Methods for identifying free thiols in proteins

Abstract

Compositions and methods for identifying free thiols in protein are provided. An exemplary method labeling peptides with a tag to identify free thiols and a tag to identify native disulfide bonds and analyzing the tags using targeted MS2. In one embodiment, the method provides complete coverage of all 32 cysteine residues in an IgG molecule. In other embodiments the method covers the 16 cysteine residues on the heavy and light chains in an IgG molecule. In another embodiment, the method covers the 5 cysteine residues on each light chain of an IgG molecule. In another embodiment, the method covers the 11 cysteine residues on each heavy chain of an IgG molecule.

1 - 20 . (canceled) 
     
     
         21 . A method for identifying free thiols in a protein drug product, comprising:
 labeling a sample comprising a protein drug product with a first label comprising a sulfhydryl-reactive iodoacetyl group, an MS-neutral spacer arm, and a first MS/MS reporter having a unique reporter ion mass;   removing excess first label;   denaturing and reducing the sample;   labeling the sample with a second label comprising a sulfhydryl-reactive iodoacetyl group, an MS-neutral spacer arm, and a second MS/MS reporter having a unique reporter ion mass;   enzymatically digesting the sample;   analyzing the sample using an UPLC-MS 2  system comprising a charged surface hybrid column and a formic acid buffer mobile phase;   equilibrating the sample; and   quantifying the first MS/MS reporter and the second MS/MS reporter, wherein the amount of the first MS/MS reporter correlates to the amount of free thiols in the protein drug product and the amount of the second MS/MS reporter correlates to the amount of bound thiols in the protein drug product.   
     
     
         22 . The method of claim  1 , wherein the first MS/MS reporter has a unique mass of 126 Da, 127 Da, 128 Da, 129 Da, 130 Da and 131 Da, and the second MS/MS reporter has a unique mass of 126 Da, 127 Da, 128 Da, 129 Da, 130 Da, or 131 Da, wherein the first and second MS/MS reporters can be differentiated on MS/MS spectra. 
     
     
         23 . The method of claim  1 , wherein the first MS/MS reporter has a mass of 128 Da and the second MS/MS reporter has a mass of 131 Da. 
     
     
         24 . The method of claim  1 , wherein the first and second labels are selected from the group consisting of 
       
         
           
           
               
               
           
         
       
     
     
         25 . The method of claim  1 , wherein the method provides complete coverage of all 32 cysteine residues in an IgG molecule. 
     
     
         26 . The method of claim  1 , wherein the method covers the 16 cysteine residues on the heavy and light chains in an IgG molecule. 
     
     
         27 . The method of claim  1 , wherein the method covers the 5 cysteine residues on each light chain of an IgG molecule. 
     
     
         28 . The method of claim  1 , wherein the method covers the 11 cysteine residues on each heavy chain of an IgG molecule. 
     
     
         29 . The method of claim  1 , wherein a threshold of 5-6% free thiols is set, and wherein a result below the threshold does not show a potential for disulfide bond breakage. 
     
     
         30 . A method of identifying disulfide heterogeneity in a protein drug product comprising the steps of:
 labeling a sample comprising a protein drug product with a first label comprising a sulfhydryl-reactive iodoacetyl group, an MS-neutral spacer arm, and a first MS/MS reporter having a unique reporter ion mass;   removing excess first label;   denaturing and reducing the sample;   labeling the sample with a second label comprising a sulfhydryl-reactive iodoacetyl group, an MS-neutral spacer arm, and a second MS/MS reporter having a unique reporter ion mass;   enzymatically digesting the sample;   analyzing the sample using an UPLC-MS 2  system comprising a charged surface hybrid column and a formic acid buffer mobile phase;   equilibrating the sample; and   quantifying the first MS/MS reporter and the second MS/MS reporter, wherein the amount of the first MS/MS reporter correlates to the amount of free thiols in the protein drug product and the amount of the second MS/MS reporter correlates to the amount of bound thiols in the protein drug product, wherein the detection of a free thiol indicates the protein drug product has disulfide heterogeneity.   
     
     
         31 . The method of  claim 30 , wherein the first and second labels are selected from the group consisting of 
       
         
           
           
               
               
           
         
       
     
     
         32 . The method of  claim 30 , wherein the method provides complete coverage of all 32 cysteine residues in an IgG molecule. 
     
     
         33 . The method of  claim 30 , wherein the method covers the 16 cysteine residues on the heavy and light chains in an IgG molecule. 
     
     
         34 . The method of  claim 30 , wherein the method covers the 5 cysteine residues on each light chain of an IgG molecule. 
     
     
         35 . The method of  claim 30 , wherein the method covers the 11 cysteine residues on each heavy chain of an IgG molecule. 
     
     
         36 . A method for selecting a protein drug product comprising the steps of:
 labeling a sample comprising a protein drug product with a first label comprising a sulfhydryl-reactive iodoacetyl group, an MS-neutral spacer arm, and a first MS/MS reporter having a unique reporter ion mass;   removing excess first label;   denaturing and reducing the sample;   labeling the sample with a second label comprising a sulfhydryl-reactive iodoacetyl group, an MS-neutral spacer arm, and a second MS/MS reporter having a unique reporter ion mass;   enzymatically digesting the sample;   analyzing the sample using an UPLC-MS 2  system comprising a charged surface hybrid column and a formic acid buffer mobile phase;   equilibrating the sample;   quantifying the first MS/MS reporter and the second MS/MS reporter, wherein the amount of the first MS/MS reporter correlates to the amount of free thiols in the protein drug product and the amount of the second MS/MS reporter correlates to the amount of bound thiols in the protein drug product; and   selecting the protein product if it contains less than 5 free thiols or no free thiols.   
     
     
         37 . The method of  claim 36 , wherein the free thiol is in a light chain of an IgG molecule. 
     
     
         38 . The method of  claim 36 , wherein the free thiol is in a heavy chain of an IgG molecule. 
     
     
         39 . The method of  claim 36 , wherein at least one free thiol is in a light chain and at least one free thiol is in a heavy chain of an IgG molecule. 
     
     
         40 . A pharmaceutical composition comprising the protein product selected in  claim 36 .