Details for Patent: 8,354,477
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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-- wherein 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 Maria (Padua, IT), Caliceti; Paolo (Padua, IT), Schiavon; Oddone (Padua, IT) |
Assignee: | Nektar Therapeutics (San Francisco, CA) |
Filing Date: | Aug 03, 2010 |
Application Number: | 12/849,683 |
Claims: | 1. A method for preparing a conjugate of a purified branched water-soluble polymer, comprising: a. providing an impure polymer composition comprising (i) a branched water-soluble polymer having the structure: ##STR00039## where Z is a moiety comprising a site suitable for interacting with ion exchange chromatography media, and mPEG.sub.a and mPEG.sub.b are each independently a methoxy polyethylene glycol, and (ii) one or more polymeric impurities selected from the group consisting of PEG diol, mPEG-OH, and activated mPEG, b. purifying the impure polymer composition by ion exchange chromatography under conditions effective to essentially remove the polymeric impurities to thereby provide a purified branched water-soluble polymer that is in essentially pure form, c. optionally reacting the purified branched water-soluble polymer with a reagent effective to transform Z to a Z-activated moiety suitable for reaction with a nucleophilic group of a biologically active molecule, and d. reacting the purified branched water soluble polymer of step b. or step c. with one or more of the nucleophilic groups of the biologically active molecule under conditions effective to form a conjugate of the purified branched water soluble polymer and the biologically active molecule. 2. The method of claim 1 wherein Z is carboxyl. 3. The method of claim 1, wherein said polymeric impurities further comprise a mono-substituted mPEG intermediate. 4. The method of claim 3, wherein the mono-substituted mPEG intermediate is mPEG-mono-substituted lysine. 5. The method of claim 1, wherein said purifying further comprises: loading the impure polymer composition onto an ion exchange chromatography medium to provide a loaded medium, washing the polymeric impurities from said loaded medium using an aqueous eluent under conditions effective to elute said impurities from said medium, adjusting the conditions of the aqueous eluent to effect elution of said branched water-soluble polymer from the medium, eluting said branched water-soluble polymer from said medium to provide an aqueous solution comprising the purified branched water-soluble polymer in essentially pure form, and recovering the purified branched water-soluble polymer from said aqueous solution. 6. The method of claim 1, wherein said branched water soluble polymer has a molecular weight ranging from about 10,000 daltons to about 50,000 daltons. 7. The method of claim 6, wherein the branched water soluble polymer has a molecular weight of about 40,000 daltons. 8. The method of claim 1, wherein mPEG.sub.a and mPEG.sub.b are the same. 9. The method of claim 1 including step c., wherein the Z-activated moiety is an active ester. 10. The method of claim 9, wherein the active ester is a succinimidyl ester. 11. The method of claim 1, including step c., wherein the Z-activated moiety is selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active carbonate, aldehyde, sulfone, vinyl sulfone, malemide, iodoacetamide, and iminoester. 12. method of claim 11, wherein the Z-activated moiety is an active carbonate selected from succinimidyl carbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate. 13. The method of claim 1, 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. 14. The method of claim 1, wherein the nucleophilic group on the biologically active molecule is selected from amino, thiol, and hydroxyl. 15. The method of claim 14, wherein the nucleophilic group on the biologically active molecule is amino. 16. The method of claim 1, wherein the biologically active molecule is an interferon. 17. The method of claim 16, wherein the interferon is an interferon-.alpha.. 18. The method of claim 17, wherein the nucleophilic group is an amino group. 19. A method according to claim 1 including step c., wherein: (i) the branched water soluble polymer in a.(i) is a mPEG-disubstituted lysine branched polymer having the structure: ##STR00040## (ii) the one or more polymeric impurities are selected from the group consisting of PEG diol, mPEG-OH, activated mPEG, and mPEG-mono-substituted lysine, (iii) the --COOH group of the purified mPEG-disubstituted lysine is transformed to an activated ester, and (iv). step d. comprises reacting the purified activated ester of step c. with amino groups of an interferon molecule under conditions effective to form an interferon mPEG-disubstituted lysine conjugate. |