Chromatographic method for high yield purification and viral inactivation of antibodies

Abstract

An improved process for the purification of antibodies from human plasma or other sources is disclosed. The process involves suspension of the antibodies at pH 3.8 to 4.5 followed by addition of caprylic acid and a pH shift to pH 5.0 to 5.2. A precipitate of contaminating proteins, lipids and caprylate forms and is removed, while the majority of the antibodies remain in solution. Sodium caprylate is again added to a final concentration of not less than about 15 mM. This solution is incubated for 1 hour at 25° C. to effect viral inactivation. A precipitate (mainly caprylate) is removed and the clear solution is diluted with purified water to reduce ionic strength. Anion exchange chromatography using two different resins is utilized to obtain an exceptionally pure IgG with subclass distribution similar to the starting distribution. The method maximizes yield and produces a gamma globulin with greater than 99% purity. The resin columns used to obtain a high yield of IgG retain IgM and IgA. IgA and IgM may be eluted from these resins in high yield and purity.

What is claimed is: 
     
       1. A method of obtaining purified IgA from a solution comprising monomeric IgA at from about 8 mg/ml to about 15 mg/ml and at from about 35% to about 55% purity, and dimeric IgA IgM, IgG, and albumin contaminants, the method comprising:
 passing the solution over a size exclusion chromatographic resin;   collecting a first IgA-containing fraction substantially free of contaminants other than IgG and dimeric IgA;   passing the first IgA-containing fraction over a protein G-affinity chromatographic resin;   collecting a second IgA-containing fraction.   
     
     
       2. A method of preparing a purified IgA preparation from a starting solution comprising immunoglobulins and other substances, the method comprising:
 adjusting the pH of the starting material to allow formation of an intermediate solution comprising dissolved immunoglobulins;   adjusting the intermediate solution to conditions of pH, temperature, and caprylate concentration such that a first precipitate and a first supernatant comprising immunoglobulins are formed;   separating the first supernatant from the first precipitate;   incubating the first supernatant under conditions of time, pH, temperature and caprylate concentration such that a second precipitate and a second supernatant comprising immunoglobulins are formed;   separating the second supernatant from the second precipitate;   contacting the second supernatant with an anion exchange resin under conditions of pH and ionic strength such that IgA binds to the anion exchange resin;   separating a fraction containing at least a portion of contaminants and immunoglobulins other than IgA from the IgA bound to the anion exchange resin;   eluting IgA from the first anion exchange resin column with a buffered solution having a conductivity in the range of that found in a solution of at least 100 mM sodium chloride;   collecting the eluted IgA to obtain a purified, IgA preparation;   passing the IgA preparation over a size exclusion chromatographic resin;   collecting a first IgA-containing fraction substantially free of contaminants other than IgG and dimeric IgA;   passing the first IgA-containing fraction over a protein G-affinity chromatographic resin; and   collecting a second IgA-containing fraction.   
     
     
       3. A method of  claim 2 , wherein the first IgA-containing fraction is substantially free of contaminants other than IgG. 
     
     
       4. A method of  claim 2 , wherein the starting solution comprises IgA at from about 35% to about 55%. 
     
     
       5. A method of  claim 2 , wherein the starting solution comprises dimeric IgA and the second IgA-containing fraction is substantially free of dimeric IgA. 
     
     
       6. A method of  claim 2 , wherein the starting solution comprises from about 8 mg/ml to about 15 mg/ml IgA. 
     
     
       7. A method of preparing a purified IgA preparation from a starting solution comprising immunoglobulins and other substances, the method comprising the steps of:
 a) adjusting the pH of the starting material to be within a range of from about 3.8 to about 4.5 to form an intermediate solution comprising dissolved immunoglobulins;   b) adjusting the intermediate solution of step a) to conditions of pH, temperature, and caprylate concentration such that a first precipitate and a first supernatant comprising immunoglobulins are formed, wherein the conditions under which the first precipitate and first supernatant form comprise a pH within a range of from about 5.0 to about 5.2 and a caprylate concentration within a range of from about 15 mM to about 25 mM;   c) separating the first supernatant from the first precipitate;   d) incubating the first supernatant under conditions of time, pH, temperature and caprylate concentration such that a second precipitate and a second supernatant comprising immunoglobulins are formed, wherein the conditions under which the second precipitate and second supernatant form comprise a pH within a range of about 5.0 to about 5.2 and a caprylate concentration within a range of about 15 mM to about 40 mM;   e) separating the second supernatant from the second precipitate;   f) contacting the second supernatant with a first anion exchange resin under conditions of pH and ionic strength such that substantially no IgG or IgM is bound to the first resin but IgA and other substances are bound to the first resin;   g) separating a fraction containing substantially all of the immunoglobulins other than IgA from the result of step f);   h) eluting IgA from the first anion exchange resin column with a buffered solution having a conductivity in the range of that found in a solution of at least 100 mM sodium chloride;   i) separating the eluted IgA to obtain a purified IgA preparation;   j) passing the IgA preparation over a size exclusion chromatographic resin;   k) collecting a first IgA-containing fraction substantially free of contaminants other than IgG and dimeric IgA;   l) passing the first IgA-containing fraction over a protein G-affinity chromatographic resin; and   m) collecting a second IgA-containing fraction.   
     
     
       8. A method of preparing a purified, virally inactivated antibody preparation from a starting solution comprising antibodies and other substances at an initial pH, the method comprising the steps of:
 (a) adding sodium caprylate to the starting solution and adjusting the pH to form a precipitate and a supernatant solution comprising antibodies, and   (b) incubating the supernatant solution under conditions of time, pH, temperature, and caprylate ion concentration to inactivate substantially all enveloped viruses to produce a purified, virally inactivated antibody preparation, wherein di- or tri-alkyl phosphates are not present in the method in amounts to substantially inactivate enveloped viruses.