Production of Conjugates

Abstract

A method of reacting a first chemical entity and a second chemical entity to form a conjugate in which the first and second chemical entities are covalently bound with respect to each other, comprises bringing into simultaneous contact the first chemical entity, the second chemical entity and a thiol generator, wherein the thiol generator reacts with the first chemical entity in a thiolation reaction resulting in formation of a free sulfhydryl group on the first chemical entity, and the free sulfhydryl group reacts with the second chemical entity to form the conjugate, and wherein the second chemical entity is polyvalent with respect to its reactivity with sulfhydryl groups. The present invention primarily differs from the prior art in that no separation step is involved between reaction of the thiol generator and first chemical entity and reaction with the second chemical entity. The invention also provides a conjugation kit.

1 . A method of reacting a first chemical entity and a second chemical entity to form a conjugate in which the first and second chemical entities are covalently bound with respect to each other, comprising bringing into simultaneous contact the first chemical entity, the second chemical entity and a thiol generator, wherein the thiol generator reacts with the first chemical entity in a thiolation reaction resulting in formation of a free sulfhydryl group on the first chemical entity, and the free sulfhydryl group reacts with the second chemical entity to form the conjugate, and wherein the second chemical entity is polyvalent with respect to its reactivity with sulfhydryl groups. 
     
     
         2 . A method according to  claim 1 , wherein the thiol generator comprises one or more of a thiolactone, an iminothiolactone, an episulfide and a thiazolidine. 
     
     
         3 . (canceled) 
     
     
         4 . A method according to  claim 2 , wherein reaction of the thiol generator with a primary amine on the first chemical entity takes place at a pH below 8. 
     
     
         5 . (canceled) 
     
     
         6 . A method according to  claim 1 , comprising a first step in which the thiol generator and the first chemical entity are combined, and a second step in which the second chemical entity is combined with the thiol generator and first chemical entity. 
     
     
         7 . A method according to  claim 1 , comprising a first step in which the first and second chemical entities are combined, and a second step in which the thiol generator is combined with the first and second chemical entities. 
     
     
         8 - 13 . (canceled) 
     
     
         14 . A method according to  claim 1 , further comprising adding a nucleophile to terminate the conjugation reaction. 
     
     
         15 . (canceled) 
     
     
         16 . A method according to  claim 1 , wherein the second chemical entity comprises a polypeptide. 
     
     
         17 . A method according to  claim 1 , wherein the second chemical entity comprises an enzyme. 
     
     
         18 . A method according to  claim 1 , wherein the second chemical entity comprises a molecule selected from the group consisting of: horseradish peroxidase, alkaline phosphatase and glucose oxidase. 
     
     
         19 . A method according to  claim 1 , wherein the second chemical entity comprises five or more sulfhydryl-reactive groups per molecule. 
     
     
         20 - 21 . (canceled) 
     
     
         22 . A method according to  claim 1 , wherein the molar ratio of second chemical entity to thiol generator is less than 2.0:1. 
     
     
         23 - 24 . (canceled) 
     
     
         25 . A method according to  claim 22 , wherein the ratio is in the range 1:10 to 1:15. 
     
     
         26 . A conjugation kit for use in the method of  claim 1 , comprising at least one sample of reagent selected from a first chemical entity, a second chemical entity and a thiol generator, and instructions for performing the method of the invention. 
     
     
         27 . A kit according to  claim 26 , comprising samples of at least two reagents selected from a first chemical entity, a second chemical entity and a thiol generator. 
     
     
         28 - 30 . (canceled) 
     
     
         31 . A kit according to  claim 26 , including at least one sample of thiol generator selected from a thiolactone, an iminothiolactone, an episulphide and a thiazolidine. 
     
     
         32 - 33 . (canceled) 
     
     
         34 . A kit according to according to an  claim 26 , wherein the reagent samples are in dried form. 
     
     
         35 . A kit according to  claim 26 , wherein the at least one sample of reagent additionally comprises a polyol. 
     
     
         36 - 37 . (canceled) 
     
     
         38 . A kit according to  claim 26 , wherein the at least one sample of reagent additionally comprises a metal ion. 
     
     
         39 - 40 . (canceled) 
     
     
         41 . A kit according to  claim 26 , wherein the at least one sample of reagent is freeze-dried and the reagent is freeze-dried from an aqueous solution comprising sodium phosphate at a pH in the range 5-6.5, preferably 5.0-6.0. 
     
     
         42 - 43 . (canceled)