Formulations with reduced oxidation

Abstract

The invention provides formulations comprising a protein in combination with a compound that prevents oxidation of the protein. The invention also provides methods for making such formulations and methods of using such formulations. The invention further provides methods of screening for compounds that prevent oxidation of a protein in a protein composition and methods of preventing oxidation of a protein in a formulation.

1 - 18 . (canceled) 
     
     
         19 . A method of making a protein formulation comprising adding an amount of a compound that prevents oxidation of a protein to the protein formulation, wherein the compound is of formula:
                       wherein R 2  is selected from hydrogen, hydroxyl, —COOH, and —CH 2 COOH;   R 3  is selected from hydrogen, hydroxyl, —COOH, —CH 2 COOH, and —CH 2 CHR 3a (NH 2 ); wherein R 3a  is COOH or hydrogen;   R 4 , R 5 , R 6 , and R 7  are independently selected from hydrogen and hydroxyl;   provided that one of R 2 , R 3 , R 4 , R 5 , R 6 , and R 7  is hydroxyl;   or a pharmaceutically acceptable salt thereof.   
     
     
         20 . A method of preventing oxidation of a protein in a protein formulation comprising adding an amount of a compound that prevents oxidation of the protein to the formulation, wherein the compound is of formula:
                       wherein R 2  is selected from hydrogen, hydroxyl, —COOH, and —CH 2 COOH;   R 3  is selected from hydrogen, hydroxyl, —COOH, —CH 2 COOH, and —CH 2 CHR 3a (NH 2 ); wherein R 3a  is COOH or hydrogen;   R 4 , R 5 , R 6 , and R 7  are independently selected from hydrogen and hydroxyl;   provided that one of R 2 , R 3 , R 4 , R 5 , R 6 , and R 7  is hydroxyl;   or a pharmaceutically acceptable salt thereof.   
     
     
         21 . The method of  claim 19 , wherein the compound is a compound of formula:
                       wherein R 2  and R 3  are independently selected from hydrogen, hydroxyl, —COOH, and —CH 2 COOH; and   R 4 , R 5 , R 6 , and R 7  are independently selected from hydrogen and hydroxyl;   provided that one of R 2 , R 3 , R 4 , R 5 , R 6 , and R 7  is hydroxyl;   or a pharmaceutically acceptable salt thereof.   
     
     
         22 . The method of  claim 19 , wherein the compound is a compound of formula:
                       wherein R 3a  is COOH or hydrogen;   R 2 , R 4 , R 5 , R 6 , and R 7  are independently selected from hydrogen and hydroxyl, provided that one of R 2 , R 4 , R 5 , R 6 , and R 7  is hydroxyl;   or a pharmaceutically acceptable salt thereof.   
     
     
         23 . The method of  claim 19 , wherein R 4 , R 5  or R 7  is hydroxyl. 
     
     
         24 . The method of  claim 19 , wherein the compound is selected from the group consisting of 5-hydroxy-tryptophan, 5-hydroxy indole, 7-hydroxy indole, and serotonin. 
     
     
         25 . The method of  claim 19 , wherein the protein formulation is a pharmaceutical formulation suitable for administration to a subject. 
     
     
         26 . The method of  claim 19 , wherein the protein formulation is aqueous. 
     
     
         27 . The method of  claim 19 , wherein the compound in the formulation has a concentration from about 0.3 mM to about 1 mM. 
     
     
         28 . The method of  claim 19 , wherein the compound prevents oxidation of tryptophan, cysteine, histidine, tyrosine, and/or methionine in the protein. 
     
     
         29 . The method of  claim 19 , wherein the compound prevents oxidation of the protein by a reactive oxygen species. 
     
     
         30 . The method of  claim 29 , wherein the reactive oxygen species is selected from the group consisting of singlet oxygen, hydrogen peroxide, a hydroxyl radical, and an alkyl peroxide. 
     
     
         31 . The method of  claim 19 , wherein the protein is susceptible to oxidation. 
     
     
         32 . The method of  claim 19 , wherein tryptophan in the protein is susceptible to oxidation. 
     
     
         33 . The method of  claim 19 , wherein the protein is an antibody. 
     
     
         34 . The method of  claim 33 , wherein the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, or antibody fragment. 
     
     
         35 . The method of  claim 19 , wherein the protein concentration in the formulation is about 1 mg/mL to about 250 mg/mL. 
     
     
         36 . The method of  claim 19 , wherein the formulation further comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, a surfactant, and a tonicity agent. 
     
     
         37 . The method of  claim 19 , wherein the formulation has a pH of about 4.5 to about 7.0. 
     
     
         38 . A method of screening a compound that prevents oxidation of a protein in a protein composition, comprising selecting a compound that has lower oxidation potential and less photosensitivity as compared to L-tryptophan, and testing the effect of the selected compound on preventing oxidation of the protein. 
     
     
         39 . The method of  claim 38 , wherein the photosensitivity is measured based on the amount of H 2 O 2  produced by the compound upon light exposure. 
     
     
         40 . The method of  claim 39 , wherein the compound that produces less than about 10% of the amount of H 2 O 2  produced by L-tryptophan is selected. 
     
     
         41 . The method of  claim 38 , wherein the oxidation potential is measured by cyclic voltammetry. 
     
     
         42 . The method of  claim 38 , wherein the selected compound is tested for the effect on preventing oxidation of the protein by reactive oxygen species generated by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), light, and/or a Fenton reagent.