Process for the production of tumor necrosis factor-binding proteins
The invention provides methods for increasing the recombinant production of polypeptides, in particular Tumor Necrosis Factor Binding Proteins, from mammalian cells at a temperature below 30° C.
1 - 16 . (canceled)
17 . A method for producing a recombinant polypeptide comprising culturing a mammalian cell line, the cell line expressing a recombinant polypeptide in a production phase at a temperature at or below 29° C.
18 . The method of claim 17 , wherein the polypeptide is a Tumor Necrosis Factor Binding Protein (TBP), or a mutein or fragment thereof.
19 . The method of claim 18 , wherein the polypeptide is recombinant human TBP-1 or TBP-2.
20 . The method of claim 19 , wherein the polypeptide is expressed by a mammalian cell line cornp rising a DNA sequence encoding a TBP-1 polypeptide selected from the group consisting of:
(a) a polypeptide comprising SEQ ID NO: 1;
(b) a mutein of (a), wherein the amino acid sequence has at least 40% or 50% or 60% or 70% or 80% or 90% identity to the sequence in (a);
(c) a mutein of (a) which is encoded by a DNA sequence, which hybridizes to the complement of the native DNA sequence encoding (a) under moderately stringent conditions or under highly stringent conditions;
(d) a mutein of (a) wherein any changes in the amino acid sequence are conservative amino acid substitutions to the amino acid sequences in (a); and
(e) a salt or an isoform, fused protein, functional derivative, active fraction or circularly permutated derivative of (a).
21 . The method of claim 19 , wherein the polypeptide is expressed by a mammalian cell line comprising a DNA sequence encoding a TBP-2 polypeptide selected from the group consisting of:
(a) a polypeptide comprising SEQ ID NO: 2;
(b) a mutein of (a), wherein the amino acid sequence has at least 40% or 50% or 60% or 70% or 80% or 90% identity to the sequence in (a);
(c) a mutein of (a) which is encoded by a DNA sequence, which hybridizes to the complemerit of the native DNA sequence encoding (a) under moderately stringent conditions or under highly stringent conditions;
(d) a mutein of (a) wherein any changes in the amino acid sequence are conservative amino acid substitutions to the amino acid sequences in (a);
(e) a salt or an isoform, fused protein, functional derivative, active fraction or circularly permutated derivative of (a).
22 . The method of claim 20 , wherein the mammalian cell line is cultured at a temperature between 20° C. and 29° C.
23 . The method of claim 21 , wherein the mammalian cell line is cultured at a temperature between 20° C. and 29° C.
24 . The method of claim 22 , wherein the mammalian cell line is cultured at a temperature of about 25 to 29° C.
25 . The method of claim 24 , wherein the mammalian cell line is cultured at a temperature of about 26° C., or about 27° C., or about 28° C.
26 . The method of claim 24 , wherein the mammalian cell line is cultured at a temperature of about 29° C.
27 . The method of claim 23 , wherein the mammalian cell line is cultured at a temperature of about 25 to 29° C.
28 . The method of claim 27 , wherein the mammalian cell line is cultured at a temperature of about 26° C., or about 27° C., or about 28° C.
29 . The method of claim 27 , wherein the mammalian cell line is cultured at a temperature of about 29° C.
30 . The method of claim 17 , wherein the mammalian cell line is a CHO cell line.
31 . The method of claim 17 , wherein the medium used during the production phase is serum free.
32 . The method of claim 17 , further comprising collecting the polypeptide from the medium.
33 . The method of claim 17 , further comprising purifying the polypeptide from medium or cell derived components.
34 . The method of claim 17 , further comprising formulating the purified polypeptide with a pharmaceutically acceptable carrier.
35 . An isolated polypeptide produced by the method of claim 17 , said polypeptide being mono-glycosylated.