N-terminally chemically modified protein compositions and methods

Abstract

Provided herein are methods and compositions relating to the attachment of water soluble polymers to proteins. Provided are novel methods for N-terminally modifying proteins or analogs thereof, and resultant compositions, including novel N-terminally chemically modified G-CSF compositions and related methods of preparation. Also provided is chemically modified consensus interferon.

What is claimed is: 
     
         1 . A substantially homogenous preparation of N-terminally chemically modified G-CSF or analog thereof, optionally in a pharmaceutically acceptable diluent, carrier or adjuvant. 
     
     
         2 . A preparation of  claim 1  where said G-CSF is chemically modified with a chemical selected from the group consisting of dextran, poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols. 
     
     
         3 . A preparation of  claim 2  where said G-CSF or analog thereof is chemically modified with polyethylene glycol. 
     
     
         4 . A preparation of  claim 3  said polyethylene glycol has a molecular weight of between about 2 kDa and 100 kDa. 
     
     
         5 . A preparation of  claim 4  wherein said polyethylene glycol has a molecular weight of between about 6 kDa and 25 kDa. 
     
     
         6 . A preparation of  claim 1  wherein said preparation is comprised of at least 90% N-terminally monopegylated G-CSF or analog thereof and at most 10% unpegylated G-CSF or analog thereof. 
     
     
         7 . A preparation of  claim 6  wherein said preparation is comprised of at least 95% N-terminally monopegylated G-CSF or analog thereof and at most 5% unpegylated G-CSF or analog thereof. 
     
     
         8 . A preparation of  claim 1  wherein said G-CSF has the sequence identified in SEQ. ID No. 1. 
     
     
         9 . A substantially homogenous preparation of N-terminally monopegylated G-CSF, optionally in a pharmaceutically acceptable diluent, carrier or adjuvant, wherein: (a) said G-CSF has the amino acid sequence identified in SEQ. ID No. 1; (b) said G-CSF is monopegylated with a polyethylene glycol moiety having a molecular weight of about 12 kDa. 
     
     
         10 . A pharmaceutical composition comprising: (a) a substantially homogenous preparation of monopegylated G-CSF, said monopegylated G-CSF consisting of a polyethylene glycol moiety having a molecular weight of about 12 kDa connected to a G-CSF moiety solely at the N-terminus thereof via an amine linkage; (b) fewer than 5% non-pegylated G-CSF molecules; and (c) a pharmaceutically acceptable diluent, adjuvant or carrier. 
     
     
         11 . A method of treating a hematopoietic disorder comprising administering a therapeutically effective dose of a preparation of any of  claims 1 - 10 . 
     
     
         12 . A method for attaching a water soluble polymer to a protein or analog thereof, wherein said water soluble polymer has a single reactive aldehyde croup, said method comprising:
 (a) reacting a protein moiety with a water soluble polymer moiety under reducing alkylation conditions, at a pH sufficiently acidic to selectively activate the α-amino group at the amino terminus of said protein moiety so that said water soluble polymer selectively attaches to said α-amino group; and   (b) obtaining the reaction product and   (c) optionally, separating the reaction products from unreacted moieties.   
     
     
         13 . A method of  claim 12  wherein said polymer is pharmaceutically acceptable. 
     
     
         14 . A method of  claim 12  wherein said water soluble polymer is selected from the group consisting of dextran, poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols. 
     
     
         15 . A method of  claim 14  wherein said polymer is polyethylene glycol. 
     
     
         16 . A method of  claim 12  wherein said reducing alkylation reaction involves the use of a reducing agent selected from sodium borohydride, sodium cyanoborohydride, dimethylamine borate, timethylamine borate and pyridine borate. 
     
     
         17 . A method for attaching a polyethylene glycol molecule to a G-CSF molecule, wherein said polyethylene glycol molecule has a single reactive aldehyde group, said method comprising:
 (a) reacting said G-CSF with said polyethylene glycol molecule under reducing alkylation conditions, at a pH sufficiently acidic to selectively activate the α-amino group at the amino terminus of said G-CSF; and   (b) obtaining the pegylated G-CSF and   (c) optionally, separating the pegylated G-CSF from non-pegylated G-CSF.   
     
     
         18 . A method of  claim 17  wherein said polyethylene glycol molecule has a molecular weight of about 6 kDa to about 25 kDa. 
     
     
         19 . The pegylated G-CSF product produced by the process of  claim 17 . 
     
     
         20 . Chemically modified consensus interferon comprised of a consensus interferon protein moiety connected to at least one water soluble polymer moiety. 
     
     
         21 . A chemically modified consensus interferon of  claim 20  wherein said consensus interferon moiety is selected from the group consisting of IFN-con 1 , IFN-con 2 , and IFN-con 3 . 
     
     
         22 . A chemically modified consensus interferon of  claim 21  wherein said water soluble polymer is pharmaceutically acceptable. 
     
     
         23 . A chemically modified consensus interferon of  claim 20  wherein said water soluble polymer is selected from the group consisting of dextran, poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols. 
     
     
         24 . A chemically modified consensus interferon according to  claim 23  wherein said water soluble polymer moiety is polyethylene glycol. 
     
     
         25 . A chemically modified consensus interferon according to  claim 20  wherein said water soluble polymer moiety is connected to said consensus interferon moiety directly without an additional linkage group. 
     
     
         26 . A chemically modified consensus interferon comprised of IFN-con 1  connected to at least one polyethylene glycol moiety. 
     
     
         27 . Pegylated consensus interferon. 
     
     
         28 . A method for attaching a water soluble polymer to consensus interferon, wherein said water soluble polymer has a single reactive aldehyde group, said method comprising:
 (a) reacting a consensus interferon moiety with a water soluble polymer moiety under reducing alkylation conditions, at a pH sufficiently acidic to selectively activate the α-amino group at the amino terminus of said consensus interferon moiety; and   (b) obtaining the reaction product and   (c) optionally, separating the reaction products from unreacted moieties.   
     
     
         29 . A method of  claim 28  wherein said polymer is pharmaceutically acceptable. 
     
     
         30 . A method of  claim 28  wherein said water soluble polymer is selected from the group consisting of dextran, poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols. 
     
     
         31 . A method of  claim 30  wherein said polymer is polyethylene glycol. 
     
     
         32 . A method of  claim 28  wherein said reducing alkylation reaction involves the use of a reducing agent selected from sodium borohydride, sodium cyanoborohydride, dimethylamine borate, timethylamine borate and pyridine borate. 
     
     
         33 . A method for attaching a polyethylene glycol molecule to a consensus interferon molecule, wherein said polyethylene glycol molecule has a single reactive aldehyde group, said method comprising:
 (a) reacting said consensus interferon with said polyethylene glycol molecule under reducing alkylation conditions, at a pH sufficiently acidic to selectively activate the α-amino group at the amino terminus of said consensus interferon; and   (b) obtaining the pegylated consensus interferon and   (c) optionally, separating the pegylated consensus interferon from non-pegylated consensus interferon.   
     
     
         34 . A method of  claim 33  wherein said polyethylene glycol molecule has a molecular weight of about 2 kDa to about 100 kDa. 
     
     
         35 . The pegylated consensus interferon product produced by the process of  claim 33 . 
     
     
         36 . A substantially homogenous preparation of monopegylated consensus interferon. 
     
     
         37 . A preparation of  claim 36  comprising about 90% monopegylated consensus interferon and about 10% unpegylated consensus interferon. 
     
     
         38 . A pharmaceutical composition comprising: (a) a substantially homogenous preparation of monopegylated consensus interferon, said monopegylated consensus interferon consisting of a polyethylene glycol moiety connected to a consensus interferon moiety solely at the N-terminus thereof via an amine linkage; (b) fewer than 5% non-pegylated consensus interferon molecules; and (c) a pharmaceutically acceptable diluent, adjuvant or carrier.