CELL CULTURE METHODS AND MEDIA COMPRISING N-ACETYLCYSTEINE
This application provides improved cell culture media and cell culture methods comprising N-acetylcysteine. These improved cell culture media and cell culture methods increase cell viability, cellular growth rate and/or reduce cell doubling time of cholesterol auxotrophic cells, myeloma cells, and hybridoma cells.
1 . A cell culture method comprising:
a. providing a cell culture medium sufficient to support cell growth, wherein the cell culture medium comprises N-acetylcysteine; and
b. culturing a cell in the cell culture medium, wherein the cell is a cholesterol auxotroph, a myeloma, or a hybridoma.
2 . A method of increasing cell viability, increasing cell growth rate or reducing cell doubling time comprising:
a. providing a cell culture medium sufficient to support cell growth, wherein the cell culture medium comprises N-acetylcysteine; and
b. culturing a cell in the cell culture medium, wherein the cell is a cholesterol auxotroph, a myeloma, or a hybridoma.
3 - 6 . (canceled)
7 . The method of claim 1 or 2 , wherein the cell culture medium is a serum free and animal-protein free medium or is a chemically-defined medium.
8 . (canceled)
9 . (canceled)
10 . The method of claim 1 or 2 , wherein the cells are murine, hamster, rat, monkey, or human or are derived from a mammal.
11 . (canceled)
12 . The method of claim 1 or 2 , wherein the cells are selected or are derived from a cell selected from the group consisting of: NS0, NS1, U937, M19, SRD-12B, SRD-13A, CHO-215, X63Ag8, Sp2/0, J558L, U266, P3U1, XG-1, XG-2, XG-3, XG-4, XG-5, XG-6, XG-7, XG-8, XG-9, U266, RPM1-8226, LP1, L363, OPM1, OPM2, and NCLH929 cells.
13 . (canceled)
14 . The method of claim 1 or 2 , wherein the cells are NS0 cells.
15 . (canceled)
16 . (canceled)
17 . The method of claim 1 or 2 , wherein the cell culture medium comprises N-acetylcysteine at a concentration of from about 0.25 mM to about 3 mM, from about 0.5 to about 2.5 mM, from about 1.0 to about 1.5 mM, about 1 mM or about 1.5 mM.
18 - 22 . (canceled)
23 . The method of claim 2 , wherein the average doubling time is shorter than in a cell culture with a control medium excluding N-acetylcysteine, or is reduced by at least about 10%, 15%, 20%, 25% or 50% compared to a cell culture medium without N-acetylcysteine.
24 - 28 . (canceled)
29 . The method of claim 23 , wherein the average cell doubling time is 60, 42, 32, 30 or 29 hours or less.
30 - 33 . (canceled)
34 . The method of claim 2 , wherein the cell viability is increased over a cell culture with a control medium excluding N-acetylcysteine, or is increased by at least about 5%, 7% or 10% compared to a cell culture medium without N-acetylcysteine, or wherein cell viability is at least about 90%, 92% or 93%.
35 - 41 . (canceled)
42 . The method of claim 1 or 2 , wherein the cells express a heterologous protein or are transformed with a heterologous nucleic acid.
43 - 46 . (canceled)
47 . The method of claim 1 or 2 , wherein the cells express a heterologous protein selected from the group consisting of:
(a) an antibody or antigen-binding fragment thereof;
(b) an IL-13 antibody or antigen-binding fragment thereof;
(c) an IL-13 antibody BAK502G9 or an antigen-binding fragment thereof;
(d) an antibody or antigen binding fragment having a heavy chain variable region comprising a sequence that is at least about 80% identical to any one of SEQ ID NOs: 1, 9, 17, or 25 and a light chain variable region comprising a sequence that is at least about 80% identical to any one of SEQ ID NOs: 2, 10, 18, or 26; and
(e) an antibody or antigen binding fragment comprising: a heavy chain variable region comprising:
i. a HC CDR1 that has one mutation compared to a sequence chosen from SEQ ID NOs: 3, 11, 19, or 27;
ii. a HC CDR2 that has one or two mutations compared to a sequence chosen from SEQ ID NOs: 4, 12, 20, and 28; or
iii. a HC CDR3 that has one or two mutations compared to a sequence chosen from SEQ ID NOs: 5, 13, 21, and 29; and
a light chain variable region comprising:
i. a LC CDR1 that has one mutation compared to a sequence chosen from SEQ ID NOs: 6, 14, 22, and 30;
ii. a LC CDR2 that has one or two mutations compared to a sequence chosen from SEQ ID NOs: 7, 15, 23, and 31; or
iii. a LC CDR3 that has one or two mutations compared to a sequence chosen from SEQ ID NOs: 8, 16, 24, and 32.
48 - 54 . (canceled)
55 . A cell culture medium comprising N-acetylcysteine, a carbohydrate source, an amino acid source, and a cholesterol source, wherein the cell culture medium comprises N-acetylcysteine at a concentration of from about 0.25 mM to about 3 mM, from about 0.5 to about 2.5 mM, from about 1.0 to about 1.5 mM, of aboutl mM or 1.5 mM.
56 - 65 . (canceled)