Granulocyte macrophage-colony stimulating factor mutants
AssigneePartner Therapeutics, Inc.
InventorsMichael FELDHAUS, Jeffrey YOST
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Abstract
The present invention is based on the discovery that amino acid substitutions in the sequence of sargramostim yield a product with simplified manufacturability.
Claims82
A composition comprising a recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF) protein, comprising an amino acid sequence having at least about 97% identity with SEQ ID NO:
- 1 or SEQ ID NO:
- 2 and having a substitution or deletion at position N37, E38 and/or T39 or a position corresponding thereto.
The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF is soluble.
The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF is functionally similar to sargramostim.
The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF comprises a plurality of molecular forms.
The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF is substantially free of hypermannosylated forms.
The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF resolves as three peaks when quantified by reversed phase high-performance liquid chromatography (RP-HPLC).
The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF provides no substantial peaks at a retention time of less than about 20 minutes when quantified by reversed phase high-performance liquid chromatography (RP-HPLC).
The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF is substantially free of hypermannosylated forms when purified without the use of organic solvents.
The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF is substantially free of hypermannosylated forms, optionally when purified without the use of a reversed phase C4 HPLC column.
A nucleic acid molecule encoding the recombinant human GM-CSF of any one of the above claims.
A pharmaceutical composition comprising a recombinant human GM-CSF of any one of the above claims and a pharmaceutically acceptable excipient or carrier.
A method of making a recombinant producing a composition comprising a recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF) comprising:
- (a) obtaining a cell transfected with a nucleic acid encoding a recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF), comprising an amino acid sequence having at least about 97% identity with SEQ ID NO:
- 2 and having a substitution or deletion at position N37, G38 and/or T39 or a position corresponding thereto, or an extract thereof;
- (b) purifying the GM-CSF from the transfected cell using one or more HPLC columns, wherein the purification is in the absence of an organic solvent;
- and
- (c) collecting the purified GM-CSF, the purified GM-CSF being substantially free of hypermannosylated GM-CSF forms.
A composition comprising a recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF) protein, comprising an amino acid sequence having at least about 97% identity with SEQ ID NO:
- 1 or SEQ ID NO:
- 2 and having a N37Q substitution, or a position corresponding thereto,
- wherein the GM-CSF is isolatable without substantial hyperglycosylated species, without the need for organic solvent purification.
Dependencies are inferred from the claim text.