CORRECTLY FOLDED ETANERCEPT IN HIGH PURITY AND EXCELLENT YIELD

• US 20190330325A1
• Filed: 06/17/2019
• Published: 10/31/2019
• Est. Priority Date: 09/11/2012
• Status: Active Grant

• Current Assignee: Coherus BioSciences Inc.
• Original Assignee: Coherus BioSciences Inc.
• Inventors: ARAKAWA, Tsutomu, FARRAR, Douglas
• Granted Patent: US 10,947,306 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 39/395 Antibodies agglutinins A61K38/36 ; as drug carriers A61K47/50; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: C07K 1/165 mixed-mode chromatography
• CPC Class Codes: C07K 14/70578 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

A mixed mode chromatography method for separating correctly folded from incorrectly folded conformations of a given protein is provided. The method is highly effective in separating correctly folded etanercept from incorrectly folded etanercept and aggregates in commercially attractive yields capable of affording etanercept preparations having very high purity in terms of correctly folded etanercept versus incorrectly folded etanercept. The invention is further directed to protein preparations and formulations comprising correctly folded proteins obtained using the present methods, and methods of treatment using the high purity preparations obtained from the mixed mode method.

Claims

1. A method of reducing formation of etanercept aggregates and fragments in an aqueous injectable pharmaceutical formulation containing about 25 to about 75 mg/ml etanercept, comprising:

a. removing incorrectly folded and aggregated etanercept from an etanercept-containing protein mixture to form an etanercept composition containing at least 90 wt. % correctly folded etanercept;

b. combining the etanercept composition with about 0.1 to 2 weight percent of sucrose and an amino acid that is not arginine and is not cysteine, one or more tonicity modifiers selected from the group consisting of sodium chloride, potassium chloride, sodium sulfate, and sodium citrate, and an aqueous buffer selected from the group consisting of phosphate, histidine, citrate, maleate, tartrate, succinate, acetate, tris-(hydroxymethyl)-aminomethane (tris), and bicarbonate to form the aqueous injectable pharmaceutical formulation containing about 25 to about 75 mg/ml etanercept,

wherein the aqueous injectable pharmaceutical formulation has a pH of about 6.2 to 7.4, and

wherein the aqueous injectable pharmaceutical formulation is less immunogenic than a commercially available etanercept-containing composition and contains less incorrectly folded etanercept than a commercially available etanercept-containing composition.

2. The method of claim 1, wherein the aqueous injectable pharmaceutical formulation comprises about 0.1 to about 2 wt. % sucrose;

about 0.1 to 2 wt. % mannitol, alanine, glycine, or lysine; and

about 10 mM to about 200 mM of one or more tonicity modifiers selected from sodium chloride, potassium chloride, sodium sulfate, and sodium citrate.

3. The method of claim 1, wherein less than 10 wt. % of the etanercept in the etanercept-containing protein mixture is incorrectly folded etanercept.

4. The method of claim 1, wherein less than 5 wt. % of the etanercept in the etanercept-containing protein mixture is incorrectly folded etanercept.

5. The method of claim 1, wherein less than 10 wt. % of the etanercept in the etanercept-containing protein mixture is incorrectly folded etanercept, fragments of etanercept, and aggregated etanercept.

6. The method of claim 1, wherein the one or more tonicity modifiers are sodium citrate and sodium chloride.

7. The method of claim 6, wherein the aqueous injectable pharmaceutical formulation comprises about 10 mM to about 200 mM of the sodium citrate and the sodium chloride.

8. The method of claim 1, wherein the aqueous injectable pharmaceutical formulation comprises about 0.1 to about 2 wt. % of a combination of sucrose, sodium chloride, and lysine.

9. The method of claim 8, wherein the aqueous injectable pharmaceutical formulation does not contain any other amino acid.

10. The method of claim 9, wherein the aqueous buffer is citrate buffer.

11. The method of claim 10, wherein less than 5 wt. % of the etanercept in the etanercept-containing protein mixture is incorrectly folded etanercept, fragments of etanercept, and aggregated etanercept.

12. A method of treating a patient in need of treatment with etanercept, comprising administering to said patient an aqueous injectable pharmaceutical formulation made according to the method of claim 1, wherein said aqueous injectable pharmaceutical formulation comprises about 25 to about 75 mg/ml of an etanercept-containing protein mixture containing at least 90 wt. % correctly folded etanercept, about 0.1 to 2 weight percent of sucrose and an amino acid that is not arginine and is not cysteine, one or more tonicity modifiers selected from the group consisting of sodium chloride, potassium chloride, sodium sulfate, and sodium citrate, and an aqueous buffer selected from the group consisting of phosphate, histidine, citrate, maleate, tartrate, succinate, acetate, tris-(hydroxymethyl)-aminomethane (tris), and bicarbonate, the aqueous pharmaceutical composition having a pH of about 6.0 to 6.6, wherein the aqueous injectable pharmaceutical formulation is less immunogenic than a commercially available etanercept-containing composition and contains less incorrectly folded etanercept than a commercially available etanercept-containing composition.

13. The method of claim 12, comprising administering 25-100 mg etanercept per dose to the subject.

14. The method of claim 12, comprising injecting the aqueous injectable pharmaceutical formulation subcutaneously or intramuscularly.

15. The method of claim 12, wherein less than 5 wt. % of the etanercept in the etanercept-containing protein mixture is incorrectly folded etanercept.

16. A method of treating a subject in need of treatment for rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Wegener'"'"'s disease (granulomatosis), Crohn'"'"'s disease (or inflammatory bowel disease), chronic obstructive pulmonary disease (COPD), Hepatitis C, endometriosis, asthma, cachexia, psoriasis, or atopic dermatitis comprising administering to the subject an aqueous pharmaceutical composition comprising about 25 to about 75 mg/ml of an etanercept-containing protein mixture containing at least 90 wt. % correctly folded etanercept, about 0.1 to 2 weight percent of sucrose and an amino acid that is not arginine and is not cysteine, one or more tonicity modifiers selected from the group consisting of sodium chloride, potassium chloride, sodium sulfate, and sodium citrate, and an aqueous buffer selected from the group consisting of phosphate, histidine, citrate, maleate, tartrate, succinate, acetate, tris-(hydroxymethyl)-aminomethane (tris), and bicarbonate, the aqueous pharmaceutical composition having a pH of about 6.2 to 7.4, wherein the aqueous injectable pharmaceutical formulation is less immunogenic than a commercially available etanercept-containing composition and contains less incorrectly folded etanercept than a commercially available etanercept-containing composition.

17. The method of claim 16, comprising administering 25-100 mg etanercept per dose to the subject.

18. The method of claim 17, wherein the administering comprises injecting the aqueous etanercept composition subcutaneously or intramuscularly.

19. A vial, syringe, or injector pen containing a stable aqueous etanercept composition comprising about 25 to about 75 mg/ml of an etanercept-containing protein mixture containing at least 90 wt. % correctly folded etanercept, about 0.1 to 2 weight percent of sucrose and an amino acid that is not arginine and is not cysteine, one or more tonicity modifiers selected from the group consisting of sodium chloride, potassium chloride, sodium sulfate, and sodium citrate, and an aqueous buffer selected from the group consisting of phosphate, histidine, citrate, maleate, tartrate, succinate, acetate, tris-(hydroxymethyl)-aminomethane (tris), wherein the stable aqueous etanercept composition is less immunogenic than a commercially available etanercept-containing composition and contains less incorrectly folded etanercept than a commercially available etanercept-containing composition.

20. The vial, syringe, or injector pen of claim 19, wherein less than 5 wt. % of the etanercept in the etanercept-containing protein mixture is incorrectly folded etanercept.