Alpha-1-antitrypsin compositions

Abstract

A streamlined method for purifying alpha-1-antitrypsin (AAT) from an AAT-containing protein mixture, such as a Cohn fraction IV precipitate, is provided. In the method of the invention, contaminating proteins are destabilized by cleavage of disulfide bonds with a reducing reagent, such as a dithiol, which does not affect AAT. The destabilized proteins are then preferentially adsorbed on a solid protein-adsorbing material, without the addition of a salt as a precipitant. Separation of the solid adsorbent from the solution leaves a purified AAT solution that is directly suitable for chromatographic purification, without the need for extensive desalting as in prior art processes. A process incorporating this method, which provides pharmaceutical grade AAT in high yield on a commercial scale, is also described.

1 .- 17 . (canceled) 
     
     
         18 . A pharmaceutical composition, comprising alpha-1-antitrypsin (AAT) isolated or purified from plasma or fractions thereof, wherein said composition further comprises:
 (a) less than 0.1% albumin; and   (b) less than 0.1% apolipoprotein A1, and   wherein percentages of albumin and apolipoprotein A1 are weight percentages based on total protein weight, and wherein total protein weight is measured using a Bradford assay or by absorbance at 280 nm using as an extinction coefficient E 1 cm,280 nm   1% =5.3.   
     
     
         19 . The composition of  claim 18 , further comprising:
 (c) less than 0.1% transferrin, and   wherein percentages of albumin, apolipoprotein A1, and transferrin are weight percentages based on total protein weight.   
     
     
         20 . The composition of  claim 18 , wherein the AAT is pasteurized and/or subjected to a viral reduction procedure. 
     
     
         21 . The composition of  claim 20 , wherein the AAT is subjected to a viral reduction procedure capable of reducing the number of enveloped viruses by at least 11 log 10  units and capable of reducing the number of non-enveloped viruses by at least 6 log 10  units. 
     
     
         22 . The composition of  claim 20 , wherein the viral reduction procedure is solvent/detergent treatment and/or nanofiltration. 
     
     
         23 . The composition of  claim 18 , wherein the apparent ratio of active to antigenic AAT is greater than 1.08 when measured by endpoint nephelometry. 
     
     
         24 . The composition of  claim 18 , wherein the specific activity of the AAT is at least 0.99 mg functional AAT per milligram of protein, when using as an extinction coefficient E 1 cm,280 nm   1% =5.3. 
     
     
         25 . The composition of  claim 24 , wherein the specific activity of the AAT is less than or equal to 1.12 mg functional AAT per milligram of protein. 
     
     
         26 . The composition of  claim 18 , wherein the composition is stable for at least 2 years when stored lyophilized at ≦25° C. 
     
     
         27 . The composition of  claim 26 , wherein the composition is stable for at least 2 years when stored lyophilized at 25° C. 
     
     
         28 . The composition of  claim 18 , wherein the composition comprises 6% or less contaminating proteins based on total protein weight as determined by SDS-PAGE. 
     
     
         29 . The composition of  claim 18 , wherein the composition is lyophilized. 
     
     
         30 . The composition of  claim 18 , wherein the albumin and apolipoprotein A1 are detected by nephelometry. 
     
     
         31 . The composition of  claim 18 , further comprising:
 (c) less than 0.8% α 1 -acid glycoprotein;   (d) less than 0.1% α2-macroglobulin;   (e) less than 0.5% antithrombin III; and   (f) less than 0.1% ceruloplasmin, and   wherein percentages of albumin, apolipoprotein A1, α 1 -acid glycoprotein, α2-macroglobulin, antithrombin III, and ceruloplasmin are weight percentages based on total protein weight.   
     
     
         32 . The composition of  claim 31 , wherein the AAT is pasteurized and/or subjected to a viral reduction procedure. 
     
     
         33 . The composition of  claim 32 , wherein the AAT is subjected to a viral reduction procedure capable of reducing the number of enveloped viruses by at least 11 log 10  units and capable of reducing the number of non-enveloped viruses by at least 6 log 10  units. 
     
     
         34 . The composition of  claim 32 , wherein the viral reduction procedure is solvent/detergent treatment and/or nanofiltration. 
     
     
         35 . The composition of  claim 31 , wherein the specific activity of the AAT is at least 0.99 mg functional AAT per milligram of protein, when using as an extinction coefficient E 1 cm,280 nm   1% =5.3. 
     
     
         36 . The composition of  claim 35 , wherein the specific activity of the AAT is less than or equal to 1.12 mg functional alpha-1-antitrypsin per milligram of protein. 
     
     
         37 . The composition of  claim 31 , wherein the composition is stable for at least 2 years when stored lyophilized at ≦25° C. 
     
     
         38 . The composition of  claim 37 , wherein the composition is stable for at least 2 years when stored lyophilized at 25° C. 
     
     
         39 . The composition of  claim 31 , wherein the composition is lyophilized. 
     
     
         40 . The composition of  claim 31 , wherein the albumin, apolipoprotein A1, α 1 -acid glycoprotein, α2-macroglobulin, antithrombin III, and ceruloplasmin are detected by nephelometry. 
     
     
         41 . A pharmaceutical composition, comprising alpha-1-antitrypsin (AAT) isolated or purified from plasma or fractions thereof, wherein the AAT is prepared by a process comprising:
 (a) contacting an AAT-containing protein mixture with a disulfide-reducing agent to produce a reduced AAT-containing protein mixture;   (b) contacting the reduced AAT-containing protein mixture with an insoluble protein-adsorbing material without the addition of a substantial amount of additional salts; and   (c) isolating the resulting AAT-containing product,   wherein, following step (b), the AAT-containing product comprises less than 0.5% albumin and less than 2.5% transferrin, and   wherein percentages of albumin and transferrin are weight percentages based on total protein weight, and wherein total protein weight is measured using a Bradford assay or by absorbance at 280 nm using as an extinction coefficient E 1 cm,280 nm   1% =5.3.