Protease Inhibitor-Containing Compositions, Compositions Comprising Same, and Methods for Producing and Using Same

Abstract

Provided herein are methods and compositions for oral administration of therapeutic proteins, improved protease inhibitor preparations, methods for producing same, and compositions comprising same.

1 .- 33 . (canceled) 
     
     
         34 . An oral pharmaceutical composition produced by a process comprising
 (a) subjecting soybean trypsin inhibitor (SBTI) to column chromatography under conditions in which fractions containing the SBTI's Bowman-Birk Inhibitor (BBI) activity elute separately from fractions containing the SBTI's Kunitz Trypsin Inhibitor (KTI) activity;   (b) eluting and combining fractions from (a) that contain the BBI activity;   (c) filtering the combined fractions from (b) that contain the BBI activity under conditions that reduce the contaminants having a molecular weight of greater than 30 KDa to be less than 0.1% of the BBI preparation, thus producing a purified BBI product characterized in that:
 (i) contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation; and 
 (ii) 1 mg of the purified BBI product has an activity of about 40 BTEE units per mg of protein; 
   (d) eluting and combining the fractions from (a) that contain the KTI activity and in which contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation thus producing a purified KTI product characterized in that:
 (i) contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation; and 
 (ii) 1 mg of the purified KTI product has an activity of about 10,000 BAEE units per mg of protein; 
   (e) combining
 (i) an oil-based liquid formulation, wherein the oil comprises fish oil, 
 (ii) a therapeutic protein having a molecular weight of up to 100 kilodalton, 
 (iii) a chelator of divalent cations, 
 (iv) the purified BBI product of part (c); and 
 (v) the purified KTI product of part (d), 
   
       such that the ratio of anti-chymotrypsin activity to anti-trypsin activity present in the pharmaceutical composition is between 1.5:1 and 1:1 inclusive, 
       wherein the composition is water-free and comprises a coating that resists degradation in the stomach. 
     
     
         35 . An oral pharmaceutical composition comprising an oil-based liquid formulation, wherein the oil in the oil-based liquid formulation comprises fish oil, and wherein the oil-based liquid formulation further comprises:
 a therapeutic protein of up to 100 kilodalton,   a chelator of divalent cations, and   a Bowman-Birk Inhibitor (BBI),   
       wherein the composition is water-free and comprises a coating that resists degradation in the stomach, 
       wherein the BBI has a ratio of anti-chymotrypsin activity to anti-trypsin activity present of between 1.5:1 and 1:1 inclusive, and 
       wherein the BBI has been produced by a process comprising:
 (a) subjecting soybean trypsin inhibitor (SBTI) to column chromatography under conditions in which fractions containing the SBTI's Bowman-Birk Inhibitor (BBI) activity elute separately from fractions containing the SBTI's Kunitz Trypsin Inhibitor (KTI) activity; 
 (b) eluting and combining the fractions from (a) that contain the BBI activity; and 
 (c) filtering the combined fractions from (b) that contain the BBI activity under conditions that reduce contaminants having a molecular weight of greater than 30 KDa to be less than 0.1% of the BBI preparation, thus producing a purified BBI product characterized in that:
 (i) contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation; and 
 (ii) 1 mg of the purified BBI product has an activity of about 40 BTEE units per mg of protein. 
 
 
     
     
         36 . A method for making a pharmaceutical composition, the method comprising
 (a) providing a preparation of Bowman-Birk Inhibitor (BBI), wherein the BBI has been produced by a process comprising   (i) subjecting soybean trypsin inhibitor (SBTI) to column chromatography under conditions in which fractions containing the SBTI's Bowman-Birk Inhibitor (BBI) activity elute separately from fractions containing the SBTI's Kunitz Trypsin Inhibitor (KTI) activity;   (ii) eluting and combining the fractions from (i) that contain the BBI activity   (iii) filtering the combined fractions from (ii) that contain the BBI activity under conditions that reduce contaminants having a molecular weight of greater than 30 KDa to be less than 0.1% of the BBI preparation, thus producing a purified BBI product characterized in that: contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation; and 1 mg of the purified BBI product has an activity of about 40 BTEE units per mg of protein; and   (b) mixing the purified BBI product of part (iii) and a therapeutic protein of up to 100 kilodaltons into an oil-based liquid formulation in which the oil comprises fish oil;   wherein the preparation of BBI that is mixed with the therapeutic protein is of a purity of at least 85% as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); and   wherein the ratio of anti-chymotrypsin activity to anti-trypsin activity present in the pharmaceutical composition is between 1.5:1 and 1:1 inclusive.   
     
     
         37 . The method of  claim 36 , wherein the oil in the formulation is fish oil. 
     
     
         38 . A method for making an oral pharmaceutical composition comprising
 (a) subjecting soybean trypsin inhibitor (SBTI) to column chromatography under conditions in which fractions containing the SBTI's Bowman-Birk Inhibitor (BBI) activity elute separately from fractions containing the SBTTs Kunitz Trypsin Inhibitor (KTI) activity;   (b) eluting and combining the fractions from (a) that contain the BBI activity;   (c) filtering the combined fractions from (a) that contain the BBI activity under conditions that reduce contaminants having a molecular weight of greater than 30 KDa to be less than 0.1% of the BBI preparation, thus producing a purified BBI product characterized in that:
 (i) contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation; and 
 (ii) 1 mg of the purified BBI product has an activity of about 40 BTEE units per mg of protein; 
   (d) eluting and combining the fractions from (a) that contain the KTI activity and in which contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation thus producing a purified KTI product characterized in that:
 (i) contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation; and 
 (ii) 1 mg of the purified KTI product has an activity of about 10,000 BAEE units per mg of protein; 
   (e) combining
 (i) an oil-based liquid formulation, wherein the oil comprises fish oil, 
 (ii) a therapeutic protein having a molecular weight of up to 100 kilodalton, 
 (iii) a chelator of divalent cations, 
 (iv) the purified BBI product of part (b); and 
 (v) the purified KTI product of part (c), 
   
       such that the ratio of anti-chymotrypsin activity to anti-trypsin activity present in the pharmaceutical composition is between 1.5:1 and 1:1 inclusive. 
     
     
         39 . The method of  claim 38 , wherein the oil in the formulation is fish oil.