Glycopegylated granulocyte colony stimulating factor

• US 9,029,331 B2
• Filed: 01/10/2006
• Issued: 05/12/2015
• Est. Priority Date: 01/10/2005
• Status: Expired due to Fees

• Current Assignee: Novo Nordisk A/S
• Original Assignee: Novo Nordisk A/S
• Inventors: DeFrees, Shawn, Clausen, Henrik, Zopf, David A., Bowe, Caryn, Taudte, Susann, Felo, Michael, Willett, Walter S.
• Primary Examiner(s): GOON, SCARLETT Y
• Application Number:
  US11/794,560
• Publication Number:
  US 20090203579A1
• Time in Patent Office: 3,409 Days
• Field of Search: 514/20.9, 514/9.7, 514/21.2
• US Class Current: 514/20.9
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/04 Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 1/18 Ion-exchange chromatography
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF

Abstract

The invention relates to methods of preparing and purifying conjugates between Granulocyte Colony Stimulating Factor and PEG moieties. The conjugates are linked via an intact glycosyl linking group that is interposed between and covalently attached to the peptide and the modifying group. The conjugates are purified using various chromatography methods.

Claims

1. A method of preparing a purified, pegylated GCSF peptide, which method comprises

(a) providing a composition comprising water, a buffer, at least one polysorbate, and 20 mM L-methionine, wherein the polysorbate is polyoxyethylene sorbitan monolaurate or polyoxvethylene sorbitan monooleate, and the composition has a pH of about 6.5;

(b) combining the composition of step (a) with reagents comprising GCSF, GalNAeT2, and UDP-GalNAc, wherein the composition of step (b) comprises 0.45 mM UDP-GalNAc;

(c) combining the composition of step (b) with MnCl₂ wherein the composition of step (c) comprises 1 mM MnCl₂;

(d) combining the composition of step (c) with ST6GalNAc1 and an aliquot of cytidine monophosphate sialic acid poly(ethylene glycol) (CMP-SA-PEG), wherein the composition of step (d) comprises 1 mM CMP-SA-PEG, and, wherein the composition of step (e) is not purified prior to step (d);

(e) combining the composition of step (d) with an aliquot of CMP-SA-PEG, wherein the composition of step (e) comprises 1.5 mM CMP-SA-PEG;

(f) combining the composition of step (e) with a 20 mM citrate buffer, wherein no additional L-methionine is added to the composition of step (e) and wherein the resulting composition has a pH of about 4, and

(g) applying the composition of step (f) to a cation exchange chromatography column, whereby purified PEG-GCSF is produced.

2. The method of claim 1, further comprising

(h) combining the composition of step (g) with a buffer mixture comprising 20 mM NaOAc and 600 mM Na₂SO₄, and applying the composition to a hydrophobic interaction chromatography column.

3. In a method of preparing a purified, pegylated GCSF peptide, wherein the method comprises a pegylation step and a purification step, the improvement comprising (a) pegylating GCSF in a composition comprising 20 mM L-methionine and at a pH of about 6.5 and (b) purifying the composition comprising the pegylated GCSF by applying the composition comprising the pegylated GCSF to a cation exchange chromatography column after adding a 20 mM citrate buffer and no additonal L-methionine to the composition comprising pegylated GCSF to provide a pH of about 4.0.

4. In the method of claim 3, the improvement further comprising pegylating GCSF in a composition comprising MnCl₂ at a concentration of 1 mM.