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US 2014/0350228 A1Published application
Use of n-hydroxysuccinimide to improve conjugate stability
AssigneeImmunoGen, Inc.
InventorsFang Liu, Godfrey W. Amphlett, Deborah H. Meshulam
Life of this patent
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Abstract
The invention provides processes for manufacturing cell-binding agent-cytotoxic agent conjugates of improved stability in the presence of exogenous NHS. In some embodiments, the inventive process comprises the addition of a molar ratio of exogenous NHS with respect to the amount of NHS generated during the modification reaction as a result of hydrolysis/aminolysis of the bifunctional linker.
Claims32
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 in the presence of exogenous N-hydroxysuccinimide (NHS) 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 cell-binding agent-cytotoxic agent conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent through a linker, which process comprises:
- (a) contacting a cell-binding agent with a bifunctional crosslinking reagent 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 to prepare a second mixture comprising (i) the cell-binding agent-cytotoxic agent conjugate comprising the cell-binding agent chemically coupled to the cytotoxic agent through the linker, (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 agent-cytotoxic agent conjugate from the other components of the second mixture and thereby prepare a purified second mixture of the cell-binding agent-cytotoxic agent conjugate,
- wherein exogenous N-hydroxysuccinimide is added during or after step (a) and prior to step (c).
2 more elements
(canceled)
(canceled)
-21. (canceled)
A process for preparing a cell-binding agent-cytotoxic agent conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent through a linker, which process comprises:
- (a) contacting a cell-binding agent with a bifunctional crosslinking reagent 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 to prepare a second mixture comprising (i) the cell-binding agent-cytotoxic agent conjugate comprising the cell-binding agent coupled to the cytotoxic agent through the linker, (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 agent-cytotoxic agent conjugate from the other components of the second mixture and thereby prepare a purified second mixture of the cell-binding agent-cytotoxic agent conjugate,
- wherein exogenous N-hydroxysuccinimide is added during of after step (a) and prior to step (b).
1 more element
-35. (canceled)
A process for preparing a cell-binding agent-cytotoxic agent conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent through a linker, which process comprises:
- (a) contacting a cell-binding agent with a cytotoxic agent-linker compound comprising a cytotoxic agent chemically coupled to a linker to covalently attach the cytotoxic agent-linker compound to the cell-binding agent and thereby prepare a mixture comprising the cell-binding agent-cytotoxic agent conjugate comprising the cell-binding agent chemically coupled to the cytotoxic agent through the linker and
- (b) subjecting the mixture comprising the cell-binding agent-cytotoxic agent conjugate to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography or a combination thereof to purify the conjugate, wherein exogenous N-hydroxysuccinimide is added during or after step (a) and prior to step (b).
-46. (canceled)
A process for preparing a cell-binding agent-cytotoxic agent conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent through a linker, which process comprises:
- (a) contacting a cell-binding agent with a bifunctional crosslinking reagent 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 the presence of exogenous N-hydroxysuccinimide to prepare a second mixture comprising (i) the cell-binding agent-cytotoxic agent conjugate comprising the cell-binding agent coupled to the cytotoxic agent through the linker, (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 agent-cytotoxic agent conjugate from the other components of the second mixture and thereby prepare a purified second mixture of cell-binding agent-cytotoxic agent conjugate.
1 more element
-58. (canceled)
A process for preparing a cell-binding agent-cytotoxic agent conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent through a linker, which process comprises:
- (a) contacting a cell-binding agent with a bifunctional crosslinking reagent 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 the presence of exogenous N-hydroxysuccinimide to prepare a second mixture comprising (i) the cell-binding agent-cytotoxic agent conjugate comprising 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 agent-cytotoxic agent conjugate from the other components of the second mixture and thereby prepare a purified second mixture of the cell-binding agent-cytotoxic agent conjugate.
-68. (canceled)
A process for preparing a cell-binding agent-cytotoxic agent conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent through a linker, which process comprises:
- (a) contacting a cell-binding agent with a bifunctional crosslinking reagent 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 to prepare a second mixture comprising (i) the cell-binding agent-cytotoxic agent conjugate comprising the cell-binding agent chemically coupled through the linker to the cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction by-products,
- (d) incubating the second mixture in the presence of exogenous N-hydroxysuccinimide;
- and
- (e) subjecting the second mixture after step (d) to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof to purify the cell-binding agent-cytotoxic agent conjugate 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.
3 more elements
-87. (canceled)
A process for preparing a conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent through a linker, which process comprises:
- (a) contacting a cell-binding agent with a bifunctional crosslinking reagent 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 to prepare a second mixture comprising (i) the cell-binding agent-cytotoxic agent conjugate comprising the cell-binding agent chemically coupled through the linker to the cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction by-products,
- (c) incubating the second mixture in the presence of exogenous N-hydroxysuccinimide;
- and
- (d) subjecting the second mixture after step (c) to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof, to purify the cell-binding agent-cytotoxic agent conjugate from the other components of the second mixture and thereby prepare a purified second mixture of the cell-binding agent-cytotoxic agent conjugate.
2 more elements
-103. (canceled)
A process for preparing a cell-binding agent-cytotoxic agent conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent through a linker, which process comprises:
- (a) contacting a cell-binding agent with a cytotoxic agent to form a first mixture comprising the cell-binding agent and the cytotoxic agent, then contacting the first mixture with a bifunctional crosslinking reagent comprising a linker, in a solution having a pH of about 4 to about 9, to provide a second mixture comprising (i) the cell-binding agent cytotoxic agent conjugate comprising the cell-binding agent chemically coupled through the linker to the cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction by-products;
- (b) incubating the second mixture in the presence of exogenous N-hydroxysuccinimide;
- and
- (c) subjecting the second mixture after step (b) to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof, to purify the cell-binding agent-cytotoxic agent conjugate from the other components of the second mixture and thereby prepare a purified second mixture of the cell-binding agent-cytotoxic agent conjugate.
1 more element
-116. (canceled)
A process for preparing a cell-binding agent-cytotoxic agent conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent through a linker, which process comprises:
- (a) contacting a cell-binding agent with a cytotoxic agent in the presence of exogenous N-hydroxysuccinimide to form a first mixture comprising the cell-binding agent and the cytotoxic agent, then contacting the first mixture with a bifunctional crosslinking reagent comprising a linker, in a solution having a pH of about 4 to about 9, to provide a second mixture comprising (i) the cell-binding agent cytotoxic agent conjugate comprising the cell-binding agent chemically coupled through the linker to the cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction by-products;
- and
- (b) 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 agent-cytotoxic agent conjugate from the other components of the second mixture and thereby prepare a purified second mixture of the cell-binding agent-cytotoxic agent conjugate.
A process for preparing a cell-binding agent-cytotoxic agent conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent through a linker, which process comprises:
- (a) contacting a cell-binding agent with a cytotoxic agent to form a first mixture comprising the cell-binding agent and the cytotoxic agent, then contacting the first mixture with a bifunctional crosslinking reagent comprising a linker in the presence of exogenous N-hydroxysuccinimide, in a solution having a pH of about 4 to about 9, to provide a second mixture comprising (i) the cell-binding agent cytotoxic agent conjugate comprising the cell-binding agent chemically coupled through the linker to the cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction by-products;
- and
- (b) 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 agent-cytotoxic agent conjugate from the other components of the second mixture and thereby prepare a purified second mixture of the cell-binding agent-cytotoxic agent conjugate.
-123. (canceled)
A process for preparing a cell-binding agent-cytotoxic agent conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent through a linker, which process comprises:
- (a) contacting a cell-binding agent with a cytotoxic agent-linker compound comprising a cytotoxic agent chemically coupled to a linker to covalently attach the cytotoxic agent-linker compound to the cell-binding agent and thereby prepare a mixture comprising the cell-binding agent-cytotoxic agent conjugate comprising the cell-binding agent chemically coupled to the cytotoxic agent through the linker;
- (b) incubating the mixture of step (a) in the presence of exogenous N-hydroxysuccinimide;
- and
- (c) subjecting the mixture after step (b) to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof, to purify the cell binding agent-cytotoxic agent conjugate from the other components of the mixture and thereby prepare a purified mixture of the cell binding agent-cytotoxic agent conjugate.
1 more element
-159. (canceled)
Dependencies are inferred from the claim text.