Additive systems for use in protein pegylation

Abstract

The present disclosure provides an additive system for use in protein PEGylation. The additive system includes p-aminobenzoic hydrazide used either alone or in combination with aromatic amines, such as 3,5-diaminobenzoic acid, or with ammonium salts such as ammonium chloride or ammonium acetate. The disclosed additive combination provides several benefits including increased reaction rates, higher yields and reduction in the aminoxy-PEG equivalents required to complete the conjugation reaction. Typical reactions can be run by combining the additive or additive system with a solution of a protein and aminoxy-PEG reagent. The solution is adjusted to pH 4 and held at 20-25° C. without stirring until completion, typically within 24 hours.

We claim: 
     
         1 ) An improved additive system for protein PEGylation reaction, said system comprising p-aminobenzoic hydrazide alone or in combination with aromatic amines or ammonium salts. 
     
     
         2 ) The system of  claim 1 , wherein said aromatic amine is selected from a group consisting of 3,5-diaminobenzoic acid, o-phenylenediamine, 1-pyridin-2-yl-ethylamine, 2-(dimethylamino)ethylhydrazine, m-phenylenediamine and 2-picolylamine. 
     
     
         3 ) The system of  claim 1 , wherein said ammonium salt is selected from a group consisting of ammonium acetate and ammonium chloride. 
     
     
         4 ) The system of  claim 1 , wherein the preferred system combination includes p-aminobenzoic hydrazide with 3,5-diaminobenzoic acid or p-aminobenzoic hydrazide with ammonium chloride. 
     
     
         5 ) The system of  claim 1 , wherein said reaction is a conjugation reaction between proteins containing the p-acetylphenylalanine residue and an aminoxy-PEG compound. 
     
     
         6 ) The system of  claim 1 , wherein the additive system augments the conjugation reaction rates, provides high yield of the conjugated product and facilitates reduction in the aminoxy-PEG equivalents required to complete the conjugation reaction. 
     
     
         7 ) A process for obtaining PEGylated protein, said process comprising steps of:
 (a) identifying a protein, PEG reagent and an additive system; and   (b) solubilizing the protein followed by combining with PEG reagent in presence of the additive system to obtain PEGylated protein with high yield.   
     
     
         8 ) The process of  claim 7 , wherein said protein is Relaxin or FGF21 containing a p-acetylphenylalanine residue. 
     
     
         9 ) The process of  claim 7 , wherein the solubilized protein solution combined with PEG reagent is maintained at a pH of about 4. 
     
     
         10 ) The process of  claim 7 , wherein reaction mixture is held at a temperature ranging from about 20° C. to about 25° C. 
     
     
         11 ) The process of  claim 7 , wherein said additive system includes p-Aminobenzoic hydrazide alone or in combination with combination with aromatic amines, such as 3,5-diaminobenzoic acid or ammonium salts such as ammonium acetate or ammonium chloride. 
     
     
         12 ) The process of  claim 11 , wherein said additive combination of high quality p-Aminobenzoic hydrazide with ammonium chloride is preferred for use at large scale production of PEGylated proteins. 
     
     
         13 ) The process of  claim 6 , wherein said PEG reagents are selected from a group comprising PEG-OA and other PEG derivatives with aminoxy group. 
     
     
         14 ) A pharmaceutical composition comprising a PEGylated protein obtained by the process of  claim 7  for use in therapy for a subject in need thereof.