N-terminally chemically modified protein compositions and methods
AssigneeAmgen Inc.
InventorsOlaf B. Kinstler, Nancy E. Gabriel, Christine E. Farrar, Randolph B. DePrince
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Abstract
Provided herein are methods and compositions relating to the attachment of water soluble polymers to proteins. Provided are novel methods for N-terminally modifying proteins or analogs thereof, and resultant compositions, including novel N-terminally chemically modified G-CSF compositions and related methods of preparation. Also provided is chemically modified consensus interferon.
Claims12
A substantially homogenous preparation of N-terminally PEGylated G-CSF or analog thereof, optionally in a pharmaceutically acceptable diluent, carrier or adjuvant, said preparation being essentially free of G-CSF or analog thereof PEGylated at sites other than the N-terminus.
A substantially homogenous preparation of N-terminally monoPEGylated G-CSF, optionally in a pharmaceutically acceptable diluent, carrier or adjuvant, wherein:
- (a) said G-CSF has the amino acid sequence identified in SEQ. ID No. 1;
- (b) said G-CSF is monoPEGylated with a polyethylene glycol moiety having a molecular weight of between about 6 kDa and about 25 kDa.
A pharmaceutical composition comprising:
- (a) a substantially homogenous preparation of monoPEGylated G-CSF, said monoPEGylated G-CSF consisting of a polyethylene glycol moiety having a molecular weight of about 6 kDa and about 25 kDa connected to a G-CSF moiety solely at the N-terminus thereof via an amine linkage;
- (b) fewer than 5% non-PEGylated G-CSF molecules;
- and (c) a pharmaceutially acceptable diluent, adjuvant or carrier.
A method for attaching a polyethylene glycol molecule to a G-CSF molecule, wherein said polyethylene glycol molecule has a single reactive aldehyde group, said method comprising:
- (a) reacting said G-CSF with said polyethylene glycol molecule under reducing alkylation conditions, at a pH sufficiently acidic to selectively activate the alpha-amino group at the amino terminus of said G-CSF;
- and
- (b) obtaining the pegylated G-CSF and
- (c) optionally, separating the PEGylated G-CSF from from non-PEGylated G-CSF.
Dependencies are inferred from the claim text.