Process for manufacturing conjugates of improved homogeneity
AssigneeImmunoGen, Inc.
InventorShengjin JIN
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Abstract
The invention provides processes for manufacturing cell-binding agent-cytotoxic agent conjugates of improved homogeneity comprising performing the modification reaction at a lower temperature. The inventive processes comprise contacting a cell-binding agent with a bifunctional crosslinking reagent at a temperature of about 15° C. or less to covalently attach a linker to the cell-binding agent and thereby prepare a mixture comprising cell-binding agents having linkers bound thereto.
Claims66
A process for preparing a cell-binding agent having a linker bound thereto, which process comprises contacting a cell-binding agent with a bifunctional crosslinking reagent at a temperature of about 15° C. or less to covalently attach a linker to the cell-binding agent and thereby prepare a mixture comprising cell-binding agents having linkers bound thereto.
A process for preparing a conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent, which process comprises:
- (a) contacting a cell-binding agent with a bifunctional crosslinking reagent at a temperature of about 15° C. or less to covalently attach a linker to the cell-binding agent and thereby prepare a first mixture comprising cell-binding agents having linkers bound thereto,
- (b) subjecting the first mixture to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof and thereby prepare a purified first mixture of cell-binding agents having linkers bound thereto,
- (c) conjugating a cytotoxic agent to the cell-binding agents having linkers bound thereto in the purified first mixture by reacting the cell-binding agents having linkers bound thereto with a cytotoxic agent in a solution having a pH of about 4 to about 9 to prepare a second mixture comprising (i) cell-binding agent chemically coupled through the linker to the cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction by-products, and
- (d) subjecting the second mixture to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof to purify the cell-binding agents chemically coupled through the linkers to the cytotoxic agent from the other components of the second mixture and thereby prepare a purified second mixture of cell-binding agents chemically coupled through the linkers to the cytotoxic agent.
A process for preparing a conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent, which process comprises:
- (a) contacting a cell-binding agent with a bifunctional crosslinking reagent at a temperature of about 15° C. or less to covalently attach a linker to the cell-binding agent and thereby prepare a first mixture comprising cell-binding agents having linkers bound thereto,
- (b) conjugating a cytotoxic agent to the cell-binding agents having linkers bound thereto in the first mixture by reacting the cell-binding agents having linkers bound thereto with a cytotoxic agent in a solution having a pH of about 4 to about 9 to prepare a second mixture comprising (i) cell-binding agent chemically coupled through the linker to the cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction by-products, and
- (c) subjecting the second mixture to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof, to purify the cell binding agents chemically coupled through the linkers to the cytotoxic agent from the other components of the second mixture and thereby prepare a purified second mixture of cell binding agents chemically coupled through the linkers to the cytotoxic agent.
Dependencies are inferred from the claim text.