Method for purifying active polypeptides or immunoconjugates

Abstract

The present invention provides methods for isolating an active polypeptide or immunoconjugate by purification of a solution containing both the active polypeptide or immunoconjugate and an acidic variant thereof, such as a deamidated variant, using anion exchange chromatography. The present invention also provides compositions, formulations, and unit dosage forms comprising the purified polypeptide or immunoconjugate.

What is claimed is: 
     
         1 . A method of purifying an active immunoconjugate, wherein said immunoconjugate is deamidated at one or more residues, and wherein said deamidation results in an inhibition of potency of said immunoconjugate, the method comprising: (a) contacting the immunoconjugate with an anion exchange AIEX chromatography matrix; and (b) eluting the bound immunoconjugate from the AIEX chromatography matrix with a high salt buffer, thereby separating the active immunoconjugate from the deamidated variant. 
     
     
         2 . A method of producing a purified polypeptide from a solution comprising the polypeptide and an acidic variant of the polypeptide, wherein said acidic variant of the polypeptide results in an inhibition of potency of said polypeptide, the method comprising: (a) contacting the polypeptide with an anion exchange (AIEX) chromatography matrix; and (b) eluting the bound polypeptide from the AIEX chromatography matrix with a high salt buffer, thereby separating said polypeptide from the acidic variant and producing a purified polypeptide. 
     
     
         3 . A method of producing a purified polypeptide or immunoconjugate from a solution comprising the polypeptide and an acidic variant of the polypeptide, the method comprising: (a) producing the polypeptide or immunoconjugate in a bacterial cell which expresses the polypeptide or immunoconjugate; (b) isolating inclusion bodies containing the polypeptide or immunoconjugate from the bacterial cells; (c) refolding the polypeptide or immunoconjugate isolated from the inclusion bodies; (d) contacting the composition containing the polypeptide or immunoconjugate with an AIEX chromatography matrix; and (b) eluting the bound polypeptide or immunoconjugate from the AIEX chromatography matrix with a high salt buffer, thereby purifying the polypeptide or immunoconjugte from the solution. 
     
     
         3 . The method of  claim 2  or  3 , wherein the acidic variant is a deamidated variant. 
     
     
         4 . The method of any of  claims 1 - 3 , wherein the AIEX matrix contains quaternary amine and tertiary amine ion exchange groups. 
     
     
         5 . The method of  claim 4 , wherein the AIEX matrix contains a quaternary amino (Q) group. 
     
     
         6 . The method of  claim 5 , wherein the AIEX matrix is Q sepharose. 
     
     
         7 . The method of any of  claims 1 - 6 , wherein the polypeptide is eluted with a linear or step salt gradient. 
     
     
         8 . The method of  claim 7 , wherein the polypeptide is eluted with a linear salt gradient. 
     
     
         9 . The method of  claim 8 , wherein the linear salt gradient is from about 150 mM NaCl in Tris/HCl, pH 8.0 to about 300 mM NaCl in Tris/HCl, pH 8.0. 
     
     
         10 . The method of  claim 9 , wherein the linear salt gradient is from about 175 mM NaCl in Tris/HCl, pH 8.0 to about 275 mM NaCl in Tris/HCl, pH 8.0. 
     
     
         11 . The method of  claim 9 , wherein the linear salt gradient is from about 192 mM NaCl in Tris/HCl, pH 8.0 to about 245 mM NaCl in Tris/HCl, pH 8.0. 
     
     
         12 . The method of any of  claims 1 - 11 , wherein between about 75 to about 99% of the acidic or deamidated variant is removed. 
     
     
         13 . The method of  claim 12 , wherein about 80% of the acidic or deamidated variant is removed. 
     
     
         14 . The method of  claim 12 , wherein about 85% of the acidic or deamidated variant is removed. 
     
     
         15 . The method of  claim 12 , wherein about 90% of the acidic or deamidated variant is removed. 
     
     
         16 . The method of  claim 12 , wherein about 95% of the acidic or deamidated variant is removed. 
     
     
         17 . The method of  claim 12 , wherein about 96%, 97%, 98%, or 99% of the acidic or deamidated variant is removed. 
     
     
         18 . The method of any of  claims 1 - 17 , wherein the polypeptide or immunoconjugate comprises an antibody or antigen binding fragment thereof. 
     
     
         19 . The method of  claim 18 , wherein the antibody or antigen binding fragment comprises a Fab, a Fab′, a F(ab′) 2 , a Fd, a single chain Fv or scFv, a disulfide linked Fv, a V-NAR domain, an IgNar, an intrabody, an IgGΔCH2, a minibody, a F(ab′) 3 , a tetrabody, a triabody, a diabody, a single-domain antibody, DVD-Ig, Fcab, mAb 2 , a (scFv) 2 , or a scFv-Fc. 
     
     
         20 . The method of  claim 18  or  19 , wherein the antibody or antigen binding fragment binds a cell surface receptor. 
     
     
         21 . The method of  claim 20 , wherein the cell surface receptor is CD22. 
     
     
         22 . The method of any of  claims 1 - 21 , wherein the polypeptide or immunoconjugate comprises a toxin. 
     
     
         23 . The method of  claim 22 , wherein the toxin is selected from the group consisting of:  Pseudomonas  exotoxin, ricin, abrin, diphtheria toxin and subunits thereof, as well as botulinum toxins A through F or variants, or derivatives thereof. 
     
     
         24 . The method of  claim 22  or  23 , wherein the toxin is a  Pseudomonas  exotoxin, or variant thereof. 
     
     
         25 . The method of  claim 24 , wherein said  Pseudomonas  exotoxin, or variant thereof has an amino acid sequence selected from the group consisting of SEQ ID NOs:16-22. 
     
     
         26 . The method of  claim 24 , wherein said  Pseudomonas  exotoxin, or variant thereof has the amino acid sequence of SEQ ID NO:22. 
     
     
         27 . The method of  claim 18  or  19 , wherein said antibody or antigen binding fragment thereof comprises a V H  and a V L  sequence. 
     
     
         28 . The method of  claim 27 , wherein said V H  sequence is selected from the group consisting of SEQ ID NOs: 6-11. 
     
     
         29 . The method of  claim 27 , wherein said V L  sequence is selected from the group consisting of SEQ ID NOs: 2, and 12-15. 
     
     
         30 . The method of any of  claims 1 - 29 , wherein the polypeptide or immunoconjugate comprises an anti-CD22 antibody or antigen binding fragment thereof and a PE or variant thereof. 
     
     
         31 . The method of  claim 30 , wherein the immunoconjugate is the CAT-8015 immunotoxin comprising the V H -PE38 subunit of SEQ ID NO:1 and the V L  subunit of SEQ ID NO:2. 
     
     
         32 . A composition comprising a purified immunoconjugate having less than between about 25% and about 1% deamidated species, wherein said immunoconjugate is purified by the method of any of  claims 1 - 31 . 
     
     
         33 . The composition of  claim 32 , wherein less than about 25% of the deamidated species is present. 
     
     
         34 . The composition of  claim 32 , wherein less than about 20% of the deamidated species is present. 
     
     
         35 . The composition of  claim 32 , wherein less than about 10% of the deamidated species is present. 
     
     
         36 . The composition of  claim 32 , wherein less than about 5% of the deamidated species is present. 
     
     
         37 . The composition of  claim 32 , wherein less than about 3% of the deamidated species is present. 
     
     
         38 . The composition of  claim 32 , wherein less than about 2% of the deamidated species is present. 
     
     
         39 . The composition of  claim 32 , wherein less than about 1% of the deamidated species is present. 
     
     
         40 . A pharmaceutical composition comprising the purified immunoconjugate of any of  claims 32 - 39  and a pharmaceutically acceptable carrier. 
     
     
         41 . A composition comprising a purified immunoconjugate having less than between about 20% and about 1% deamidated species, wherein said immunoconjugate comprises an anti-CD22 antibody or antigen binding fragment thereof and a PE toxin or variant thereof. 
     
     
         42 . The composition of  claim 41 , wherein said immunoconjugate comprises the V H -PE38 subunit of SEQ ID NO:1 and the V L  subunit of SEQ ID NO:2. 
     
     
         43 . The composition of  claim 41 , wherein said purified immunoconjugate has less than 20% of deamidated species. 
     
     
         44 . A unit dosage form of a purified immunoconjugate in the range of 0.1 mg to 6 mg, wherein said immunoconjugate comprises an anti-CD22 antibody or antigen binding fragment thereof and a PE toxin or variant thereof. 
     
     
         45 . The unit dosage form of  claim 44 , wherein said immunoconjugate comprises the V H -PE38 subunit of SEQ ID NO:1 and the V L  subunit of SEQ ID NO:2. 
     
     
         46 . A formulation comprising the composition of any of  claims 32 - 43  and at least one excipient selected from the group consisting of sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium hydroxide and water. 
     
     
         47 . A formulation comprising the composition of any of  claims 32 - 43  comprising 1 mg/mL CAT-8015 in 25 mM sodium phosphate, 4% sucrose, 8% glycine, 0.02% polysorbate 80 (PS80), pH 7.4. 
     
     
         48 . The formulation of claim any one of  claims 41  to  43  that is further lyophilized. 
     
     
         49 . A method of modifying the bioactivity of a polypeptide solution comprising a polypeptide and a deamidated variant, the method comprising separating the polypeptide from the deamidated variant by linear elution AIEX chromatography; and combining the purified polypeptide and deamidated variant in fixed quantities to obtain the desired bioactivity of the polypeptide solution. 
     
     
         50 . The method of any of  claims 24 - 27 , wherein said one or more deamidated residues are present within the  Pseudomonas  exotoxin or variant thereof. 
     
     
         51 . The method of  claim 47 , wherein said immunoconjugate is encoded by the amino acid sequence of SEQ ID NO:1 and is deamidated at position 358 of SEQ ID NO:1. 
     
     
         52 . The method of  claim 3 , wherein refolding the polypeptide or immunoconjugate isolated from the inclusion bodies comprises solubilization of inclusion bodies at a pH in a range of about pH 9.0 to about pH 10.5. 
     
     
         53 . The method of  claim 3  or  52 , wherein refolding the polypeptide or immunoconjugate isolated from the inclusion bodies comprises solubilization of inclusion bodies at a pH of 10.5, a pH of 10.0, a pH of 9.5, or pH of 9.0.