Calicheamicin derivative-carrier conjugates

A method for preparing a composition comprising monomeric calicheamicin derivative/antibody conjugates with reduced low conjugated fraction (LCF) having the formula, Pr(—X—W)m, wherein:

• Pr is an antibody;
• W is a calicheamicin derivative comprising a calicheamicin;
• X is a hydrolyzable linker that links the antibody to the calicheamicin derivative and that is capable of releasing the calicheamicin derivative from the calicheamicin derivative/antibody conjugate after binding and entry into target cells;
• m is the average loading for a purified conjugation product such that the calicheamicin constitutes 4-10% of the calicheamicin derivative/antibody conjugate by weight;
• the method comprising the steps of:
• (1) adding the calicheamicin derivative to the antibody wherein the calicheamicin derivative is 4.5-11% by weight of the antibody;
• (2) incubating calicheamicin derivatives and antibodies in a non-nucleophilic, protein-compatible, buffered solution having a pH in the range from about 7 to 9 to produce a composition comprising the monomeric calicheamicin derivative/antibody conjugates, wherein the solution further comprises (a) an organic cosolvent, and (b) an additive comprising at least one C6-C18 carboxylic acid or its salt, and wherein the incubation is conducted at a temperature ranging from about 30° C. to about 35° C. for a period of time ranging from about 15 minutes to 24 hours;
• and
• (3) subjecting the composition produced in step (2) to a chromatographic separation process to separate monomeric calicheamicin derivative/antibody conjugates with a loading in the range of 4-10% by weight calicheamicin and with low conjugated fraction (LCF) below 10 percent from unconjugated antibody, calicheamicin derivative, and aggregated conjugates.