Production of anti-abeta
First Claim
1. A method of producing an anti-ABeta antibody in a large-scale production cell culture comprising the steps of:
- providing a cell culture comprising;
mammalian cells that contain a gene encoding the anti-ABeta antibody, which gene is expressed under condition of cell culture; and
a medium containing glutamine and having a medium characteristic selected from the group consisting of;
(i) a cumulative amino acid amount per unit volume greater than 70 mM, (ii) a molar cumulative glutamine to cumulative asparagine ratio of less than 2, (iii) a molar cumulative glutamine to cumulative total amino acid ratio of less than 0.2, (iv) a molar cumulative inorganic ion to cumulative total amino acid ratio between about 0.4 to 1, (v) a combined cumulative amount of glutamine and asparagine per unit volume of greater than 16 mM, and combinations thereof;
maintaining said culture in an initial growth phase under a first set of culture conditions for a first period of time sufficient to allow said cells to reproduce to a viable cell density within a range of about 20%-80% of the maximal possible viable cell density if said culture were maintained under the first set of culture conditions;
changing at least one of the culture conditions, so that a second set of culture conditions is applied;
maintaining said culture for a second period of time under the second set of conditions and for a second period of time so that the anti-ABeta antibody accumulates in the cell culture.
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Accused Products
Abstract
An improved system for large scale production of proteins and/or polypeptides in cell culture, particularly in media characterized by one or more of: i) a cumulative amino acid concentration greater than about 70 mM; ii) a molar cumulative glutamine to cumulative asparagine ratio of less than about 2; iii) a molar cumulative glutamine to cumulative total amino acid ratio of less than about 0.2; iv) a molar cumulative inorganic ion to cumulative total amino acid ratio between about 0.4 to 1; or v) a combined cumulative glutamine and cumulative asparagine concentration between about 16 and 36 mM, is provided. The use of such a system allows high levels of protein production and lessens accumulation of certain undesirable factors such as ammonium and/or lactate. Additionally, culture methods including a temperature shift, typically including a decrease in temperature when the culture has reached about 20-80% of it maximal cell density, are provided. Alternatively or additionally, the present invention provides methods such that, after reaching a peak, lactate and/or ammonium levels in the culture decrease over time.
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Citations
108 Claims
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1. A method of producing an anti-ABeta antibody in a large-scale production cell culture comprising the steps of:
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providing a cell culture comprising; mammalian cells that contain a gene encoding the anti-ABeta antibody, which gene is expressed under condition of cell culture; and a medium containing glutamine and having a medium characteristic selected from the group consisting of;
(i) a cumulative amino acid amount per unit volume greater than 70 mM, (ii) a molar cumulative glutamine to cumulative asparagine ratio of less than 2, (iii) a molar cumulative glutamine to cumulative total amino acid ratio of less than 0.2, (iv) a molar cumulative inorganic ion to cumulative total amino acid ratio between about 0.4 to 1, (v) a combined cumulative amount of glutamine and asparagine per unit volume of greater than 16 mM, and combinations thereof;maintaining said culture in an initial growth phase under a first set of culture conditions for a first period of time sufficient to allow said cells to reproduce to a viable cell density within a range of about 20%-80% of the maximal possible viable cell density if said culture were maintained under the first set of culture conditions; changing at least one of the culture conditions, so that a second set of culture conditions is applied; maintaining said culture for a second period of time under the second set of conditions and for a second period of time so that the anti-ABeta antibody accumulates in the cell culture. - View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108)
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2. A method of producing an anti-ABeta antibody in a large-scale production cell culture comprising the steps of:
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providing a cell culture comprising; mammalian cells that contain a gene encoding the anti-ABeta antibody, which gene is expressed under condition of cell culture; and a medium containing a cumulative amino acid amount per unit volume greater than 70 mM; and said medium containing glutamine; and said medium having two medium characteristics selected from the group consisting of;
(i) a molar cumulative glutamine to cumulative asparagine ratio of less than 2, (ii) a molar cumulative glutamine to cumulative total amino acid ratio of less than 0.2, (iii) a molar cumulative inorganic ion to cumulative total amino acid ratio between about 0.4 to 1, (iv) a combined cumulative amount of glutamine and asparagine per unit volume of greater than 16 mM, and combinations thereof;maintaining said culture in an initial growth phase under a first set of culture conditions for a first period of time sufficient to allow said cells to reproduce to a viable cell density within a range of about 20%-80% of the maximal possible viable cell density if said culture were maintained under the first set of culture conditions; changing at least one of the culture conditions, so that a second set of culture conditions is applied; maintaining said culture for a second period of time under the second set of conditions and for a second period of time so that the anti-ABeta antibody accumulates in the cell culture.
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3. A method of producing an anti-ABeta antibody in a large-scale production cell culture comprising the steps of:
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providing a cell culture comprising; mammalian cells that contain a gene encoding the anti-ABeta antibody, which gene is expressed under condition of cell culture; and a medium containing a molar cumulative glutamine to cumulative asparagine ratio of less than 2; and said medium containing glutamine; and said medium having two medium characteristics selected from the group consisting of;
(i) a medium containing a cumulative amino acid amount per unit volume greater than 70 mM, (ii) a molar cumulative glutamine to cumulative total amino acid ratio of less than 0.2, (iii) a molar cumulative inorganic ion to cumulative total amino acid ratio between about 0.4 to 1, (iv) a combined cumulative amount of glutamine and asparagine per unit volume of greater than 16 mM, and combinations thereof;maintaining said culture in an initial growth phase under a first set of culture conditions for a first period of time sufficient to allow said cells to reproduce to a viable cell density within a range of about 20%-80% of the maximal possible viable cell density if said culture were maintained under the first set of culture conditions; changing at least one of the culture conditions, so that a second set of culture conditions is applied; maintaining said culture for a second period of time under the second set of conditions and for a second period of time so that the anti-ABeta antibody accumulates in the cell culture.
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4. A method of producing an anti-ABeta antibody in a large-scale production cell culture comprising the steps of:
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providing a cell culture comprising; mammalian cells that contain a gene encoding the anti-ABeta antibody, which gene is expressed under condition of cell culture; and a medium containing a molar cumulative glutamine to cumulative total amino acid ratio of less than 0.2; and said medium containing glutamine; and said medium having two medium characteristics selected from the group consisting of;
(i) a medium containing a cumulative amino acid amount per unit volume greater than 70 mM, (ii) a molar cumulative glutamine to cumulative asparagine ratio of less than 2, (iii) a molar cumulative inorganic ion to cumulative total amino acid ratio between about 0.4 to 1, (iv) a combined cumulative amount of glutamine and asparagine per unit volume of greater than 16 mM, and combinations thereof;maintaining said culture in an initial growth phase under a first set of culture conditions for a first period of time sufficient to allow said cells to reproduce to a viable cell density within a range of about 20%-80% of the maximal possible viable cell density if said culture were maintained under the first set of culture conditions; changing at least one of the culture conditions, so that a second set of culture conditions is applied; maintaining said culture for a second period of time under the second set of conditions and for a second period of time so that the anti-ABeta antibody accumulates in the cell culture.
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5. A method of producing an anti-ABeta antibody in a large-scale production cell culture comprising the steps of:
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providing a cell culture comprising; mammalian cells that contain a gene encoding the anti-ABeta antibody, which gene is expressed under condition of cell culture; and a medium containing a molar cumulative inorganic ion to cumulative total amino acid ratio between about 0.4 to 1; and said medium containing glutamine; and said medium having two medium characteristics selected from the group consisting of;
(i) a medium containing a cumulative amino acid amount per unit volume greater than 70 mM, (ii) a molar cumulative glutamine to cumulative asparagine ratio of less than 2, (iii) a molar cumulative glutamine to cumulative total amino acid ratio of less than 0.2, (iv) a combined cumulative amount of glutamine and asparagine per unit volume of greater than 16 mM, and combinations thereof;maintaining said culture in an initial growth phase under a first set of culture conditions for a first period of time sufficient to allow said cells to reproduce to a viable cell density within a range of about 20%-80% of the maximal possible viable cell density if said culture were maintained under the first set of culture conditions; changing at least one of the culture conditions, so that a second set of culture conditions is applied; maintaining said culture for a second period of time under the second set of conditions and for a second period of time so that the anti-ABeta antibody accumulates in the cell culture.
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6. A method of producing an anti-ABeta antibody in a large-scale production cell culture comprising the steps of:
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providing a cell culture comprising; mammalian cells that contain a gene encoding the anti-ABeta antibody, which gene is expressed under condition of cell culture; and a medium containing a combined cumulative amount of glutamine and asparagine per unit volume of greater than 16 mM; and said medium containing glutamine; and said medium having two medium characteristics selected from the group consisting of;
(i) a medium containing a cumulative amino acid amount per unit volume greater than 70 mM, (ii) a molar cumulative glutamine to cumulative asparagine ratio of less than 2, (iii) a molar cumulative glutamine to cumulative total amino acid ratio of less than 0.2, (iv) a molar cumulative inorganic ion to cumulative total amino acid ratio between about 0.4 to 1, and combinations thereof;maintaining said culture in an initial growth phase under a first set of culture conditions for a first period of time sufficient to allow said cells to reproduce to a viable cell density within a range of about 20%-80% of the maximal possible viable cell density if said culture were maintained under the first set of culture conditions; changing at least one of the culture conditions, so that a second set of culture conditions is applied; maintaining said culture for a second period of time under the second set of conditions and for a second period of time so that the anti-ABeta antibody accumulates in the cell culture.
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70. A method of producing an anti-ABeta antibody in a large-scale production cell culture comprising steps of;
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providing a cell culture comprising; mammalian cells that contain a gene encoding the anti-ABeta antibody, which gene is expressed under condition of cell culture; and a defined medium containing glutamine and having at least two medium characteristics selected from the group consisting of;
i) a starting amino acid concentration greater than 70 mM, ii) a molar glutamine to asparagine ratio of less than 2, iii) a molar glutamine to total amino acid ratio of less than 0.2, iv) a molar inorganic ion to total amino acid ratio between about 0.4 to 1, and v) a combined glutamine and asparagine concentration greater than 16 mM;maintaining said culture in an initial growth phase under a first set of culture conditions for a first period of time sufficient to allow said cells to reproduce within a range of about 20%-80% of the maximal possible viable cell density if said culture were maintained under the first set of culture conditions; changing at least one of the culture conditions, so that a second set of culture conditions is applied; maintaining said culture for a second period of time under the second set of conditions and for a second period of time so that the anti-ABeta antibody accumulates in the cell culture.
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71. A method of producing an anti-ABeta antibody in a large-scale production cell culture comprising steps of;
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providing a cell culture comprising; mammalian cells that contain a gene encoding the anti-ABeta antibody, which gene is expressed under condition of cell culture; and a defined medium containing glutamine and having at least three medium characteristic selected from the group consisting of;
i) a starting amino acid concentration greater than 70 mM, ii) a molar glutamine to asparagine ratio of less than 2, iii) a molar glutamine to total amino acid ratio of less than 0.2, iv) a molar inorganic ion to total amino acid ratio between about 0.4 to 1, and v) a combined glutamine and asparagine concentration greater than 16 mM;maintaining said culture in an initial growth phase under a first set of culture conditions for a first period of time sufficient to allow said cells to reproduce within a range of about 20%-80% of the maximal possible viable cell density if said culture were maintained under the first set of culture conditions; changing at least one of the culture conditions, so that a second set of culture conditions is applied; maintaining said culture for a second period of time under the second set of conditions and for a second period of time so that the anti-ABeta antibody accumulates in the cell culture.
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72. A method of producing an anti-ABeta antibody in a large-scale production cell culture comprising steps of;
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providing a cell culture comprising; mammalian cells that contain a gene encoding the anti-ABeta antibody, which gene is expressed under condition of cell culture; and a defined medium containing glutamine and having at least four medium characteristic selected from the group consisting of;
i) a starting amino acid concentration greater than 70 mM, ii) a molar glutamine to asparagine ratio of less than 2, iii) a molar glutamine to total amino acid ratio of less than 0.2, iv) a molar inorganic ion to total amino acid ratio between about 0.4 to 1, and v) a combined glutamine and asparagine concentration greater than 16 mM;maintaining said culture in an initial growth phase under a first set of culture conditions for a first period of time sufficient to allow said cells to reproduce within a range of about 20%-80% of the maximal possible viable cell density if said culture were maintained under the first set of culture conditions; changing at least one of the culture conditions, so that a second set of culture conditions is applied; maintaining said culture for a second period of time under the second set of conditions and for a second period of time so that the anti-ABeta antibody accumulates in the cell culture.
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73. A method of producing an anti-ABeta antibody in a large-scale production cell culture comprising steps of;
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providing a cell culture comprising; mammalian cells that contain a gene encoding the anti-ABeta antibody, which gene is expressed under condition of cell culture; and a defined medium containing glutamine, characterized by;
i) a starting amino acid concentration greater than 70 mM, ii) a molar glutamine to asparagine ratio of less than 2, iii) a molar glutamine to total amino acid ratio of less than 0.2, iv) a molar inorganic ion to total amino acid ratio between about 0.4 to 1, and v) a combined glutamine and asparagine concentration greater than 16 mM;maintaining said culture in an initial growth phase under a first set of culture conditions for a first period of time sufficient to allow said cells to reproduce within a range of about20%-80% of the maximal possible viable cell density if said culture were maintained under the first set of culture conditions; changing at least one of the culture conditions, so that a second set of culture conditions is applied; maintaining said culture for a second period of time under the second set of conditions and for a second period of time so that the anti-ABeta antibody accumulates in the cell culture.
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74. A method of producing an anti-ABeta antibody in a large-scale production cell culture comprising the steps of:
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providing a cell culture comprising; mammalian cells that contain a gene encoding the anti-ABeta antibody, which gene is expressed under condition of cell culture; and a medium containing glutamine and having a combined cumulative amount of glutamine and asparagine per unit volume of greater than 16 mM; maintaining said culture in an initial growth phase under a first set of culture conditions for a first period of time sufficient to allow said cells to reproduce within a range of about 20%-80% of the maximal possible viable cell density if said culture were maintained under the first set of culture conditions; changing at least one of the culture conditions, so that a second set of culture conditions is applied; maintaining said culture for a second period of time under the second set of conditions and for a second period of time so that the anti-ABeta antibody accumulates in the cell culture.
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Specification