Method of conjugation of Cys-mAbs
AssigneeAmgen Inc.
InventorsMichal Achmatowicz, Dante Romanini, James R. Falsey, Bradley J. Herberich, Oliver R. Thiel
Life of this patent
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Abstract
The present disclosure relates to a method of capping, reducing, and oxidizing cys-mAbs in order to provide homogenous material for subsequent conjugation reactions. The present method demonstrates robust ways to manufacture conjugates of cysteine-engineered antibodies that offer high yield and consistent product quality.
Claims24
A method for preparing an antibody conjugate, the method comprising the steps of:
- a) exposing an intact antibody to cysteamine, wherein the cysteamine forms a mixed-disulfide bond with at least one cysteine residue of a heavy chain of the intact antibody, wherein the at least one cysteine residue is at the amino acid position relative to position E276 of SEQ ID NO:
- 8;
- b) contacting the intact antibody, after step a), with trisodium triphenylphosphine-3, 3′,3″-trisulfonate (“TPPTS”) to reduce the mixed-disulfide bond to form a reduced intact antibody;
- c) contacting the reduced intact antibody with an oxidizing agent to form an oxidized intact antibody;
- and
- d) contacting the oxidized intact antibody with an activated chemical moiety to form the antibody conjugate.
A method for preparing an antibody-GLP-1 receptor agonist conjugate, the method comprising the steps of:
- a) exposing an intact antibody to cysteamine, wherein the cysteamine forms a mixed-disulfide bond with at least one cysteine residue of a heavy chain of the intact antibody, wherein the at least one cysteine residue is at the amino acid position relative to position E276 of SEQ ID NO:
- 8;
- b) contacting the intact antibody, after step a), with trisodium triphenylphosphine-3, 3′,3″-trisulfonate (“TPPTS”) to reduce the mixed-disulfide bond to form a reduced intact antibody;
- c) contacting the reduced intact antibody with an oxidizing agent to form an oxidized intact antibody;
- and
- d) contacting the oxidized intact antibody with a bromoacetylated GLP-1 receptor agonist to form the antibody-GLP-1 receptor agonist conjugate.
A method for preparing an anti-GIPR antibody conjugate, the method comprising the steps of:
- a) exposing an intact anti-GIPR antibody to cysteamine, wherein the cysteamine forms a mixed-disulfide bond with at least one cysteine residue of a heavy chain of the intact anti-GIPR antibody;
- b) contacting the intact anti-GIPR antibody, after step a), with trisodium triphenylphosphine-3, 3′,3′'-trisulfonate (“TPPTS”) to reduce the mixed-disulfide bond to form a reduced intact anti-GIPR antibody;
- c) contacting the reduced intact anti-GIPR antibody with an oxidizing agent to form an oxidized intact anti-GIPR antibody;
- and
- d) contacting the oxidized intact anti-GIPR antibody with an activated chemical moiety to form the anti-GIPR antibody conjugate.
A method for preparing an anti-GIPR antibody-GLP-1 receptor agonist conjugate, the method comprising the steps of;
- a) exposing an intact anti-GIPR antibody to cysteamine, wherein the cysteamine forms a mixed-disulfide bond with at least one cysteine residue of a heavy chain of the intact anti-GIPR antibody;
- b) contacting the intact anti-GIPR antibody, after step a), with trisodium triphenylphosphine-3, 3′,3″-trisulfonate (“TPPTS”) to reduce the mixed-disulfide bond to form a reduced intact anti-GIPR antibody;
- c) contacting the reduced intact anti-GIPR antibody with an oxidizing agent to form an oxidized intact anti-GIPR antibody;
- and
- d) contacting the oxidized intact anti-GIPR antibody with a bromoacetylated GLP-1 receptor agonist to form the anti-GIPR antibody-GLP-1 receptor agonist conjugate.
A method for preparing an anti-GIPR antibody-GLP-1 receptor agonist conjugate, the method comprising the steps of:
- a) obtaining an anti-GIPR antibody comprising two light chains and two heavy chains, wherein each light chain comprises a complementarity determining region 1 (LCDR1), a LCDR2, and LCDR3 of SEQ ID NOs:
- 156-158, respectively, and each heavy chain comprises a complementary determining region 1 (HCDR1), a HCDR2, and a HCDR3 of SEQ ID NOs:
- 162-164, respectively;
- b) exposing the anti-GIPR antibody to cysteamine, wherein the cysteamine forms a mixed-disulfide bond with a cysteine residue in each heavy chain of the anti-GIPR antibody;
- c) contacting the anti-GIPR antibody, after step b), with trisodium triphenylphosphine-3, 3′,3″'-trisulfonate to reduce the mixed-disulfide bond to form a reduced anti-GIPR antibody;
- d) contacting the reduced anti-GIPR antibody with an oxidizing agent to form an oxidized anti-GIPR antibody;
- and
- e) contacting the oxidized anti-GIPR antibody with a bromoacetylated GLP-1 receptor agonist of SEQ ID NO:
- 129 C-terminally linked to a peptide linker of SEQ ID NO:
- 29, to form the anti-GIPR antibody-GLP-1 receptor agonist conjugate.
Dependencies are inferred from the claim text.