US 2011/0034672 A1Published application

Purification of not-glycosylated polypeptides

OwnerRoberto Falkenstein, Birgit Weydanz +8inventors

InventorsRoberto Falkenstein, Birgit Weydanz, Nicole Fuehrler, Claudia Giessel, Sybille Greithanner, Adelbert Grossmann, Friederike Hesse, Marc Pompiati, Andreas Schaubmar, Brigitte Kraemer

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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.

Claims14

Dependent claims

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).

19 more elements

Dependencies are inferred from the claim text.

Facts

Application: 12/811,397Filed 15 Jan 2009
Priority: EP 08000884.018 Jan 2008
Published: 10 Feb 2011
Term ends: Not estimatedThe source records don’t support an estimate.

Classification11 codes

C07K 1/1077
By covalent attachment of residues or functional groups: by covalent attachment of residues other than amino acids or peptide residues, e.g.…
A61K 47/60
Obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes: the organic…
C07K 14/56
Interferons [IFN]: iFN-alpha
C07K 14/65
Hormones: insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2