Purification of not-glycosylated polypeptides

Abstract

The current invention reports a method for the purification of a not-glycosylated, heterologous polypeptide, which has been recombinantly produced in a prokaryotic cell, wherein the method comprises three chromatography steps of which the first chromatography step selected from i) hydrophobic charge induction chromatography, or ii) hydrophobic interaction chromatography, or iii) affinity chromatography, or iv) ion exchange chromatography, the second chromatography step is selected from i) anion exchange chromatography, or ii) cation exchange chromatography, or iii) hydroxylapatite chromatography, or iv) hydrophobic interaction chromatography, and the a third chromatography step is selected from i) hydrophobic charge induction chromatography, or ii) anion exchange chromatography, or iii) cation exchange chromatography, or iv) hydrophobic interaction chromatography, whereby the first chromatography step is an affinity chromatography in case of polypeptides capable of interacting with metal ligands, the second chromatography step is not a hydroxylapatite chromatography step in case of polypeptides with an isoelectric point below 6.0, and the third chromatography step can be performed in flow-through mode with polypeptides having a low or high isoelectric point.

1 . A method for the purification of a not-glycosylated, heterologous polypeptide, which has been recombinantly produced in an  E. coli  cell, wherein said not-glycosylated, heterologous polypeptide is selected from growth factor agonists or antagonists, or interferons or interferon variants, comprising the following steps in the following order:
 a) providing a not-glycosylated, heterologous polypeptide, which has been recombinantly produced in an  E. coli  cell,   b) a first chromatography step selected from the group consisting of
 i) hydrophobic charge induction chromatography, 
 ii) hydrophobic interaction chromatography, 
 iii) metal chelating chromatography, or 
 iv) ion exchange chromatography, 
   c) a second chromatography step selected from the group consisting of
 i) anion exchange chromatography, 
 ii) cation exchange chromatography, 
 iii) hydroxylapatite chromatography, 
 iv) hydrophobic interaction chromatography, or 
 v) hydrophobic charge induction chromatography, and 
   d) a third chromatography step selected from the group consisting of
 i) hydrophobic charge induction chromatography, 
 ii) anion exchange chromatography, 
 iii) cation exchange chromatography, or 
 iv) hydrophobic interaction chromatography, 
   whereby
 said first chromatography step is an affinity chromatography or a hydrophobic charge induction chromatography in case of polypeptides capable of interacting with metal ligands, 
 said second chromatography step is not a hydroxylapatite chromatography step in case of polypeptides with an isoelectric point below 6.0, 
 said third chromatography step can be performed in flow-through mode with polypeptides having a low or high isoelectric point, 
   and the purified non-glycosylated, heterologous polypeptide is obtained after step d).   
     
     
         2 .- 4 . (canceled) 
     
     
         5 . The method of  claim 1 , characterized in that said method comprises an additional step after step d) which is
 e) PEGylating said polypeptide.   
     
     
         6 . The method of  claim 5 , characterized in that said steps b) and c) are cation exchange chromatography steps. 
     
     
         7 . A method for the recombinant production of a not-glycosylated heterologous polypeptide in a prokaryotic cell, characterized in that said method comprises the following steps:
 a) cultivating a prokaryotic cell comprising a nucleic acid encoding said heterologous polypeptide under conditions suitable for the expression of said heterologous polypeptide, wherein the prokaryotic cell is an  E. coli  cell,   b) recovering said heterologous polypeptide from the culture medium or the prokaryotic cells, and   c) purifying said heterologous polypeptide with the method of  claim 1 .   
     
     
         8 . A method for the recombinant production of a not-glycosylated heterologous polypeptide in a prokaryotic cell via inclusion bodies, characterized in that said method comprises the following steps:
 a) cultivating said prokaryotic cell comprising a nucleic acid encoding said heterologous polypeptide under conditions suitable for the expression of said heterologous polypeptide and formation of inclusion bodies containing said heterologous polypeptide, wherein the prokaryotic cell is an  E. coli  cell,   b) recovering said inclusion bodies from the prokaryotic cells,   c) solubilizing and renaturating said heterologous polypeptide from said inclusion bodies, and   d) purifying said heterologous polypeptide with the method of  claim 1 .   
     
     
         9 . The method of  claim 1 , characterized in that content of endotoxins, and/or  E. coli  DNA, and/or  E. coli  cell proteins is reduced in the polypeptide solution obtained after the third chromatography step compared to the content prior to the first chromatography step. 
     
     
         10 . The method of  claim 1 , wherein the not-glycosylated, heterologous polypeptide is IGF-1 or an IGF-1 variant, whereby the first chromatography step is a hydrophobic charge induction chromatography, the second chromatography step is selected from hydroxylapatite chromatography or cation exchange chromatography, and the third chromatography step is selected from hydrophobic charge induction chromatography or anion exchange chromatography. 
     
     
         11 . The method of  claim 1 , wherein the not-glycosylated, heterologous polypeptide is IFNα-2a, whereby the first chromatography step is a hydrophobic charge induction chromatography, the second chromatography step is an anion exchange chromatography step, and the third chromatography step is an hydrophobic charge induction chromatography. 
     
     
         12 . The method of  claim 1 , wherein the not-glycosylated, heterologous polypeptide is IFNα-2a, whereby the first chromatography step is a hydrophobic interaction chromatography, the second chromatography step is a cation exchange chromatography step, and the third chromatography step is an hydrophobic interaction chromatography. 
     
     
         13 . A method for producing a not-glycosylated, PEGylated, heterologous polypeptide, which has been recombinantly produced in a prokaryotic cell comprising the following steps in the following order:
 a) providing a not-glycosylated, heterologous polypeptide, which has been recombinantly produced in a prokaryotic cell,   b) a first chromatography step selected from
 i) hydrophobic charge induction chromatography, 
 ii) hydrophobic interaction chromatography, 
 iii) affinity chromatography, or 
 iv) ion exchange chromatography, 
   c) a second chromatography step selected from
 i) anion exchange chromatography, 
 ii) cation exchange chromatography, 
 iii) hydroxylapatite chromatography, or 
 iv) hydrophobic interaction chromatography, 
   d) a third chromatography step selected from
 i) hydrophobic charge induction chromatography, 
 ii) anion exchange chromatography, 
 iii) cation exchange chromatography, or 
 iv) hydrophobic interaction chromatography, 
   whereby said not-glycosylated, heterologous polypeptide is PEGylated after step d).   
     
     
         14 . The method of  claim 13 , wherein the non-glycosylated PEGylated heterologous polypeptide is IFNα-2a, whereby the first chromatography step is selected from hydrophobic interaction chromatography or metal affinity chromatography, the second chromatography step is a cation exchange chromatography, and the third chromatography step is an anion exchange chromatography and wherein after the third chromatography step the purified not-glycosylated and not-PEGylated IFNα-2a is PEGylated. 
     
     
         15 . The method of  claim 13 , wherein the non-glycosylated PEGylated heterologous polypeptide is PEGylated interferon, whereby the first chromatography step is hydrophobic interaction chromatography, the second chromatography step is a cation exchange chromatography, and the third chromatography step is an hydrophobic charge induction chromatography and wherein after the third chromatography step the purified not-glycosylated and not-PEGylated IFN is PEGylated. 
     
     
         16 . A method for the purification of a not-glycosylated, heterologous polypeptide, which has been recombinantly produced in a prokaryotic cell, wherein said not-glycosylated, heterologous polypeptide is selected from growth factor agonists or antagonists, or interferons or interferon variants, characterized in that said method comprises the following steps in the following order:
 a) a first chromatography step selected from
 i) hydrophobic charge induction chromatography, 
 ii) hydrophobic interaction chromatography, 
 iii) affinity chromatography, or 
 iv) ion exchange chromatography, 
   b) a second chromatography step selected from
 i) anion exchange chromatography, 
 ii) cation exchange chromatography, 
 iii) hydroxylapatite chromatography, 
 iv) hydrophobic interaction chromatography, or 
 v) hydrophobic charge induction chromatography, and 
   c) a third chromatography step selected from
 i) hydrophobic charge induction chromatography, 
 ii) anion exchange chromatography, 
 iii) cation exchange chromatography, or 
 iv) hydrophobic interaction chromatography, 
   whereby
 said first chromatography step is an affinity chromatography or a hydrophobic charge induction chromatography in case of polypeptides capable of interacting with metal ligands, 
 said second chromatography step is not a hydroxylapatite chromatography step in case of polypeptides with an isoelectric point below 6.0, 
 said third chromatography step can be performed in flow-through mode with polypeptides having a low or high isoelectric point, 
   and whereby at least two different sequences of three chromatographic steps yield a purified not-glycosylated, heterologous polypeptide with comparable purity.