Deamidated interferon-beta

Abstract

Interferon-β protein analogs in which the asparagine at position 25, numbered in accordance with native interferon-β, is deamidated exhibit a biological activity of native human interferon-β at an increased level and do not require HA for protein stabilization. The deamidated product is suitable for large scale manufacturing for incorporation in HA-containing or HA-free therapeutics for treatment of diseases including multiple sclerosis. An endoproteinase-C peptide map technique that produces a fingerprint profile for proteins using an enzymatic digest followed by RP-HPLC is also useful in quality control as an ID and/or quantitative test for the deamidated products.

1 . A purified and isolated synthetic human interferon-β protein analog, wherein,
 the asparagine at position 25, numbered in accordance with native interferon-β, is deamidated, and,   said protein analog exhibits a biological activity of native human interferon-β.   
     
     
         2 . The synthetic protein analog of  claim 1 , wherein the cysteine at position 17, numbered in accordance with native interferon-β, is deleted or replaced by a neutral amino acid. 
     
     
         3 . The synthetic protein analog of  claim 2 , wherein said cysteine residue has been replaced by a serine residue. 
     
     
         4 . The synthetic protein analog of  claim 3 , wherein said asparagine residue has been replaced by a residue selected from the group consisting of aspartate, iso-aspartate and cyclic imide. 
     
     
         5 . The synthetic protein analog of  claim 4 , wherein the protein analog is unglycosylated. 
     
     
         6 . The synthetic protein analog of  claim 5 , wherein the protein analog has an N-terminal methionine deletion. 
     
     
         7 . The synthetic protein analog of  claim 4 , wherein the protein analog has a biological activity greater than IFN-β ser17 . 
     
     
         8 . A therapeutic composition having IFN-β activity comprising a therapeutically effective amount of the synthetic protein analog of  claims 4  admixed with a pharmaceutically acceptable carrier medium. 
     
     
         9 . The composition of  claim 8 , wherein at least 50% of the synthetic protein analog is deamidated at position 25, numbered in accordance with native interferon-β. 
     
     
         10 . The composition of  claim 8 , wherein substantially all of the synthetic protein analog is deamidated at position 25, numbered in accordance with native interferon-β. 
     
     
         11 . The composition of  claim 8 , wherein the composition is HA-free. 
     
     
         12 . A method of making deamidated INF-β analog, comprising:
 incubating an IFN-β protein under conditions suitable at moderate to high temperature of about 25-60° C.;   purifying and isolating the deamidated protein analog.   
     
     
         13 . The method of  claim 12 , comprising:
 incubation at a temperature of about 40° C. for about 14 days.   
     
     
         14 . The method of  claim 13 , further comprising wherein the incubation is conducted at a pH of at least 4. 
     
     
         15 . The method of  claim 14 , comprising: incubation at a pH of 7-14. 
     
     
         16 . The method of  claim 15 , wherein the incubation is at pH 8-9 for about 14 days. 
     
     
         17 . A method of treating a patient comprising administering to said patient an effective amount of the composition of  claim 8 . 
     
     
         18 . The method of  claim 17 , wherein the treatment is for regulating cell growth in the patient and the effective amount is a cell growth regulating amount of the composition. 
     
     
         19 . The method of  claim 17 , wherein the treatment is for a viral disease of the patient and the effective amount is a viral disease inhibiting amount of the composition. 
     
     
         20 . The method of  claim 17 , wherein the treatment is for stimulating natural killer cell activity in the patient and the effective amount is a natural killer cell stimulating amount of the composition. 
     
     
         21 . The method of  claim 17 , wherein the treatment is for multiple sclerosis in the patient and the effective amount is a therapeutically effective amount of the composition. 
     
     
         22 . The method of  claim 21 , wherein the treatment comprises reducing the frequency of multiple sclerosis flare-ups. 
     
     
         23 . The method of  claim 22 , wherein said multiple sclerosis is relapsing remitting type. 
     
     
         24 . A peptide mapping method, comprising:
 incubating a protein sample in a buffered solution below pH 8 containing a reducing agent;   digesting the incubated sample with endoproteinase-C; and   resolving the peptide fragments of the digest by liquid chromatography.   
     
     
         25 . The method of  claim 24 , wherein the reducing agent is tris-(2-carboxyethyl) phosphine (TCEP). 
     
     
         26 . The method of  claim 25 , wherein the incubation is conducted at a pH of about 4. 
     
     
         27 . The method of  claim 26 , wherein the liquid chromatography is RP-HPLC. 
     
     
         28 . The method of  claim 27 , wherein the protein sample is a deamidated IFN-β.