Details for Patent: 8,546,493

Title:	Multi-armed, monofunctional, and hydrolytically stable derivatives of poly(ethylene glycol) and related polymers for modification of surfaces and molecules
Abstract:	Multi-armed, monofunctional, and hydrolytically stable polymers are described having the structure ##STR00001## wherein Z is a moiety that can be activated for attachment to biologically active molecules such as proteins and wherein P and Q represent linkage fragments that join polymer arms poly.sub.a and poly.sub.b, respectively, to central carbon atom, C, by hydrolytically stable linkages in the absence of aromatic rings in the linkage fragments. R typically is hydrogen or methyl, but can be a linkage fragment that includes another polymer arm. A specific example is an mPEG disubstituted lysine having the structure ##STR00002## where mPEG.sub.a and mPEG.sub.b have the structure CH.sub.3O--(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2-- where n may be the same or different for poly.sub.a- and poly.sub.b- and can be from 1 to about 1,150 to provide molecular weights of from about 100 to 100,000.
Inventor(s):	Harris; J. Milton (Huntsville, AL), Veronese; Francesco M. (Padua, IT), Caliceti; Paolo (Padua, IT), Schiavon; Oddone (Padua, IT)
Assignee:	Nektar Therapeutics (San Francisco, CA)
Filing Date:	Dec 14, 2012
Application Number:	13/714,917
Claims:	1. A method for preparing a branched, monofunctional, water-soluble polymer, the method comprising: (i) reacting a monomethoxypoly(ethylene glycol)polymer comprising a single reactive moiety, W, and having the structure mPEG-W, where W is the single reactive moiety, with a linker molecule comprising from 1 to 10 carbon atoms and having the structure X--C(Y)(R)--Z, where X and Y each independently comprise reactive groups that can react with W to form hydrolytically stable linkage fragments P and Q, respectively, Z is a moiety that (a) comprises a single ionizable reactive site that is capable of reacting with ion exchange chromatography media, and (b) is not reactive with either X or Y, and R is H, methyl or another alkyl group, under conditions effective to provide an impure branched monofunctional, water-soluble polymer having the structure: ##STR00039## "mPEG-P--C(R)(Z)-Q-mPEG", and one or more polymeric impurities, where neither the linker molecule nor the resulting linkage fragments P or Q comprises aromatic or ester groups, and (ii) purifying the impure branched monofunctional, water-soluble polymer from step (i) by ion exchange chromatography under conditions effective to essentially remove the polymeric impurities to thereby provide purified mPEG-P--C(R)(Z)-Q-mPEG in essentially pure form. 2. The method of claim 1, wherein mPEG-P--C(R)(Z)-Q-mPEG is formed in step (i) in a single reaction step. 3. The method of claim 1, wherein the conditions effective to form mPEG-P--C(R)(Z)-Q-mPEG comprise at least two reaction steps. 4. The method of claim 3, wherein the at least two reaction steps comprise reaction of mPEG-W with the linker molecule, X--C(Y)(R)--Z, to form a mono-substituted intermediate polymer product, mPEG-P--C(R)(Z)-Q, followed by reaction of mPEG-P--C(R)(Z)-Q with mPEG-W', a monomethoxypoly(ethylene glycol)polymer comprising a single reactive moiety, W', where W' can be the same or different from W, to form mPEG-P--C(R)(Z)-Q-mPEG. 5. The method of claim 4, wherein prior to reaction of mPEG-P--C(R)(Z)-Q with mPEG-W', mPEG-P--C(R)(Z)-Q is recovered. 6. The method of claim 1, wherein mPEG-W has a molecular weight from about 100 to 100,000 daltons. 7. The method of claim 1, wherein Z comprises a single ionizable reactive site that is selected from carboxyl, hydroxyl, and amino. 8. The method of claim 1, wherein the linker molecule is lysine or is a diamino alcohol. 9. The method of claim 8, wherein the linker molecule is lysine. 10. The method of claim 1, wherein the one or more polymeric impurities are selected from PEG diol, mPEG-OH, and mPEG-W. 11. The method of claim 1, wherein the single reactive moiety, W, is selected from the group consisting of active ester, active carbonate, aldehyde, vinyl sulfone, and iodoacetamide. 12. The method of claim 10, wherein the one or more polymeric impurities further comprise mPEG-P--C(R)(Z)-Q. 13. The method of claim 1, wherein the single reactive moiety W is an active ester and the linker molecule is lysine. 14. The method of claim 1, wherein the purifying step comprises: loading the impure branched monofunctional, water-soluble polymer and one or more polymeric impurities onto an ion exchange chromatography medium to provide a loaded medium, washing the polymeric impurities from the loaded medium using an aqueous eluent under conditions effective to elute the impurities from the medium, adjusting the conditions of the aqueous eluent to effect elution of the branched monofunctional water-soluble polymer from the medium, eluting the branched monofunctional water-soluble polymer from the medium to provide an aqueous solution comprising the purified branched monofunctional water-soluble polymer in essentially pure form, and recovering the purified branched monofunctional water-soluble polymer from the aqueous solution. 15. The method of claim 1, wherein Z is a carboxyl group. 16. The method of claim 1, further comprising (iii) recovering the branched, monofunctional, water-soluble polymer in essentially pure form. 17. A branched, monofunctional, water-soluble polymer in essentially pure form prepared by the method of claim 1. 18. A method of preparing a conjugate of a branched, monofunctional water-soluble polymer, the method comprising reacting the branched, monofunctional, water-soluble polymer of claim 1 in essentially pure form, mPEG-P--C(R)(Z)-Q-mPEG, where Z is optionally transformed to a Z-activated group suitable for reaction with a nucleophilic group of a biologically active molecule, with one or more nucleophilic groups of a biologically active molecule under conditions effective to form a conjugate of the branched, monofunctional water-soluble polymer and the biologically active molecule. 19. The method of claim 18, wherein Z is a carboxyl group, the Z-activated group is an active ester, and the nucleophilic group of the biologically active molecule is selected from amino, thiol, and hydroxyl. 20. The method of claim 18, wherein the biologically active molecule is selected from the group consisting of a peptide, a protein, a nucleotide, a polynucleotide, a lipid and a small molecule drug.