Method of activating protein
A method of producing a protein having free cysteine with the use of a serum-free medium, characterized in that the protein is produced in the activated state; a method of producing a protein by culturing in a serum-free medium in accordance with the above method; a method of activating a protein having free cysteine which has been produced in the inactivated state; and a protein obtained by any of the above methods. This protein shows physicochemical or biological properties comparable to a protein obtained by using a serum medium.
1 . A method of producing a protein having free cysteine with the use of a serum-free medium, characterized in that the protein having free cysteine is produced in an activated state.
2 . A method according to claim 1 , characterized in that the method is carried out in the presence of a reducing agent having capacity low enough not to reduce a disulfide linkage in the protein molecule upon production of the protein but high enough to reduce the modification of free cysteine.
3 . A method according to claim 2 , characterized in that the reducing agent is cysteine.
4 . A method according to claim 1 , characterized in that the method is performed in the presence of an antioxidant.
5 . A method according to claim 4 , characterized in that the antioxidant has capacity enough to inhibit polymerization of proteins via the free cysteine.
6 . A method according to claim 4 , characterized in that the antioxidant is ascorbic acid.
7 . A method according to claim 1 , characterized in that the method is carried out in the presence of a substance facilitating the reduction of the modification of free cysteine.
8 . A method according to claim 7 , characterized in that the substance facilitating the reduction of the modification of free cysteine is sodium chloride or potassium chloride.
9 . A method according to claim 1 , characterized in that the protein has, in the molecule thereof, free cysteine which has not formed a disulfide linkage and the protein has binding activity derived from the thiol group of the cysteine.
10 . A method according to claim 1 , characterized in that the protein is a recombinant protein.
11 . A method according to claim 1 , characterized in that the protein is an antibody.
12 . A method according to claim 11 , characterized in that the antibody has, in the variable region thereof, free cysteine.
13 . A method according to claim 11 , characterized in that the antibody is a human monoclonal antibody.
14 . A method according to claim 13 , characterized in that the human monoclonal antibody has, in the heavy-chain hypervariable regions thereof, amino acid sequences of SEQ. ID NOS. 1, 2 and 3 of Sequence Listing and, in the light-chain hypervariable regions, amino acid sequences of SEQ. ID NOS. 4, 5 and 6 of Sequence Listing.
15 . A method according to claim 13 , characterized in that the human monoclonal antibody has heavy-chain hypervariable regions containing an amino acid sequence of SEQ. ID No. 7 of Sequence Listing and light-chain hypervariable regions containing an amino acid sequence of SEQ. ID No. 8 of Sequence Listing.
16 . A method according to claim 11 , characterized in that the antibody is a F(ab′) 2 fragment antibody.
17 . A method of producing a protein by culturing in a serum-free medium in accordance with a method as claimed in claim 1 .
18 . A method according to claim 17 , characterized in that the protein has, in the molecule thereof, free cysteine which has not formed a disulfide linkage and the protein has binding activity derived from a thiol group of cysteine.
19 . A method according to claim 17 , characterized in that the protein is a recombinant protein.
20 . A method according to claim 17 , characterized in that the protein is an antibody.
21 . A method according to claim 20 , characterized in that the antibody has, in the variable region thereof, free cysteine.
22 . A method according to claim 20 , characterized in that the antibody is a human monoclonal antibody
23 . A method according to claim 20 , characterized in that the antibody has, in the heavy-chain hypervariable regions thereof, amino acid sequences of SEQ. ID NOS. 1, 2 and 3 of Sequence Listing and, in the light-chain hypervariable regions, amino acid sequences of SEQ. ID NOS. 4, 5 and 6 of Sequence Listing.
24 . A method according to claim 20 , characterized in that the antibody has heavy-chain hypervariable regions containing an amino acid sequence of SEQ. ID No. 7 of Sequence Listing and light-chain hypervariable regions containing an amino acid sequence of SEQ. ID No. 8 of Sequence Listing.
25 . A method according to claim 20 , characterized in that the antibody is a F(ab′) 2 fragment antibody.
26 . A method of activating a protein which has been produced in an inactivate state and having free cysteine, characterized in that the method is carried out in the presence of a reducing agent, an antioxidant and a substance facilitating the reduction of the modification of the free cysteine.
27 . A method according to claim 26 , characterized in that the method is carried out in the presence of a reducing agent having capacity low enough not to reduce a disulfide linkage in the molecule of the protein thus produced but high enough to reduce the modification of free cysteine.
28 . A method according to claim 26 , characterized in that the reducing agent is cysteine.
29 . A method according to claim 26 , characterized in that the antioxidant has capacity high enough to inhibit polymerization of proteins via the free cysteine.
30 . A method according to claim 26 , characterized in that the antioxidant is ascorbic acid.
31 . A method according to claim 26 , characterized in that the substance facilitating the reduction of the modification of the free cysteine is sodium chloride or potassium chloride.
32 . A method according to claim 26 , characterized in that the protein has, in the molecule thereof, free cysteine which has not formed a disulfide linkage and the protein has binding activity derived from a thiol group of free cysteine.
33 . A method according to claim 26 , characterized in that the protein is a recombinant protein.
34 . A method according to claim 26 , characterized in that the protein is an antibody.
35 . A method according to claim 34 , characterized in that the antibody has, in the variable region thereof, free cysteine.
36 . A method according to claim 34 , characterized in that the antibody is a human monoclonal antibody.
37 . A method according to claim 36 , characterized in that the human monoclonal antibody has, in the heavy-chain hypervariable regions thereof, amino acid sequences of SEQ. ID NOS. 1, 2 and 3 of Sequence Listing and, in the light-chain hypervariable regions, amino acid sequences of SEQ. ID NOS. 4, 5 and 6 of Sequence Listing.
38 . A method according to claim 36 , characterized in that the human monoclonal antibody has heavy-chain hypervariable regions containing an amino acid sequence of SEQ. ID No. 7 of Sequence Listing and light-chain hypervariable regions containing an amino acid sequence of SEQ. ID No. 8 of Sequence Listing.
39 . A method according to claim 34 , characterized in that the antibody is a F(ab′) 2 fragment antibody.
40 . A method according to claim 26 , characterized in that the reducing agent, antioxidant or substance facilitating the reduction of the modification of free cysteine is added when a whole antibody after purification exists.
41 . A protein obtained by a method as claimed in claim 1 .
42 . A protein according to claim 41 , wherein the protein exhibits physicochemical or biological properties comparable to those of a protein available by using a serum medium.
43 . A pharmaceutical composition comprising a protein as claimed in claim 41 .
44 . A pharmaceutical composition according to claim 43 , characterized in that the composition is an antitumor agent.