Homogenous antibody drug conjugates via enzymatic methods

A method of making an antibody drug conjugate comprising an antibody specifically conjugated to a conjugate moiety comprising a small molecule handle and an active moiety comprising:

• a) contacting an antibody composition comprising the antibody with the small molecule handle in the presence of a transglutaminase under a condition sufficient to generate an intermediate conjugate comprising the antibody specifically conjugated to the small molecule handle, wherein the transglutaminase is a wildtype transglutaminase and the concentration of the transglutaminase in the reaction mixture is about 0.1 mg/ml to about 1 mg/ml, and
• b) contacting the intermediate conjugate with the active moiety thereby obtaining the antibody drug conjugate, wherein at least about 50% of the antibody in the antibody composition is glycosylated in the Fc-region, wherein the conjugate moiety is conjugated to an endogenous acceptor glutamine residue on the antibody, and wherein the active moiety is selected from the group consisting of a moiety that improves the pharmacokinetic property of the antibody composition, a therapeutic moiety, and a diagnostic moiety.

A method of making an antibody drug conjugate comprising an antibody specifically conjugated to a conjugate moiety comprising:

• contacting an antibody composition comprising the antibody with the conjugate moiety in the presence of a transglutaminase under a condition sufficient to generate the antibody drug conjugate, wherein at least about 50% of the antibody in the antibody composition is glycosylated in the Fc-region, wherein the conjugate moiety is conjugated to an endogenous acceptor glutamine residue on the antibody, and wherein the transglutaminase is a wildtype transglutaminase with an added proline residue at the N-terminus, and optionally a purification tag, and the concentration of the transglutaminase in the reaction mixture is about 0.1 mg/ml to about 1 mg/ml.