Method for refolding antibody, process for producing refolded antibody, refolded antibody, and uses thereof
AssigneeNational University Corporation Kyoto Institute of Technology
InventorsYoichi Kumada, Yasuyuki Ishikawa, Yusuke Fujiwara, Michimasa Kishimoto
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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.
Claims17
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.
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.
Dependencies are inferred from the claim text.