Process for concentration of a polypeptide

Abstract

The present invention comprises a method of concentrating a composition comprising a polypeptide of interest and the use of such a concentrated composition for the treatment of diseases in mammals, in particular by subcutaneous injection.

1 - 28 . (canceled) 
     
     
         29 . A method of concentrating a composition comprising porphobilinogen deaminase comprising:
 a) performing centrifugation and/or filtration of a composition comprising porphobilinogen deaminase; and   b) concentrating the supernatant or filtrate, respectively, obtained from step a).   
     
     
         30 . The method of  claim 29 , wherein the porphobilinogen deaminase comprises an amino acid selected from the group consisting of:
 i) an amino acid sequence as defined by SEQ ID NO. s: 14 or 15;   ii) a functionally equivalent part of an amino acid sequence as defined in i); and   iii) a functionally equivalent analogue of an amino acid sequence as defined in i) or ii), the amino acid sequence of said analogue being at least 85% identical to an amino acid sequence as defined in i) or ii).   
     
     
         31 . The method according to  claim 29 , wherein step b) is performed by freeze-drying, evaporation, ultrafiltration, tangential flow filtration or with a centrifugal device. 
     
     
         32 . The method according to  claim 29 , wherein the composition comprising porphobilinogen deaminase further comprises one or more of the components selected from the group consisting of: glycine, L-serine, sucrose and mannitol. 
     
     
         33 . The method according to  claim 29 , wherein the composition comprising porphobilinogen deaminase further comprises one or more buffers selected from the group consisting of: TRIS-HCL, Na-citrate and Na 2 HPO 4 . 
     
     
         34 . The method according to  claim 29 , wherein said method further comprises sterilizing the concentrated composition comprising porphobilinogen deaminase obtained from step b). 
     
     
         35 . The method according to  claim 29 , wherein said method further comprises freeze-drying the concentrated composition comprising porphobilinogen deaminase obtained from step b). 
     
     
         36 . The method according to  claim 29 , wherein the filter used for the filtration in step a) has a pore-size in the range of 0.20 to 5.0 micrometer. 
     
     
         37 . The method according to  claim 29 , wherein said method further comprises one or more of the following prior to step a):
 i) recombinant expression of porphobilinogen deaminase;   ii) purification of porphobilinogen deaminase by one or more chromatographic separations; or   iii) exchange of the formulation buffer.   
     
     
         38 . The method according to  claim 37 , wherein the one or more chromatographic separations in step ii) is selected from the group consisting of: affinity chromatography, ion exchange chromatography and hydroxyapatite chromatography. 
     
     
         39 . The method according to  claim 29 , wherein said method comprises the following prior to step a):
 i) recombinant expression of porphobilinogen deaminase;   ii) subjecting the composition comprising a porphobilinogen deaminase from step i) to affinity chromatography; and   iii) subjecting the composition comprising a porphobilinogen deaminase of step ii) to ion exchange chromatography.   
     
     
         40 . The method according to  claim 29 , wherein said method comprises the following prior to step a):
 i) recombinant expression of a porphobilinogen deaminase;   ii) subjecting the composition comprising a porphobilinogen deaminase from step i) to affinity chromatography;   iii) subjecting the composition comprising a porphobilinogen deaminase from step ii) to ion exchange chromatography; and   subjecting the composition comprising porphobilinogen deaminase from step iii) to a hydroxyapatite column.   
     
     
         41 . The method according to  claim 29 , wherein said method comprises recombinant expression of a nucleic acid sequence comprising a sequence selected from the group consisting of:
 i) a nucleic acid sequence as defined by any of SEQ ID NOs.: 1-13; and   ii) a nucleic acid sequence which is at least 75% identical to a nucleic acid sequence as defined in i).   
     
     
         42 . The method according to  claim 29 , wherein said method further comprises dilution or diafiltration of the composition comprising porphobilinogen deaminase obtained from step ii). 
     
     
         43 . The method according to  claim 29 , wherein the porphobilinogen deaminase is selected from the group consisting of recombinant porphobilinogen deaminase (rPBGD) and/or functionally equivalent parts and analogues hereof, recombinant human porphobilinogen deaminase (rhPBGD) and/or functionally equivalent parts and analogues hereof and a fusion protein of porphobilinogen deaminase. 
     
     
         44 . The method according to  claim 29 , wherein the amount of porphobilinogen deaminase and functionally equivalent parts and analogues hereof present as aggregates constitute less than 5% of the total amount of porphobilinogen deaminase and functionally equivalent parts and analogues hereof in the composition. 
     
     
         45 . A composition comprising at least 10 mg/ml of porphobilinogen deaminase and functionally equivalent parts and analogues hereof. 
     
     
         46 . The composition according to  claim 45 , wherein the porphobilinogen deaminase comprises an amino acid selected from the group consisting of:
 i) an amino acid sequence as defined by SEQ ID NOs.: 14 or 15;   ii) a functionally equivalent part of an amino acid sequence as defined in i); and   iii) a functionally equivalent analogue of an amino acid sequence as defined in i) or ii), the amino acid sequence of said analogue being at least 75% identical to an amino acid sequence as defined in i) or ii).   
     
     
         47 . The composition according to  claim 45 , wherein the porphobilinogen deaminase is selected from the group consisting of recombinant porphobilinogen deaminase (rPBGD) and/or functionally equivalent parts and analogues hereof, recombinant human porphobilinogen deaminase (rhPBGD) and/or functionally equivalent parts and analogues hereof and a fusion protein of porphobilinogen deaminase. 
     
     
         48 . The composition according to  claim 45 , wherein the amount of porphobilinogen deaminase and functionally equivalent parts and analogues hereof present as aggregates constitute less than 5% of the total amount of porphobilinogen deaminase and functionally equivalent parts and analogues hereof in the composition. 
     
     
         49 . A method of treating or inhibiting Acute Intermittent  Porphyria  in a mammal comprising administering a composition comprising 50-300 mg/ml porphobilinogen deaminase and functionally equivalent parts and analogues hereof to a mammal in need thereof. 
     
     
         50 . The method according to  claim 47 , wherein the amount of porphobilinogen deaminase and functionally equivalent parts and analogues hereof present as aggregates constitute less than 5% of the total amount of porphobilinogen deaminase and functionally equivalent parts and analogues hereof in the composition. 
     
     
         51 . The method according to  claim 47 , wherein the porphobilinogen deaminase is selected from the group consisting of recombinant porphobilinogen deaminase (rPBGD) and/or functionally equivalent parts and analogues hereof, recombinant human porphobilinogen deaminase (rhPBGD) and/or functionally equivalent parts and analogues hereof and a fusion protein of porphobilinogen deaminase.