COMPOSITIONS COMPRISING CONJUGATES AND MALEAMIC ACID-TERMINATED, WATER-SOLUBLE POLYMERS
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.