Method for refolding antibody, process for producing refolded antibody, refolded antibody, and uses thereof

Patent metadata

Field	Value
Publication number	US-9850316-B2
Application number	US-201414767373-A
Country	US
Kind code	B2
Filing date	Feb 3, 2014
Priority date	Feb 15, 2013
Publication date	Dec 26, 2017
Grant date	Dec 26, 2017

Abstract

A method for refolding an antibody, a process for producing a refolded antibody, a refolded antibody, and uses thereof are provided. A method for refolding an antibody in a liquid phase comprises the steps of denaturing an inactive antibody binding directly or through a linker to a peptide, the peptide having an isoelectric point lower than the isoelectric point of the inactive antibody, and dispersing in a liquid phase the peptide-binding inactive antibody denatured in the step above. Also provided is a process for producing a refolded antibody.

First claim

The invention claimed is:

1. A method for improving the refolding efficiency of an antibody in a liquid phase, the method comprising the steps of:
(A) denaturing an inactive antibody binding directly or indirectly through a linker to a peptide, wherein the peptide has an isoelectric point lower than the isoelectric point of the inactive antibody; and
(B) dispersing the peptide-bound inactive antibody denatured in step (A) above in a buffer having a pH that is 1 to 4.5 units higher than the isoelectric point of the peptide-bound inactive antibody denatured in step (A) to obtain a refolded antibody,
wherein a refolding efficiency of the refolded antibody is higher than a refolding efficiency obtained when the peptide represented by SEQ ID NO: 10 is bound to the inactive antibody, instead of the peptide of step (A) above, and
wherein the peptide is selected from the amino acid sequence of any one of 5 (D5), 10 (D10) or 15(D15) aspartic acid residues, SEQ ID NOS: 1 to 4, or a variant of SEQ ID NOS: 1 to 4, wherein one to fifteen of the amino acids of SEQ ID NOS: 1 to 4 is (are) deleted or substituted with aspartic acid and/or glutamic acid residues and wherein the peptide has 4 to 20 more acidic amino acids than basic amino acids.

2. The refolding method according to claim 1 , wherein the antibody is at least one member selected from the group consisting of single-chain antibodies, Fab fragments, F(ab′)2 fragments, single-domain antibodies, multivalent single-chain antibodies, single-chain antibodies fused with a constant region, and full-length antibodies.

3. The refolding method according to claim 1 , wherein the isoelectric point of the peptide is 8.5 or lower.

4. A process for producing a refolded antibody, the process comprising the steps of:
improving the refolding efficiency of an antibody in a liquid phase comprising:
(A) denaturing an inactive antibody binding directly or indirectly through a linker to a peptide, wherein the peptide has an isoelectric point lower than the isoelectric point of the inactive antibody; and
(B) dispersing the peptide-bound inactive antibody denatured in step (A) above in a buffer having a pH that is 1 to 4.5 units higher than the isoelectric point of the peptide-bound inactive antibody denatured in step (A) to obtain a refolded antibody,
wherein a refolding efficiency of the refolded antibody is higher than a refolding efficiency obtained when the peptide represented by SEQ ID NO: 10 is bound to the inactive antibody, instead of the peptide of step (A) above, and
wherein the peptide is selected from the amino acid sequence of any one of 5 (D5), 10 (D10) or 15(D15) aspartic acid residues, SEQ ID NOS: 1 to 4, or a variant of SEQ ID NOS: 1 to 4, wherein one to fifteen of the amino acids of SEQ ID NOS: 1 to 4 is (are) deleted or substituted with aspartic acid and/or glutamic acid residues and wherein the peptide has 4 to 20 more acidic amino acids than basic amino acids.

5. The production process according to claim 4 , wherein the antibody is at least one member selected from the group consisting of single-chain antibodies, Fab fragments, F(ab′)2 fragments, single-domain antibodies, multivalent single-chain antibodies, single-chain antibodies fused with a constant region, and full-length antibodies.

6. The production process according to claim 4 , wherein the isoelectric point of the peptide is 8.5 or lower.

7. The production process according to claim 4 , wherein the linker is a peptide having an affinity for a substrate.

8. The refolding method according to claim 1 , wherein the peptide is 5 to 10 amino acid residues in length.

9. The refolding method according to claim 1 , wherein the amino acid residues of the peptide consist of aspartic acid residues, glutamic acid residues, or a combination thereof.

10. The production process according to claim 4 , wherein the peptide is 5 to 10 amino acid residues in length.

11. The production process according to claim 4 , wherein the amino acid residues of the peptide consist of aspartic acid residues, glutamic acid residues, or a combination thereof.

12. The refolding method according to claim 1 , wherein the peptide is selected from SEQ ID NO: 4 or 5(D5), 10(D10) or 15(D15) aspartic acid residues.

13. The production process according to claim 4 , wherein the peptide is selected from SEQ ID NO: 4 or 5(D5), 10(D10) or 15(D15) aspartic acid residues.

14. The refolding method according to claim 1 , wherein the refolding efficiency is at least 88%.

15. The production process according to claim 4 , wherein the refolding efficiency is at least 88%.

16. The refolding method according to claim 1 , wherein the buffer is selected from the group consisting of
a buffer comprising 50mM 2-(N-morpholino)ethanesulfonic acid at pH 6,
a buffer comprising 50mM N-(2-acetamido)iminodiacetic acid at pH 6.5,
a buffer comprising 50mM 3-(N-morpholino)propanesulfonic acid at pH 7,
a buffer comprising 50 mM 2-[4-(2-Hydroxyethyl) -1-piperazinyl]ethanesulfonic acid at pH7.5,
a buffer comprising 50mM N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid at pH 8, and
a buffer comprising 50mM N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid at pH 8.5.

17. The production process according to claim 4 , wherein the buffer is selected from the group consisting of
a buffer comprising 50mM 2-(N-morpholino)ethanesulfonic acid at pH 6,
a buffer comprising 50mM N-(2-acetamido)iminodiacetic acid at pH 6.5,
a buffer comprising 50mM 3-(N-morpholino)propanesulfonic acid at pH 7,
a buffer comprising 50 mM 2-[4-(2-Hydroxyethyl) -1-piperazinyl]ethanesulfonic acid at pH7.5,
a buffer comprising 50 mM N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid at pH 8, and
a buffer comprising 50mM N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid at pH 8.5.

Assignees

• Nat Univ Corp Kyoto Inst Technology

Inventors

• Kumada Yoichi
• Ishikawa Yasuyuki
• Fujiwara Yusuke
• Kishimoto Michimasa

Classifications

• C08F122/10

  Esters · CPC title

• C07K2317/622

  Single chain antibody (scFv) · CPC title

• C07K16/3007

  Carcino-embryonic Antigens · CPC title

• C07K2319/35

  containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin · CPC title

• C07K16/4241 Primary

  against anti-human or anti-animal Ig · CPC title