration of lactate is 60% ⁇ 5% lower than the control.
  • the ammonia secretion is expected to be lower by 40% ⁇ 10% in comparison to the control.

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Abstract

La présente invention concerne des procédés de culture cellulaire et des compositions de ceux-ci qui, selon un aspect, améliorent le rendement et la qualité d'un produit biologique. Les procédés de culture cellulaire comprennent l'expression d'un polynucléotide d'intérêt pour améliorer le rendement et la qualité d'un produit biologique produit dans une culture de cellules de mammifère. Selon un aspect, l'invention concerne un procédé de production d'un produit biologique d'intérêt dans une population cellulaire, le procédé comprenant : la mise à disposition d'une population cellulaire exprimant un polynucléotide; la réunion de conditions permettant la production d'un produit biologique d'intérêt dans la population cellulaire; le polynucléotide étant choisi dans le groupe constitué d'un polynucléotide codant pour un transporteur d'aspartate à travers la membrane cellulaire, d'un polynucléotide codant pour une protéine impliquée dans la biosynthèse de l'aspartate, ou d'une combinaison de ceux-ci, l'expression du polynucléotide entraînant une augmentation de la quantité d'aspartate dans la cellule par comparaison avec une population cellulaire n'exprimant pas le polynucléotide, l'augmentation de la quantité d'aspartate réduisant la production de lactate dans la population cellulaire, ce qui permet d'obtenir un procédé pour la production d'un produit biologique d'intérêt dans la population cellulaire.
PCT/EP2022/080777 2021-11-05 2022-11-04 Culture cellulaire avec production réduite de lactate WO2023079058A1 (fr)

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Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
EP0117058A2 (fr) 1983-01-19 1984-08-29 Genentech, Inc. Procédés de production de protéines matures dans des cellules hôtes de vertébrés
EP0117060A2 (fr) 1983-01-19 1984-08-29 Genentech, Inc. Méthodes de criblage et d'amplification dans des cellules eucaryotiques hôtes, et séquences de nucléotides et vecteurs d'expression à utiliser dans celles-ci
EP0171496A2 (fr) 1984-08-15 1986-02-19 Research Development Corporation of Japan Procédé pour la production d'un anticorps monoclonal chimérique
EP0173494A2 (fr) 1984-08-27 1986-03-05 The Board Of Trustees Of The Leland Stanford Junior University Récepteurs chimériques par liaison et expression de l'ADN
EP0239400A2 (fr) 1986-03-27 1987-09-30 Medical Research Council Anticorps recombinants et leurs procédés de production
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
GB2177096B (en) 1984-09-03 1989-05-17 Celltech Ltd Production of chimeric antibodies
WO1992006193A1 (fr) 1990-10-05 1992-04-16 Scott David Gorman Anticorps a efficacite antigoniste a l'antigene cd3
US5149792A (en) 1989-12-19 1992-09-22 Amgen Inc. Platelet-derived growth factor B chain analogs
US5272064A (en) 1989-12-19 1993-12-21 Amgen Inc. DNA molecules encoding platelet-derived growth factor B chain analogs and method for expression thereof
WO1994010308A1 (fr) 1992-10-23 1994-05-11 Immunex Corporation Procede de preparation de proteines oligomeres solubles
US5658754A (en) 1989-10-05 1997-08-19 Optein, Inc. Cell-free synthesis and isolation of novel genes and polypeptides
US6235883B1 (en) 1997-05-05 2001-05-22 Abgenix, Inc. Human monoclonal antibodies to epidermal growth factor receptor
US6291159B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for producing polymers having a preselected activity
US6657103B1 (en) 1990-01-12 2003-12-02 Abgenix, Inc. Human antibodies derived from immunized xenomice
KR20160085666A (ko) * 2015-01-08 2016-07-18 삼성바이오에피스 주식회사 Got1 과발현에 의한 재조합 폴리펩타이드의 생산

Patent Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
EP0117058A2 (fr) 1983-01-19 1984-08-29 Genentech, Inc. Procédés de production de protéines matures dans des cellules hôtes de vertébrés
EP0117060A2 (fr) 1983-01-19 1984-08-29 Genentech, Inc. Méthodes de criblage et d'amplification dans des cellules eucaryotiques hôtes, et séquences de nucléotides et vecteurs d'expression à utiliser dans celles-ci
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
EP0171496A2 (fr) 1984-08-15 1986-02-19 Research Development Corporation of Japan Procédé pour la production d'un anticorps monoclonal chimérique
EP0173494A2 (fr) 1984-08-27 1986-03-05 The Board Of Trustees Of The Leland Stanford Junior University Récepteurs chimériques par liaison et expression de l'ADN
GB2177096B (en) 1984-09-03 1989-05-17 Celltech Ltd Production of chimeric antibodies
EP0239400A2 (fr) 1986-03-27 1987-09-30 Medical Research Council Anticorps recombinants et leurs procédés de production
US6291159B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for producing polymers having a preselected activity
US5658754A (en) 1989-10-05 1997-08-19 Optein, Inc. Cell-free synthesis and isolation of novel genes and polypeptides
US5149792A (en) 1989-12-19 1992-09-22 Amgen Inc. Platelet-derived growth factor B chain analogs
US5272064A (en) 1989-12-19 1993-12-21 Amgen Inc. DNA molecules encoding platelet-derived growth factor B chain analogs and method for expression thereof
US6657103B1 (en) 1990-01-12 2003-12-02 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1992006193A1 (fr) 1990-10-05 1992-04-16 Scott David Gorman Anticorps a efficacite antigoniste a l'antigene cd3
WO1994010308A1 (fr) 1992-10-23 1994-05-11 Immunex Corporation Procede de preparation de proteines oligomeres solubles
US6235883B1 (en) 1997-05-05 2001-05-22 Abgenix, Inc. Human monoclonal antibodies to epidermal growth factor receptor
KR20160085666A (ko) * 2015-01-08 2016-07-18 삼성바이오에피스 주식회사 Got1 과발현에 의한 재조합 폴리펩타이드의 생산

Non-Patent Citations (38)

* Cited by examiner, † Cited by third party
Title
"Methods in Enzymology Series", vol. 182, 1997, ACADEMIC PRESS, article "Guide to Protein Purification : Methods in Enzymology"
"Protein Expression: A Practical Approach", 1999, OXFORD UNIV PRESS
ANDERSON ET AL., NATURE, vol. 392, 1998, pages 25 - 30
BOSHART ET AL., CELL, vol. 41, 1985, pages 521
DATABASE EMBL [online] 15 February 2008 (2008-02-15), "Cricetulus griseus glutamate and aspartate transporter (GLAST) mRNA, complete cds.", XP002808641, retrieved from EBI accession no. EM_STD:EF155647 Database accession no. EF155647 *
DATABASE EMBL [online] 6 September 2005 (2005-09-06), "Mus musculus 16 days embryo heart cDNA, RIKEN full-length enriched library, clone:I920001F12 product:glutamate oxaloacetate transaminase 1, soluble, full insert sequence.", XP002808642, retrieved from EBI accession no. EM_HTC:AK146445 Database accession no. AK146445 *
DATABASE EMBL [online] 9 March 2001 (2001-03-09), "Homo sapiens glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2), mRNA (cDNA clone MGC:8599 IMAGE:2961181), complete cds.", XP002808643, retrieved from EBI accession no. EM_STD:BC000525 Database accession no. BC000525 *
DIJKEMA ET AL., EMBO J., vol. 4, 1985, pages 761
DOCHEN-KIANG, CYTOKINE GROWTH FACTOR REV., vol. 13, no. 1, 2002, pages 19 - 25
FIONA HARTLEY ET AL: "Mechanisms driving the lactate switch in Chinese hamster ovary cells", BIOTECHNOLOGY AND BIOENGINEERING, JOHN WILEY, HOBOKEN, USA, vol. 115, no. 8, 10 April 2018 (2018-04-10), pages 1890 - 1903, XP071115602, ISSN: 0006-3592, DOI: 10.1002/BIT.26603 *
FLOTTE, J. BIOENERG. BIOMEMB, vol. 25, 1993, pages 37 - 42
GETHING ET AL., NATURE, vol. 293, 1981, pages 620 - 625
GORMAN ET AL.: "79", PROC. NATL. ACAD. SCI. USA, 1982, pages 6777
GRAHAMVAN DER ERB, VIROLOGY, vol. 52, 1978, pages 456 - 457
GUPTA SANJEEV KUMAR ET AL: "Metabolic engineering of CHO cells for the development of a robust protein production platform", PLOS ONE, vol. 12, no. 8, 1 August 2017 (2017-08-01), pages e0181455, XP093024922, DOI: 10.1371/journal.pone.0181455 *
HAKANSSON ET AL., STRUCTURE, vol. 7, 1999, pages 255 - 64
HARBURY ET AL., NATURE, vol. 371, 1994, pages 80 - 83
HARBURY ET AL., SCIENCE, vol. 262, 1993, pages 1401 - 1293
JOLLY, CANC. GENE THER., vol. 1, 1994, pages 51 - 64
JONES ET AL., NATURE, vol. 321, 1986, pages 522 - 525
KEOWN ET AL., METHODS IN ENZYMOLOGY, 1989
KEOWN ET AL., METHODS IN ENZYMOLOGY, vol. 185, 1990, pages 527 - 537
KIRSHENBAUM ET AL., J. CLIN. INVEST, vol. 92, 1993, pages 381 - 387
KOZBOR ET AL., IMMUNOLOGY TODAY, vol. 4, 1983, pages 7279
MANTEI ET AL., NATURE, vol. 281, 1979, pages 40 - 46
MILLERROSMAN, BIOTECHNIQUES, vol. 7, 1992, pages 980 - 990
MORRISON ET AL., PROC. NATL. ACAD SCI. U.S.A., vol. 81, 1985, pages 6851
OKAYAMA ET AL., MOL. CELL BIOL, vol. 5, 1985, pages 1136 - 1142
OLSSON ET AL., METH. ENZYMOL, vol. 92, 1982, pages 3 - 16
PRESTA, CURR. OP. STRUCT. BIOL, vol. 2, 1992, pages 593 - 596
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329
SCOPES: "Protein Purification Principles and Practice", 1987, SPRINGER-VERLAG
TAKEDA ET AL., NATURE, vol. 314, 1985, pages 452
TENG ET AL., PROC. NATL. ACAD SCI. U.S.A., vol. 80, 1983, pages 7308 - 7312
THOMAS ET AL., CELL, vol. 51, 1987, pages 503 - 512
VERMASOMIA, NATURE, vol. 389, 1997, pages 239 - 242
WILSON, NEW ENGL. J. MED., vol. 334, 1996, pages 1185 - 1187
WLASCHIN ET AL: "Engineering cell metabolism for high-density cell culture via manipulation of sugar transport", JOURNAL OF BIOTECHNOLOGY, ELSEVIER, AMSTERDAM NL, vol. 131, no. 2, 6 August 2007 (2007-08-06), pages 168 - 176, XP022184732, ISSN: 0168-1656, DOI: 10.1016/J.JBIOTEC.2007.06.006 *

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