Methods for making recombinant proteins
View Patent ↗The present invention provides methods and compositions for making proteins, preferably antibodies, more preferably anti-tumor necrosis factor alpha antibodies, and most preferably adalimumab. The present invention further provides methods and compositions for mammalian cell culture, preferably Chinese Hamster Ovary cells.
1. A method of producing adalimumab in a mammalian cell culture comprising:
a) inoculating a fed-batch bioreactor containing a cell culture production medium with mammalian cells comprising a nucleic acid encoding adalimumab or a fragment thereof wherein the mammalian cells are at a concentration from about 0.1 million to about 2 million cells/milliliter, wherein the production medium comprises glucose at an initial concentration from about 0.1 to about 0.9 grams/liter and at least one other hexose selected from the group consisting of galactose, mannose, fructose, maltose, or a combination thereof at an initial concentration from about 0.1 to about 20 grams/liter, such that adalimumab is produced;
b) maintaining the glucose in the production medium at a concentration from about 0.1 to about 0.9 grams/liter;
c) optionally, maintaining the at least one other hexose in the production medium at a concentration from about 0.1 to about 20 grams/liter;
d) optionally, supplementing the production medium with at least one additional feed absent glucose; and
e) harvesting the mammalian cell culture at a cell viability from about 20% to about 100%; and
f) deactivation, inactivation, or removal of virus from the cell culture.
2. The method of claim 1 wherein the mammalian cells are Chinese Hamster Ovary cells.
3. The method of claim 1 or 2 , wherein the adalimumab or the fragment thereof is secreted into the production medium.
4. The method of claim 3 further comprising purifying the adalimumab or the fragment thereof from a production medium.
5. The method of claim 4 , further comprising formulating the purified adalimumab or the fragment thereof into a pharmaceutical composition.
6. The method of claim 1 , wherein the cell viability is from about 50% to about 80%.
7. A method of producing adalimumab in a mammalian cell culture comprising:
a) a growth phase comprising:
i) inoculating a perfusion bioreactor, containing a cell culture growth medium, with mammalian cells comprising a nucleic acid encoding adalimumab or a fragment thereof wherein the mammalian cells are at a concentration from about 0.1 million to about 5 million cells/milliliter, wherein the growth medium comprises glucose at a concentration from about 0.1 to about 20 grams/liter and at least one other hexose selected from the group consisting of galactose, mannose, fructose, maltose, or a combination thereof at a concentration from about 0.1 to about 20 grams/liter; and
ii) allowing the mammalian cells to propagate resulting in an inoculum comprising from about 1 to about 50 million cells/milliliter; and
b) a production phase comprising:
i) inoculating a fed-batch bioreactor, containing a cell culture production medium, with the inoculum comprising from about 3 million to about 20 million cells/milliliter, wherein the production medium comprises glucose at an initial concentration from out 0.1 to about 0.9 grams/liter and at least one other hexose selected from the group consisting of galactose, mannose, fructose, maltose, or a combination thereof at an initial concentration from about 0.1 to about 20 grams/liter, such that adalimumab is produced;
ii) maintaining the glucose in the production medium at a concentration from about 0.1 to about 0.9 grams/liter;
iii) optionally, maintaining the at least one other hexose in the production medium at a concentration from about 0.1 to about 20 grams/liter;
iv) optionally, supplementing the production medium with at least one additional feed absent glucose; and
v) harvesting the mammalian cell culture at a cell viability from about 20% to about 100%.
8. The method of claim 7 wherein the cell viability is from about 50% to about 80%.
9. A method of producing adalimumab in a mammalian cell culture comprising:
a) inoculating a fed-batch bioreactor containing a cell culture production medium with mammalian cells comprising a nucleic acid encoding adalimumab or a fragment thereof wherein the mammalian cells are at a concentration from about 0.1 million to about 2 million cells/milliliter, wherein the production medium comprises glucose at an initial concentration from about 0.1 to about 0.9 grams/liter and at least one other hexose selected from the group consisting of galactose, mannose, fructose, maltose, or a combination thereof at an initial concentration from about 0.1 to about 20 grams/liter, such that adalimumab is produced;
b) maintaining the glucose in the production medium at a concentration from about 0.1 to about 0.9 grams/liter;
c) optionally, maintaining the at least one other hexose in the production medium at a concentration from about 0.1 to about 20 grams/liter;
d) optionally, supplementing the production medium with at least one additional feed absent glucose; and
e) harvesting the mammalian cell culture at a cell viability from about 20% to about 100%.
10. The method of claim 9 wherein the cell viability is from about 50% to about 80%.
11. A method of producing adalimumab in a mammalian cell culture comprising:
a) a first cycle comprising:
i) inoculating a fed-batch bioreactor containing a first cell culture production medium, with mammalian cells comprising a nucleic acid encoding adalimumab or a fragment thereof wherein the mammalian cells are at a concentration from about 0.1 million to about 2 million cells/milliliter, wherein the first production medium comprises glucose at an initial concentration from about 0.1 to about 0.9 grams/liter and at least one other hexose selected from the group consisting of galactose, mannose, fructose, maltose, or a combination thereof at an initial concentration from about 0.1 to about 20 grams/liter;
ii) maintaining the glucose in the first production medium at a concentration from about 0.1 to about 0.9 grams/liter;
iii) optionally, maintaining the at least one other hexose in the first production medium at a concentration from about 0.1 to about 20 grams/liter;
iv) optionally, supplementing the first production medium with at least one additional feed absent glucose; and
v) harvesting 90% mammalian cell culture comprising a cell viability from about 80% to about 100%, and
b) a second cycle comprising:
i) removing the first production medium from the remaining 10% of the mammalian cell culture;
ii) adding a second production medium comprising glucose at an initial concentration from about 0.1 to about 0.9 grams/liter and at least one other hexose selected from the group consisting of galactose, mannose, fructose, maltose, or a combination thereof at an initial concentration from about 0.1 to about 20 grams/liter;
iii) maintaining the glucose in the second production medium at a concentration from about 0.1 to about 0.9 grams/liter;
iv) optionally, maintaining the at least one other hexose in the second production medium at a concentration from about 0.1 to about 20 grams/liter;
v) optionally, supplementing the second production medium with at least one additional feed absent glucose; and
vi) harvesting 90% mammalian cell culture comprising a cell viability from about 80% to about 100%; and
c) optionally, repeating the first and second cycle, such that adalimumab is produced.
12. A method of producing adalimumab in a mammalian cell culture comprising:
a) a growth phase comprising:
i) inoculating a perfusion bioreactor containing a cell culture growth medium with mammalian cells comprising a nucleic acid encoding adalimumab or a fragment thereof wherein the mammalian cells are at a concentration from about 0.1 million to about 5 million cells/milliliter, wherein the growth medium comprises glucose at an initial concentration from about 0.1 to about 0.9 grams/liter and at least one other hexose selected from the group consisting of galactose, mannose, fructose, maltose, or a combination thereof at an initial concentration from about 0.1 to about 20 grams/liter; and;
ii) allowing the mammalian cells to propagate resulting in the mammalian cell culture comprising from about 10 to about 40 million cells/milliliter; and
b) a production phase comprising:
i) removing the growth medium;
ii) adding a production medium comprising glucose at a concentration from about 0.1 to about 0.9 grams/liter and at least one other hexose selected from the group consisting of galactose, mannose, fructose, maltose, or a combination thereof at an initial concentration from about 0.1 to about 20 grams/liter;
iii) maintaining the glucose in the production medium at a concentration from about 0.1 to about 0.9 grams/liter;
iv) optionally, maintaining the at least one other hexose in the production medium at a concentration from about 0.1 to about 20 grams/liter;
v) optionally, supplementing the production medium with at least one additional feed absent glucose; and
vi) harvesting the mammalian cell culture comprising a cell viability from about 20% to about 100%, such that adalimumab is produced.
13. The method of claim 12 wherein the cell viability is from about 50% to about 80%.
14. The method of claim 7 , 9 , 11 , or 12 wherein the mammalian cells are Chinese Hamster Ovary Cells.