Modified Human Growth Hormone

Abstract

Modified growth hormone polypeptide and uses thereof are provided.

1 - 54 . (canceled) 
     
     
         55 . A method of making a GH linked via an oxime bond to a water-soluble polymer comprising contacting a GH that comprises a NEAA comprising a carbonyl group with a PEG oxyamine under conditions suitable for formation of an oxime bond. 
     
     
         56 . The method of  claim 55  wherein the NEAA contains a ketone group. 
     
     
         57 . The method of  claim 56  wherein the NEAA is para-acetylphenylalanine. 
     
     
         58 . The method of  claim 57  wherein the para-acetylphenylalanine is substituted at a position in the GH, e.g., hGH corresponding to amino acid 35 in SEQ ID NO: 2. 
     
     
         59 . The method of  claim 55  wherein the PEG oxyamine is a monomethoxyPEG (MPEG) oxyamine. 
     
     
         60 . The method of  claim 59  wherein the MPEG oxyamine is linear. 
     
     
         61 . The method of  claim 60  wherein the MW of the PEG is about 20-40 kDa. 
     
     
         62 . The method of  claim 61  wherein the MW of the PEG is about 30 kDa. 
     
     
         63 . The method of  claim 62  wherein the MPEG oxyamine is a linear 30 kD monomethoxy-PEG-2-aminooxy ethylamine carbamate hydrochloride. 
     
     
         64 . The method of  claim 55  further comprising making the GH comprising an NEAA by a process comprising introducing
 (i) a nucleic acid encoding a GH wherein the nucleic acid has been modified to provide a signal for incorporation of the NEAA; and 
 (ii) the NEAA; to an organism that is capable of incorporating the NEAA into a protein in response to the signal of the nucleic acid of (i). 
 
     
     
         65 . The method of  claim 55  wherein the conditions comprise
 (i) mixing the MPEG and GH to produce a MPEG-GH mixture with a MPEG:GH ratio of about 5 to 10, 
 (ii) a pH of about 4 to 6; and 
 (iii) gentle stirring of the MPEG-GH mixture for about 10 to 50 hours at room temperature. 
 
     
     
         66 . The method of  claim 55  further comprising purifying the GH. 
     
     
         67 . The method of  claim 56  wherein the GH produced by the process is at least about 99% pure. 
     
     
         68 - 79 . (canceled)