# US 8,784,794 B2 · Long-term storage of non-glycosylated recombinant human G-CSF

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- Number: US 8,784,794 B2
- Document ID: US8784794B2
- Status: Granted
- Kind: B2
- Application number: 13/805,180
- Filed: 2011-06-22
- Published: 2013-06-27
- Granted: 2014-07-22
- Earliest claimed: 2010-06-22
- Assignees: Sandoz Ltd.
- Applicants: Klaus Graumann; Helmut Lerch; Thomas Lauber
- Inventors: Klaus Graumann; Helmut Lerch; Thomas Lauber
- Web page: https://patentgrep.com/patent/US8784794B2

## CPC

- A61K 38/193: {Colony stimulating factors [CSF]}
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 9/08: Solutions {(composition of solutions A61K47/00)}
- A61K 47/26: Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 35/00: Antineoplastic agents
- C07K 14/535: Granulocyte CSF; Granulocyte-macrophage CSF
- 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)
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention provides a method for stable long-term storage of non-glycosylated recombinant human Granulocyte-Colony Stimulating Factor (“G-CSF”), wherein an aqueous acetate or glutamate buffered G-CSF composition containing the non-glycosylated recombinant human G-CSF and sorbital is cooled to a temperature of −15° C. or below to obtain a frozen G-CSF composition, which frozen composition is then stored in the frozen state and then increased in temperature to a temperature within the range of from 2° C. to 8° C. for a period of time adjusted to allow the composition to thaw and to obtain a liquid composition having a G-CSF content of at least 95% of the G-CSF content of the original composition.

## Claims

### Claim 1

A method for stable long-term storage of non-glycosylated recombinant human Granulocyte-Colony Stimulating Factor (G-CSF), said method comprising the steps of:
(a) providing an aqueous acetate or glutamate buffered G-CSF composition containing a non-glycosylated recombinant human G-CSF and sorbitol, wherein the amount of G-CSF is in the range of from 0.1 mg/ml to 8.0 mg/ml and wherein said composition has a volume of from 100 ml to 8 l;
(b) cooling the G-CSF-formulation provided in step (a) to a temperature of −15° C. or below to obtain a frozen G-CSF formulation;
(c) storing the G-CSF-formulation obtained in step (b) in the frozen state over a period of at least one month;
(d) increasing the temperature of the frozen G-CSF composition of step (c) to a temperature within the range of from 2° C. to 8° C. over a period of time of at least 6 hours to allow the composition to thaw and to obtain a liquid composition having a G-CSF content of at least 95% of the G-CSF content of the composition provided in step (a).

### Claim 2 (depends on 1)

The method of claim 1, wherein the amount of G-CSF in the buffered G-CSF formulation of step (a) is in the range of from 0.25 mg/ml to 6.5 mg/ml.

### Claim 3 (depends on 1)

The method of claim 1, wherein the buffered G-CSF composition has a pH of from 3.5 to 5.

### Claim 4 (depends on 1)

The method of claim 1, wherein the acetate or glutamate concentration of the buffered G-CSF composition is in the range of from 0.5 mM to 100 mM.

### Claim 5 (depends on 1)

The method of claim 1, wherein the amount of sorbitol in the G-CSF composition is in the range of from 10 to 100 mg/ml.

### Claim 6 (depends on 1)

The method of claim 1, wherein the aqueous acetate or glutamate buffered G-CSF composition provided in step (a) has a volume of from 100 ml to 4 l.

### Claim 7 (depends on 1)

The method of claim 1, wherein the G-CSF composition in step (b) is cooled to a temperature of between −15° C. and −25° C. or a temperature of between −60° C. and −80° C.

### Claim 8 (depends on 1)

The method of claim 1, wherein the frozen G-CSF composition in step (c) is stored over a period of at least three months.

### Claim 9 (depends on 1)

The method of claim 1, wherein temperature increase of the frozen G-CSF composition to a temperature within the range of from 2° C. to 8° C. in step (d) is effected by gradually or linearly increasing temperature over a period of time of at least 6 hours.

### Claim 10 (depends on 1)

The method of claim 1, wherein temperature increase of the frozen G-CSF composition to a temperature in step (d) is effected by transferring the frozen composition to a temperature within the range of from 2° C. to 8° C. and maintaining the composition at said temperature for a period of time of at least 12 hours.

### Claim 11 (depends on 1)

The method of claim 1, wherein the G-CSF composition is free of surfactant.

### Claim 12 (depends on 1)

The method of claim 1, wherein the G-CSF composition comprises a surfactant.

### Claim 13

A method of providing a pharmaceutical composition of non-glycosylated recombinant human G-CSF, said method comprising the steps of:
(a) formulating non-glycosylated recombinant human G-CSF with an acetate or glutamate buffer and sorbitol to obtain an aqueous buffered G-CSF composition, wherein the amount of G-CSF is in the range of from 0.1 mg/ml to 8.0 mg/ml and wherein said composition has a volume of from 100 ml to 8 l;
(b) cooling the G-CSF composition of step (a) to a temperature of −15° C. or below to obtain a frozen G-CSF composition;
(c) storing the G-CSF composition obtained in step (b) in the frozen state over a period of at least one month;
(d) increasing the temperature of the frozen G-CSF composition of step (c) to a temperature within the range of from 2° C. to 8° C. over a period of time of at least 6 hours to allow the composition to thaw and to obtain a liquid composition having a G-CSF content of at least 95% of the G-CSF content of the composition provided in step (a); and
(e) filling the liquid composition obtained in step (d) into primary packagings for parenteral use.