Compositions comprising conjugates and maleamic acid-terminated, water-soluble polymers

Abstract

Methods for preparing conjugates using polymeric reagents bearing a maleimide are provided. Also provided are compositions comprising the conjugates.

1 . A composition comprising conjugates and maleamic acid-terminated water-soluble polymers, wherein
 (i) the composition contains less than four percent of the maleamic acid-terminated, water-soluble polymers,   (ii) the composition is substantially free of furan, and   (iii) the conjugates are prepared by a method comprising combining, under conjugation conditions, a thiol-containing biologically active agent with a plurality of maleimide-terminated, water-soluble polymers, the plurality of maleimide-terminated, water-soluble polymers having been prepared by:   a) combining a composition comprising a plurality of amine-terminated, water-soluble polymers, each amine-terminated, water-soluble polymer in the plurality encompassed by the structure,
   POLY-(X 2 ) c —NH 2 ,
 
   
       wherein
 POLY is a water-soluble polymer, 
 (c) is zero or one, and 
 X 2 , when present, is a spacer moiety, 
 
       with a composition comprising a plurality of maleimide reagents, each maleimide reagent in the plurality encompassed by the structure, 
       
         
           
           
               
               
           
         
       
       wherein
 Y 1  is O or S, 
 y 2  is O or S, 
 (a) is an integer from 1 to 20, 
 R 1 , in each instance, is independently H or an organic radical, 
 R 2 , in each instance, is independently H or an organic radical, 
 R 3 , in each instance, is independently H or an organic radical, and 
 R 4 , in each instance, is independently H or an organic radical, under substantially nonaqueous conditions to form a composition comprising a plurality of substituted maleamic acid-terminated, water-soluble polymers, each substituted maleamic acid-terminated, water-soluble polymer in the plurality encompassed by the structure, 
 
       
         
           
           
               
               
           
         
       
       wherein
 POLY is a water-soluble polymer, 
 (b) is zero or one, 
 X 1 , wherein present, is a spacer moiety, 
 Y 1  is O or S, 
 Y 2  is O or S, 
 (a) is an integer from 1 to 20, 
 R 1 , in each instance, is independently H or an organic radical, 
 R 2 , in each instance, is independently H or an organic radical, 
 R 3 , in each instance, is independently H or an organic radical, and 
 R 4 , in each instance, is independently H or an organic radical; 
 b) isolating the substituted maleamic acid-terminated, water-soluble polymer to form a composition wherein at least about 90% of all polymer species in the composition are substituted maleamic acid-terminated, water-soluble polymers; 
 c) forming a dissolved substituted maleamic acid-terminated, water-soluble polymer composition by dissolving substituted maleamic acid-terminated, water-soluble polymers contained within the substituted maleamic acid-terminated, water-soluble polymer composition; and 
 d) exposing the dissolved substituted maleamic acid-terminated, water-soluble polymer composition to elimination conditions to thereby result in a composition comprising a plurality of maleimide-terminated, water-soluble polymers, each maleimide-terminated, water-soluble polymer in the plurality encompassed by the structure, 
 
       
         
           
           
               
               
           
         
       
       wherein,
 POLY is a water-soluble polymer, 
 (b) is zero or one, 
 X 1 , wherein present, is a spacer moiety, 
 R 3 , in each instance, is independently H or an organic radical, and 
 R 4 , in each instance, is independently H or an organic radical, 
 
       wherein the elimination conditions comprise conditions selected from the group consisting of heating, addition of a drying agent, and combinations thereof. 
     
     
         2 . The composition of  claim 1 , wherein isolating the substituted maleamic acid-terminated, water-soluble polymer is effected by precipitation to form the substituted maleamic acid-terminated, water-soluble polymer composition. 
     
     
         3 . The composition of  claim 2 , wherein precipitation is effected by adding an excess of an agent selected from the group consisting of isopropyl alcohol, diethyl ether, MTBE, heptane, THF, hexane, and mixtures thereof. 
     
     
         4 . The composition of  claim 1 , wherein the method for preparing the conjugates is carried out in an organic solvent. 
     
     
         5 . The composition of  claim 4 , wherein the organic solvent is selected from the group consisting of halogenated aliphatic hydrocarbons, alcohols, aromatic hydrocarbons, halogenated aromatic hydrocarbons, amides, nitriles, ketones, acetates, ethers, cyclic ethers, and combinations thereof. 
     
     
         6 . The composition of  claim 4 , wherein the organic solvent is selected from the group consisting of dichloromethane, chloroform, acetonitrile, toluene, methyl t-butyl ether, tetrahydrofuran, octanol, ethyl acetate, diethylcarbonate, acetone, cyclohexane and combinations thereof. 
     
     
         7 . The composition of  claim 6 , wherein the organic solvent is dichloromethane or acetonitrile. 
     
     
         8 . The composition of  claim 1 , wherein the step of combining the composition comprising a plurality of amine-terminated, water-soluble polymers with the composition comprising a plurality of maleimide reagents under substantially nonaqueous conditions to form the composition comprising a plurality of substituted maleamic acid-terminated, water-soluble polymers is carried out in the presence of basic catalyst. 
     
     
         9 . The composition of  claim 1 , wherein the step of exposing the dissolved substituted maleamic acid-terminated, water-soluble polymer composition to elimination conditions is carried out in an organic solvent in the presence of a base. 
     
     
         10 . The composition of  claim 9 , wherein the base is selected from the group consisting of sodium bicarbonate, potassium bicarbonate, sodium carbonate, or potassium carbonate. 
     
     
         11 . The composition of  claim 9 , wherein the step of exposing the dissolved substituted maleamic acid-terminated, water-soluble polymer composition to elimination conditions is carried out at a temperature of 10 to 60° C. 
     
     
         12 . The composition of  claim 1 , wherein the step of exposing the dissolved substituted maleamic acid-terminated, water-soluble polymer composition to elimination conditions is carried out in the presence of a non-nucleophilic amine catalyst. 
     
     
         13 . The composition of  claim 12 , wherein the non-nucleophilic amine catalyst is selected from the group consisting of diisopropylethylamine, triethylamine, n-methyl morpholine, pyridine, N,N-Dimethyl-4-aminopyridine, 1,8-Diazabicyclo[5.4.0]undec-7-ene, and 1,4-diazabicyclo[2.2.2]octane. 
     
     
         14 . The composition of  claim 1 , wherein the step of exposing the dissolved substituted maleamic acid-terminated, water-soluble polymer composition to elimination conditions comprises performing the step in the presence of a drying agent. 
     
     
         15 . The composition of  claim 14 , wherein the drying agent is selected from the group consisting of NaHCO 3 , Na 2 CO 3 , CaCl 2 , CaSO 4 , MgSO 4 , KOH, Na 2 SO 4 , K 2 CO 3 , KHCO 3 , molecular sieves and combinations thereof. 
     
     
         16 . The composition of  claim 1 , wherein the water-soluble polymer is a poly(ethylene glycol). 
     
     
         17 . The composition of  claim 16 , wherein the poly(ethylene glycol) has a molecular weight of about 100 to about 100,000 Daltons. 
     
     
         18 . The composition of  claim 1 , wherein the water-soluble polymer is a linear water-soluble polymer. 
     
     
         19 . The composition of  claim 1 , wherein the water-soluble polymer is CH 3 O—(CH 2 CH 2 O) n —CH 2 CH 2  a is one, and further wherein n is 2 to 4000. 
     
     
         20 . The composition of  claim 1 , wherein the water-soluble polymer is branched. 
     
     
         21 . The composition of  claim 1 , wherein the substantially nonaqueous conditions represents a reaction medium having less than 1000 parts per million of water. 
     
     
         22 . The composition of  claim 20 , wherein the substantially nonaqueous conditions represents a reaction medium having less than 100 parts per million of water. 
     
     
         23 . The composition of  claim 20 , wherein the substantially nonaqueous conditions represents a reaction medium having less than 60 parts per million of water. 
     
     
         24 . The composition of  claim 1 , wherein the thiol-containing biologically active agent is a protein bearing a thiol group.