Quantification of impurities for release testing of peptide products

Abstract

The present invention relates to a method for the quantitative determination of an impurity present in a peptide product, wherein the impurity cannot be separated from other impurities or the main product. The method particularly involves the use of high resolution mass spectrometry (MS) detection with or without high performance liquid chromatography (HPLC). The method can be used for the investigation of the quality of peptides and proteins, particularly of pharmaceutical peptides and proteins.

1 . A method for the quantitative determination of an impurity present in a peptide product composition, comprising the steps:
 (a) providing a peptide product composition comprising a peptide product and an unknown amount of at least one impurity, wherein said impurity cannot be separated from the peptide product or another ingredient of the composition by a chromatographic procedure,   (b) providing at least one sample of the peptide product composition without said impurity added and optionally at least one further sample of the peptide product composition with a known amount of said impurity added,   (c) quantitatively determining said impurity in said sample from step (b) by mass spectrometry, and   (d) calculating the amount of said impurity in the peptide product composition based on the results of (c).   
     
     
         2 . The method according to  claim 1  wherein step (b) comprises providing at least three samples of the peptide product composition, wherein a first sample comprises the peptide product composition without said impurity added, and wherein at least two further samples comprise the peptide product composition each with a different known amount of said impurity added. 
     
     
         3 . The method of  claim 1 , wherein the peptide product has a length of from 5-100 amino acids. 
     
     
         4 . The method of  claim 1 , wherein the peptide product has been chemically synthesized, particularly by a solid phase synthesis procedure, or produced by recombinant DNA processes. 
     
     
         5 . The method of  claim 1 , wherein the peptide product composition is a pharmaceutical formulation or a composition intended for the manufacture of a pharmaceutical formulation. 
     
     
         6 . The method of  claim 1 , wherein the peptide product is an exendin peptide, particularly Lixisenatide (AVE0010). 
     
     
         7 . The method of  claim 1 , wherein the impurity is a peptide impurity. 
     
     
         8 . The method of  claim 1 , wherein the impurity cannot be quantitatively separated from the peptide product or from another ingredient of the composition by an HPLC procedure, particularly by a Reverse Phase HPLC procedure. 
     
     
         9 . The method of  claim 1 , wherein the peptide product comprises unknown amounts of at least 2 impurities which cannot be quantitatively separated from the peptide product or from another ingredient of the composition by a chromatographic procedure. 
     
     
         10 . The method of  claim 1 , wherein the impurity is added to at least one peptide product composition sample from a stock preparation, preferably from at least two stock preparations comprising different concentrations of the impurity. 
     
     
         11 . The method of  claim 1 , wherein at least 3 or 4 further samples with different known amounts added impurity are provided and subjected to mass spectrometry determination. 
     
     
         12 . The method of  claim 1 , wherein the mass spectrometry is high resolution mass spectrometry. 
     
     
         13 . The method of  claim 1 , wherein the calculation comprises a linear regression analysis. 
     
     
         14 . The method of  claim 13 , wherein the calculation is carried out according to the equation:
     y=ax+b      wherein a=slope
 y=determined peak area of the impurity in a sample 
 x=added amount of the impurity in a sample 
 b=intercept 
   and the unknown amount of the impurity x t  is obtained as follows:
     x   t   =b·a   −1    
   
     
     
         15 . The method of  claim 12 , wherein said high resolution mass spectrometry comprises Fourier Transform mass spectrometry.