Patent ID: 8686122

Claim:
A method for forming a conjugate comprising an antibody and an enzyme, the method comprising: oxidizing an antibody form a plurality of aldehyde groups in a glycosylated Fc portion of the antibody; reacting a molar excess of hydrazide thiol linkers with the antibody, whereby hydrazide groups of the hydrazide thiol linkers react with the aldehyde groups in the glycosylated Fc portion of the antibody to form a thiolated antibody comprising about 3-5 hydrazide thiol linkers covalently bonded to the Fc portion of the antibody, each hydrazide thiol linker comprising one or more linking atoms positioned between a hydrazide group and a thiol group, and wherein reacting comprises reacting under conditions where the thiol group of each hydrazide thiol linker is substantially present in its neutral acid form; reacting a molar excess of an N-hydroxysuccinimide (NHS)-polyethylene glycol (PEG)-maleimide linker with an enzyme to form a maleimido-enzyme comprising about 17-25 maleimide groups; and forming the conjugate by reacting the thiolated antibody with the maleimido-enzyme in about a 1:1 molar ratio at a pH greater than 7 to form a covalent bond between the thiol group of one of the hydrazide thiol linkers covalently bonded to the glycosylated Fc portion of the antibody and one maleimide group of the maleimido-enzyme.