Purification Of Recombinantly Produced Polypeptides
AssigneeMedlmmune, LLC
InventorsDavid Edwin Gruber, Richard Edward Turner, Jares Samuel Bee, Christopher Samuel Afdahi, Liu Tie
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Abstract
Described herein is a method for separating a recombinantly produced polypeptide from host cell protein. The method includes a step of loading a clarified cell culture supernatant that includes the recombinantly produced polypeptide and the HCP onto a Protein A chromatography column and washing the Protein A chromatography column with a wash buffer comprising a fatty acid having a chain length of at least about 6 carbon atoms, or a fatty acid salt thereof to remove HCP and then recovering the recombinantly produced polypeptide.
Claims72
A method of separating a recombinantly produced polypeptide from host cell protein (HCP), the method comprising:
- providing a clarified cell culture supernatant comprising the HCP and the recombinantly produced polypeptide;
- loading the clarified cell culture supernatant onto a Protein A chromatography column;
- washing the Protein A chromatography column with a wash buffer comprising a fatty acid having a chain length of at least about 6 carbon atoms, or a fatty acid salt thereof to remove HCP;
- and
- recovering the recombinantly produced polypeptide.
A method of separating a recombinantly produced polypeptide from host cell protein (HCP), the method comprising:
- equilibrating a Protein A chromatography column with an equilibration buffer;
- loading the clarified cell culture supernatant onto the Protein A chromatography column;
- re-equilibrating the loaded Protein A chromatography column with the equilibration buffer;
- washing the loaded Protein A chromatography column with a first wash buffer comprising a fatty acid having a chain length of at least about 6 carbon atoms, or a fatty acid salt thereof to remove HCP;
- washing the loaded Protein A chromatography column with a second wash buffer;
- and
- eluting the recombinantly produced polypeptide with an elution buffer.
A method of reducing protease contamination in a formulation comprising a recombinantly produced polypeptide, the method comprising:
- providing a clarified cell culture supernatant comprising the HCP and the recombinantly produced polypeptide;
- loading the clarified cell culture supernatant onto a Protein A chromatography column;
- washing the Protein A chromatography column with a wash buffer comprising a fatty acid having a chain length of at least about 6 carbon atoms, or a fatty acid salt thereof to remove HCP;
- recovering the recombinantly produced polypeptide;
- and
- preparing the formulation.
A method of increasing stability of a recombinantly produced polypeptide, the method comprising:
- providing a clarified cell culture supernatant comprising the recombinantly produced polypeptide and one or more host cell proteins (HCP);
- loading the clarified cell culture supernatant onto a Protein A chromatography column;
- washing the Protein A chromatography column with a wash buffer comprising a fatty acid having a chain length of at least about 6 carbon atoms, or a fatty acid salt thereof to remove HCP;
- recovering the recombinantly produced polypeptide;
- and
- preparing a formulation comprising the recovered recombinantly produced polypeptide.
A method of reducing host cell protein level in a composition comprising a recombinantly produced polypeptide, the method comprising:
- providing a clarified cell culture supernatant comprising the recombinantly produced polypeptide and one or more host cell proteins (HCP);
- loading the clarified cell culture supernatant onto a Protein A chromatography column;
- washing the Protein A chromatography column with a wash buffer comprising a fatty acid having a chain length of at least about 6 carbon atoms, or a fatty acid salt thereof to remove HCP.
A method of reducing cathepsin-D in a composition comprising a recombinantly produced polypeptide, the method comprising:
- providing a clarified cell culture supernatant comprising the recombinantly produced polypeptide and cathepsin-D;
- loading the clarified cell culture supernatant onto a Protein A chromatography column;
- washing the Protein A chromatography column with a wash buffer comprising a fatty acid having a chain length of at least about 6 carbon atoms, or a fatty acid salt thereof to remove cathepsin-D.
The method of any preceding claim, wherein the wash buffer comprises between about 25 mM to about 200 mM fatty acid.
The method of any preceding claim, wherein the wash buffer comprises between about 50 mM to about 100 mM fatty acid.
The method according to any preceding claim, wherein the wash buffer comprises sodium chloride.
The method of any preceding claim, wherein the wash buffer has a pH between about 7 to about 9.
The method of any preceding claim, wherein the wash buffer has a pH between about 8 to about 9.
The method of any preceding claim, wherein the wash buffer has a pH between about 8.5 to about 9.
The method of any preceding claim, wherein the wash buffer has a pH of about 9.
The method of any preceding claim, wherein the wash buffer comprises between about 50 mM and about 100 mM sodium caprylate at a pH between about 8 to about 9.
The method of any preceding claim, wherein the wash buffer comprises between about 50 mM and about 100 mM sodium caprylate at a pH between about 8 to about 9 and between about 2.0M to about 2.5 M sodium chloride.
The method of any preceding claim, wherein the wash buffer comprises about 100 mM sodium caprylate in 100 mM Tris at a pH of about 9.0.
The method of any preceding claim, wherein the wash buffer comprises about 100 mM sodium caprylate in 100 mM Tris at a pH of about 9.0 and about 2.5M sodium chloride.
The method according to any preceding claim, wherein the recombinantly produced polypeptide is an antibody, or fragment thereof.
The method of any preceding claim, comprising a step of clarifying the cell culture harvest to obtain a clarified cell culture harvest and loading the clarified cell culture harvest onto the Protein A chromatography column.
The method of any preceding claim, further comprising equilibrating the loaded Protein A column with an equilibration buffer prior to washing the column with the wash buffer.
The method of any preceding claim, wherein recovering the recombinant protein comprises eluting the recombinant protein from the Protein A column with an elution buffer.
A wash buffer for separating a recombinantly produced polypeptide from host cell protein (HCP) in a Protein A chromatography column, the wash buffer comprising a fatty acid or a fatty acid salt having a chain length of at least 6 carbon atoms.
Dependencies are inferred from the claim text.