Method for the preparation of substantiallly monomeric calicheamicin derivative/carrier conjugates

A method for preparing monomeric calicheamicin derivative/carrier conjugates with higher drug loading/yield and decreased aggregation having the formula,

• Pr(--X--S--S--W).sub.m
• wherein:
• Pr is a proteinaceous carrier,
• X is a linker that comprises a product of any reactive group that can react with a proteinaceous carrier,
• W is the calicheamicin residue formed when a calicheamicin is cleaved at a methyl trisulfide linkage;
• and
• m is from 0.1 to 15,
• said method comprising the steps of:
• (1) incubating a calicheamicin derivative (X--S--S--W) and a proteinaceous carrier (Pr) in a phosphate buffered solution which solution further comprises a cosolvent t-butanol, wherein the incubation is conducted at a temperature ranging from about 25.degree. C. to about 37.degree. C. for a period of time ranging from about 2 hours to about 18 hours to produce a calicheamicin derivative/carrier conjugate;
• and
• (2) purifying the calicheamicin derivative/carrier conjugate produced in step (1) to produce a monomeric calicheamicin derivative/carrier conjugate.

A method for preparing monomeric calicheamicin derivative/carrier conjugates with higher drug loading/yield and decreased aggregation having the formula,

• Pr(--X--S--S--W).sub.m
• wherein:
• Pr is a murine monoclonal antibody,
• X is a linker that comprises a product of any reactive group that can react with Pr,
• W is the calicheamicin residue formed when a calicheamicin is cleaved at a methyl trisulfide linkage, and
• m is from 0.1 to 15;
• said method comprising the steps of:
• (1) incubating a calicheamicin derivative (X--S--S--W) and a murine monoclonal antibody (Pr) in a phosphate buffered solution which solution further comprises cosolvents propylene glycol and t-butanol, wherein the incubation is conducted at a temperature ranging from about 25.degree. C. to about 37.degree. C. for a period of time ranging from about 3 hours to about 20 hours to produce a calicheamicin derivative/carrier conjugate;
• and
• (2) purifying the calicheamicin derivative/carrier conjugate produced in step (1) to produce a monomeric calicheamicin derivative/carrier conjugate.