Charged depth filtration of antigen- binding proteins

Abstract

Methods of producing an aqueous formulation of an antigen-binding protein or enhancing re-oxidation of an antigen-binding protein are disclosed. The methods comprise (a) contacting an aqueous solution comprising antigen-binding protein molecules with a charged depth filter under conditions sufficient to enhance re-oxidation of the antigen-binding protein molecules and achieve a decrease in the percentage of reduced antigen-binding protein molecules, compared to the percentage of reduced anti-gen-binding protein molecules observed prior to step (a); and (b) optionally, measuring the amount or relative amount of reduced an¬tigen-binding protein molecules. Formulations comprising a re-oxidized antigen-binding protein are also described.

What is claimed: 
     
         1 . A method of producing an aqueous formulation of an antigen-binding protein comprising
 (a) contacting an aqueous solution comprising antigen-binding protein molecules with a charged depth filter under conditions sufficient to achieve at least a 20% decrease in the percentage of reduced antigen-binding protein molecules, compared to the percentage of reduced antigen-binding protein molecules observed prior to step (a); and   (b) optionally, measuring the amount or relative amount of reduced antigen-binding protein molecules.   
     
     
         2 . A method of enhancing re-oxidization of an antigen-binding protein comprising
 (a) contacting an aqueous solution comprising antigen-binding protein molecules with a charged depth filter under conditions sufficient to enhance re-oxidation of the antigen-binding protein molecules, optionally wherein the re-oxidation of the antigen-binding protein molecules is increased at least two-fold following the contacting step; and   (b) optionally, measuring the amount or relative amount of reduced antigen-binding protein molecules.   
     
     
         3 . The method of  claim 1  or  2 , wherein the total amount of reduced antigen-binding protein molecules after contact with the charged depth filter in step (a) is 10% or less than the total amount of antigen-binding protein molecules. 
     
     
         4 . The method according to any of  claims 1 - 3 , wherein step (a) is followed by subjecting the solution of antigen-binding protein molecules to Protein A chromatography 
     
     
         5 . The method according to any of  claims 1 - 4 , wherein step (a) is preceded by subjecting the solution of antigen-binding protein molecules to Protein A chromatography. 
     
     
         6 . The method according to any of  claims 1 - 5 , further comprising a step of inactivating one or more viruses in said solution of antigen-binding protein molecules. 
     
     
         7 . The method according to any of  claims 1 - 6 , wherein the amount of reduced disulfide bonds in the antigen-binding protein molecules is decreased by at least 3-fold following the charged depth filtration compared to before the charged depth filtration. 
     
     
         8 . The method according to any of  claims 1 - 7 , wherein the percentage of reduced antigen-binding protein molecules continues to decrease for at least 1 hours, at least 2 hours, at least 3 hours, or at least 4 hours following step (a). 
     
     
         9 . The method according to any of  claims 1 - 8 , wherein the contacting occurs at room temperature. 
     
     
         10 . The method according to any of  claims 1 - 8 , wherein the contacting occurs at a temperature of 2 degrees to 8 degrees Celsius. 
     
     
         11 . The method according to any of  claims 1 - 10 , further comprising a step of sparging air or oxygen through the solution of antigen-binding protein molecules. 
     
     
         12 . The method according to any of  claims 1 - 11 , further comprising contacting the solution of antigen-binding protein molecules with a positive ion selected from the group consisting of sodium, calcium, magnesium, mercury, molybdenum, chromium, cadmium, aluminum, potassium, cobalt, iron, manganese, titanium, zinc, nickel, copper, and combinations thereof. 
     
     
         13 . The method according to any of  claims 1 - 12 , wherein the solution of antigen-binding protein molecules is contacted with more than one charged depth filter. 
     
     
         14 . The method according to any of  claims 1 - 13 , wherein the charged depth filter comprises a diatomaceous earth layer. 
     
     
         15 . The method according to  claim 14 , wherein the charged depth filter further comprises a cellulose layer and an inorganic layer. 
     
     
         16 . The method according to  claim 15 , wherein the inorganic layer comprises a polyamine resin. 
     
     
         17 . The method according to any of  claims 1 - 16 , wherein the charged depth filter comprises a positively charged ion. 
     
     
         18 . The method according to any of  claims 1 - 17 , wherein the charged depth filter comprises a metal selected from the group consisting of sodium, calcium, magnesium, mercury, chromium, cadmium, aluminum, potassium, lead, arsenic, cobalt, iron, manganese, titanium, zinc, nickel, copper, and combinations thereof. 
     
     
         19 . The method according to any of  claims 1 - 18 , wherein the contacting occurs at a throughput of between 250 L/m2 and 850 L/m2. 
     
     
         20 . The method according to any of  claims 1 - 19 , wherein the antigen-binding protein is an IgG antibody. 
     
     
         21 . The method according to  claim 20 , wherein the antibody is an IgG1 or IgG2 antibody. 
     
     
         22 . The method according to  claim 21 , wherein the antibody is an IgG1 antibody with a Kappa light chain. 
     
     
         23 . The method according to  claim 21 , wherein the antibody is an IgG1 antibody with a Lambda light chain. 
     
     
         24 . The method according to any of  claims 1 - 23 , wherein the antigen-binding protein binds an antigen selected from the group consisting of RANKL, tumor necrosis factor alpha, epidermal growth factor receptor, CD20, calcitonin gene-related peptide, sclerostin, and platelet glycoprotein IIb/IIIa. 
     
     
         25 . The method according to any of  claims 1 - 24 , wherein the antigen-binding protein is selected from the group consisting of abciximab, adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, brentuximab vedotin, canakinumab, cetuximab, certolizumab pegol, daclizumab, denosumab, eculizumab, efalizumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, muromonab-CD3, natalizumab, nivolumab, ofatumumab, omalizumab, palivizumab, panitumumab, ranibizumab, rituximab, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab, and a bio similar of any of the foregoing. 
     
     
         26 . The method according to any of  claims 1 - 25 , wherein the antigen-binding protein comprises an antigen-binding region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-8. 
     
     
         27 . The method according to any of  claims 1 - 26 , wherein the amount of reduced antigen-binding protein molecules is measured using non-reduced capillary electrophoresis with sodium dodecyl sulfate. 
     
     
         28 . The method according to any of  claims 1 - 27 , further comprising a step of cation exchange chromatography. 
     
     
         29 . The method of any of  claims 1 - 28 , comprising (1) a Protein A chromatography step, optionally followed by charged depth filtration; (2) a viral inactivation step, optionally followed by charged depth filtration; and (3) a cation exchange chromatography step, optionally followed by charged depth filtration; further optionally comprising one or more of (4) a salt-intolerant interaction chromatography step, optionally followed by charged depth filtration; (5) a virus filtration step, optionally followed by charged depth filtration; and (5) ultrafiltration and/or diafiltration, optionally followed by charged depth filtration 
     
     
         30 . The method of any of  claims 1 - 29 , wherein following contact with a charged depth filter, the filtrate is incubated for at least about 4 hours. 
     
     
         31 . The method of any of  claims 1 - 30 , wherein the amount or relative amount of reduced antigen-binding protein molecules is determined using non-reduced capillary electrophoresis with sodium dodecyl sulfate (nrCE-SDS). 
     
     
         32 . A method of producing an aqueous formulation of an antigen-binding protein comprising contacting an aqueous solution comprising antigen-binding protein molecules with a charged depth filter under conditions sufficient to achieve at least a 20% decrease in the percentage of reduced antigen-binding protein molecules, compared to the percentage of reduced antigen-binding protein molecules observed prior to the contacting step, wherein the at least 20% decrease is determined using non-reduced capillary electrophoresis with sodium dodecyl sulfate (nrCE-SDS). 
     
     
         33 . A method of enhancing re-oxidization of an antigen-binding protein comprising contacting an aqueous solution comprising antigen-binding protein molecules with a charged depth filter under conditions sufficient to achieve at least a two-fold increase in re-oxidation of the antigen-binding protein molecules following the contacting step, wherein the at least two¬fold increase is determined using non-reduced capillary electrophoresis with sodium dodecyl sulfate (nrCE-SDS). 
     
     
         34 . An aqueous formulation comprising a re-oxidized antigen-binding protein prepared using the method of any of  claims 1 - 33 .