Prevention of protein disulfide bond reduction

Abstract

Disclosed are methods for testing the presence and/or activity of glutathione system components and thioredoxin system components during the manufacturing process of disulfide bond-containing proteins. Also disclosed are methods for mitigating reduction of disulfide bonds during the manufacturing process, and for lowering the reduction potential of disulfide bond-containing proteins. Provided are compositions, kits, and methods for mitigating reduction and diminishing reduction potential of disulfide bond-containing proteins during protein manufacturing processes.

What is claimed is: 
     
         1 . A method for increasing the yield of an intact disulfide bond-containing protein of interest in a cell culture or solution, comprising manufacturing the disulfide bond-containing protein of interest in the presence of a glutathione reductase inhibitor, a thioredoxin reductase inhibitor, or both a glutathione reductase inhibitor and a thioredoxin reductase inhibitor, whereby the amount of intact disulfide bond-containing protein is higher in the cell culture or solution manufactured in the presence of a glutathione reductase inhibitor, a thioredoxin reductase inhibitor, or a glutathione reductase inhibitor and a thioredoxin reductase inhibitor as compared to the amount of intact disulfide bond-containing protein in a cell culture or solution not manufactured in the presence of a glutathione reductase inhibitor, a thioredoxin reductase inhibitor, or a glutathione reductase inhibitor and a thioredoxin reductase inhibitor. 
     
     
         2 . The method of  claim 1  where increasing the yield of an intact disulfide bond-containing protein of interest in a cell culture or solution is controlled via the online measurement of the cell culture redox potential. 
     
     
         3 . The method of  claim 1  where increase of the yield of an intact disulfide bond-containing protein of interest in a cell culture or solution is monitored online via measurement of the cell culture redox potential. 
     
     
         4 . The method of  claim 1 , further comprising: detecting the presence of glutathione reductase and/or thioredoxin reductase in the culture and or the solution containing the disulfide bond-containing protein of interest, and adding a glutathione reductase and/or thioredoxin reductase inhibitor to mitigate reduction of the disulfide bond-containing protein of interest. 
     
     
         5 . The method of  claim 4 , further comprising determining the activity of the glutathione reductase and/or thioredoxin reductase. 
     
     
         6 . The method of  claim 5 , wherein determining the activity of the glutathione reductase and/or thioredoxin reductase comprises:
 adding 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) to a sample obtained during the manufacturing process;   adding at least one of thioredoxin reductase inhibitor and glutathione reductase inhibitor to a portion of the sample containing DTNB; and   monitoring reduction of DTNB at a wavelength of 412 nm in the sample;   
       wherein a higher reduction of DTNB in the samples without at least one of thioredoxin reductase inhibitor and glutathione reductase inhibitor, indicates activity of thioredoxin reductase and/or glutathione reductase. 
     
     
         7 . The method of  claim 6 , wherein NADPH, oxidized glutathione, and buffer are added to the sample prior to monitoring reduction. 
     
     
         8 . A method for increasing the yield of an intact disulfide bond-containing protein of interest or diminishing the reduction potential of the disulfide containing protein of interest in a cell culture or fermentation process comprising maintaining the extracellular cystine levels above 0 and/or maintaining the extracellular Cu levels above 0 and/or adding EDTA to the bioreactor during the cell culture or fermentation process. 
     
     
         9 . A method for increasing the yield of an intact disulfide bond-containing protein of interest or diminishing the reduction potential of the disulfide containing protein of interest in a cell culture or fermentation process comprising maintaining the extracellular cystine levels above 0 and/or maintaining the extracellular Cu levels above 0 and/or adding EDTA to the solution containing the disulfide bond-containing protein of interest. 
     
     
         10 . The method of  claim 8  or  9 , wherein the intact disulfide bond-containing protein of interest is released into the cell culture fluid (CCF). 
     
     
         11 . The method of  claim 10 , wherein the cell culture or fermentation process comprises maintaining the cells in CCF for at least 2 days, at least 8 days, at least 10 days, at least 12 days, at least 14 days, or at least 16 days. 
     
     
         12 . The method of  claim 11 , wherein the cell culture process is a mammalian or insect cell culture process, and the fermentation process is a bacterial, yeast, or fungi fermentation process. 
     
     
         13 . The method of any one of  claims 8  to  12 , wherein maintaining the extracellular cystine levels above 0 during the cell culture or fermentation process prevents disulfide bond reduction in the disulfide bond-containing protein of interest. 
     
     
         14 . The method of  claim 13 , wherein the potential for disulfide bond reduction of the disulfide bond-containing protein of interest is decreased relative to the potential for disulfide bond reduction of the disulfide bond-containing protein of interest produced in a cell culture or fermentation process in which an effective amount of cystine is not maintained above 0 in the CCF. 
     
     
         15 . A method for improving the stability of a purified disulfide bond-containing protein of interest, comprising using a manufacturing process that maintains the protein of interest in a form with minimal free thiols, thereby mitigating reduction or reduction potential of the protein of interest during the manufacturing process. 
     
     
         16 . A method for improving the stability of a purified disulfide bond-containing protein of interest, comprising using a manufacturing process that mitigates reduction or reduction potential of the protein of interest during the manufacturing process, thereby resulting in lower aggregate formation during the purification process. 
     
     
         17 . The method of  claim 15  or  15 , wherein the manufacturing process comprises a cell culture phase, a harvest phase, at least one hold phase, a purification phase, or any combination thereof. 
     
     
         18 . The method of  claim 17 , wherein the hold phase comprises storing the material in any of the phases during the manufacturing process. 
     
     
         19 . The method of  claim 18 , wherein the hold phase comprises storing harvested cell culture fluid (HCCF) for a period of up to one day, at least four days, at least one week, at least 10 days, at least two weeks, at least one month, or at least three months following the harvest phase and prior to the purification phase. 
     
     
         20 . The method any one of  claims 15  to  19 , wherein maintaining the protein of interest in a form with minimal free thiols comprises mitigating disulfide bond reduction throughout the manufacturing process. 
     
     
         21 . The method of  claim 19  or  20 , wherein the HCCF is stored at 2-8° C. in airtight bags with or without air or other vessels with or without headspace during the hold phase. 
     
     
         22 . The method of any one of  claims 15  to  21 , wherein the free thiol of the purified disulfide bond-containing protein of interest is minimized, thereby stabilizing the protein of interest at the drug substance stage. 
     
     
         23 . The method of any one of  claims 1 - 21 , wherein the disulfide bond-containing protein of interest is an antibody or antigen-binding fragment thereof. 
     
     
         24 . The method of any one of  claims 16 - 24  where the minimization of disulfide bond reduction and/or disulfide bond reduction potential and/or free thiols is achieved by increasing Cu, Cystine, EDTA, or redox potential (by adding redox modifiers including metal ions (including Zn 2+ , Mn 2+ , Fe 3+ , Cu 2+ , Se 2+ ), cystine, dissolved oxygen, beta mercaptoethanol, glutathione, or a combination thereof) during any step of the manufacturing process (including bioreactor and harvest). 
     
     
         25 . A method for increasing the yield of an intact disulfide bond-containing protein of interest in a cell culture or solution, comprising manufacturing the disulfide bond-containing protein of interest by increasing the metal ion concentration, cystine, beta-mercapto ethanol, glutathione, dissolved oxygen concentration, or other redox modifiers during manufacturing processes.