WO2020009594A1 - A crystalline, zinc-free form of insulin glargine and the method of its preparation - Google Patents
A crystalline, zinc-free form of insulin glargine and the method of its preparation Download PDFInfo
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- WO2020009594A1 WO2020009594A1 PCT/PL2019/050038 PL2019050038W WO2020009594A1 WO 2020009594 A1 WO2020009594 A1 WO 2020009594A1 PL 2019050038 W PL2019050038 W PL 2019050038W WO 2020009594 A1 WO2020009594 A1 WO 2020009594A1
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- WIPO (PCT)
- Prior art keywords
- insulin glargine
- crystallization
- concentration
- zinc
- mixture
- Prior art date
Links
- 108010057186 Insulin Glargine Proteins 0.000 title claims abstract description 34
- 229960002869 insulin glargine Drugs 0.000 title claims abstract description 33
- COCFEDIXXNGUNL-RFKWWTKHSA-N Insulin glargine Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(=O)NCC(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 COCFEDIXXNGUNL-RFKWWTKHSA-N 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 238000002425 crystallisation Methods 0.000 claims description 37
- 230000008025 crystallization Effects 0.000 claims description 37
- 239000000203 mixture Substances 0.000 claims description 24
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 239000012895 dilution Substances 0.000 claims description 9
- 238000010790 dilution Methods 0.000 claims description 9
- 239000003960 organic solvent Substances 0.000 claims description 9
- 239000001509 sodium citrate Substances 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 230000000977 initiatory effect Effects 0.000 claims description 6
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 abstract description 2
- 239000013078 crystal Substances 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 238000002156 mixing Methods 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000011701 zinc Substances 0.000 description 4
- 229910052725 zinc Inorganic materials 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 239000003929 acidic solution Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- 150000001484 arginines Chemical class 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000001479 atomic absorption spectroscopy Methods 0.000 description 1
- 239000003637 basic solution Substances 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000002178 crystalline material Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 1
- 239000004026 insulin derivative Substances 0.000 description 1
- 229940060975 lantus Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention relates to a method for the preparation of a crystalline form of zinc-free insulin glargine, which should find applications in making pharmaceutical preparations intended for the treatment of diabetes.
- Insulin glargine (also known as Lantus®) is an analog of human insulin in which the aspartic acid present at position 21 of A-chain has been replaced with glycine Gly (A21) and the B chain sequence has been extended by an additional two amino acids, i.e. by adding two arginines, Arg (B31) and Arg (B32) .
- the introduced modifications change the isoelectric point of the obtained analog from pH 5.4 to pH 6.7, thanks to which it has better solubility in the solution with an acidic pH (especially of about 4) than in the solution with a physiological pH (about 7.4) .
- an acidic solution of this analog e.g. with pH about 4
- insulin glargine is used for the production of sustained release insulin preparations intended for the treatment of diabetes.
- insulin and its analogs in crystalline form For practical reasons, such as improved stability and durability of the protein, it is particularly desirable to obtain insulin and its analogs in crystalline form. This applies in particular to insulin glargine.
- WO2015084694 describes a method for crystallization of insulin glargine from a basic solution (at pH higher by at least 1 than the isoelectric point of an insulin analog) containing a crystals stabilizing agent (phenolic compound) and an organic solvent miscible with water (such as ethanol, methanol, acetone or isopropanol), wherein the crystallization is initiated by the addition of a zinc salt.
- a crystals stabilizing agent phenolic compound
- an organic solvent miscible with water such as ethanol, methanol, acetone or isopropanol
- WO2014122653 describes a method for crystallization of insulin glargine from a 30% solution of isopropanol using pH 7 and zinc ions to initiate it.
- the known methods of obtaining crystalline insulin glargine are associated with the problem of the content of zinc at the levels from 0.2 to 0.6% in the active substance obtained by these methods, which necessitates taking into account its content when preparing the ready form of the drug.
- the object of the invention is associated with the problem of the content of zinc at the levels from 0.2 to 0.6% in the active substance obtained by these methods, which necessitates taking into account its content when preparing the ready form of the drug.
- the object of the invention is to provide a crystalline form of zinc-free insulin glargine produced without the use of phenol at the crystallization stage, meeting the pharmacopoeial requirements for this active substance, stable for at least 12 months within the pharmacopoeial requirements of this active and readily soluble substance.
- the invention relates to a solid form of zinc—free insulin glargine, preferably a crystalline form of zinc-free insulin glargine .
- Another object of the invention is a method for obtaining a crystalline insulin glargine, characterized in that a crystallization mixture is prepared comprising an aqueous solution containing isopropanol, sodium citrate and insulin glargine, and then, by a gradual dilution of the crystallization mixture, crystallization process is initiated and continued, after which the resulting crystalline form of insulin glargine is separated, preferably in a zinc-free form.
- the method of the invention consists in preparing a mixture in which insulin glargine is dissolved and then initiating the crystallization by diluting the mixture with water.
- the mixture under initial conditions is an aqueous solution of the following components:
- pH in the range of 6 to 9 is used.
- the crystallization is initiated by gradual addition of water.
- the dilution is carried out until the organic solvent concentration is below 70% of its concentration value at the time of initiation of the crystallization, preferably not higher than 50% of the initial concentration.
- the crystallization is carried out in the temperature range of 10 °C to 20 °C.
- the process of crystallization a process of gradual dilution of the crystallization mixture to achieve the established composition, pH and temperature of the mixture, aimed at obtaining insulin glargine in a crystalline form.
- Determination of the zinc content in the resulting crystalline insulin glargine preparation was performed by atomic absorption spectroscopy in accordance with the European Pharmacopoeia (2.2.23 Method I) .
- the zinc content has been determined in % by mass, where the detection limit (LOD) for this method is 0.01%.
- the crystallization mixture is obtained by mixing, at a specified temperature, an aqueous protein solution with an organic solvent and salt. After adjusting pH to the desired value, the mixture is ready to initiate the crystallization.
- insulin glargine concentration pH, organic solvent concentration, salt concentration, temperature of the mixture, but the parameters, such as the intensity of mixing during the crystallization process and the dilution rate with water, may also influence the quality of the obtained results.
- the insulin glargine concentration in the crystallization mixture should be between 5 and 20 mg/ml.
- Isopropanol at a concentration of 20 to 40% was used as the organic solvent.
- Sodium citrate at a concentration of 0.1 to 0.5 M was used as the salt.
- the crystallization temperature affects the moment of crystallization initiation and its efficiency and should be between 10 and 20 °C.
- the pH value should be between 6.0 and 9.0. Too high pH value, especially above 9, can significantly accelerate the degradation of insulin glargine.
- the crystallization mixture before the dilution is clear - this prevents the precipitation of protein from the crystallization mixture in an amorphous form, which may no longer transform into the crystalline form, reducing efficiency and/or making it difficult to filter the crystals .
- the process of mixing can also influence the course of the crystallization process.
- the crystalline form is formed both with and without mixing.
- the crystallization is preferably carried out with stirring. Mixing speeds up making the composition of the mixture uniform throughout the entire volume during the addition of water, which results in the crystalline material obtained being more homogeneous and its crystallization proceeds faster.
- the duration of crystallization depends on the dilution rate and the time needed to achieve a balance between the crystal form and the dissolved protein.
- aqueous solution containing protein at a concentration of approx. 8 mg/ml, isopropanol at concentration of 20% and 0.5 M sodium citrate, with pH 8.5 and temperature ca . 20 °C water was added in portions to a final protein concentration of approx. 4 mg/ml.
- insulin glargine was obtained in a crystalline form.
- Fig. 1 presents their microscope image obtained with a Nikon Eclipse 80i microscope with a Nikon Plan Apo 100x/1.4 objective.
- aqueous solution containing protein at a concentration of approx. 16 mg/ml, isopropanol at concentration of 20% and 0.1M sodium citrate, with pH 6.5 and temperature of about 10 °C water was added in portions to a final protein concentration of approx. 8 mg/ml.
- insulin glargine was obtained in a crystalline form.
- Fig. 2 presents their microscope image obtained with a Nikon Eclipse 80i microscope with a Nikon Plan Apo 100x/1.4 objective.
- Example 2 The crystals of Example 2 were filtered, washed with water and dried in a vacuum. The obtained precipitate was weighed in an amount of 77 mg and 3 ml of water with pH of about 3 was added. The precipitate dissolved to give a clear solution with a concentration of approx. 25 mg/ml.
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
A crystalline form of zinc-free insulin glargine and a method for its preparation without the use of phenol are disclosed.
Description
A crystalline, zinc-free form of insulin glargine and the method of its preparation
Field of the invention
The invention relates to a method for the preparation of a crystalline form of zinc-free insulin glargine, which should find applications in making pharmaceutical preparations intended for the treatment of diabetes.
State of the art
Insulin glargine (also known as Lantus®) is an analog of human insulin in which the aspartic acid present at position 21 of A-chain has been replaced with glycine Gly (A21) and the B chain sequence has been extended by an additional two amino acids, i.e. by adding two arginines, Arg (B31) and Arg (B32) . The introduced modifications change the isoelectric point of the obtained analog from pH 5.4 to pH 6.7, thanks to which it has better solubility in the solution with an acidic pH (especially of about 4) than in the solution with a physiological pH (about 7.4) . As a result, after a subcutaneous injection of an acidic solution of this analog (e.g. with pH about 4), it precipitates at the injection site forming the so-called depot, from which the gradual release of protein into the body occurs. Therefore, insulin glargine is used for the production of sustained release insulin preparations intended for the treatment of diabetes.
For practical reasons, such as improved stability and durability of the protein, it is particularly desirable to obtain insulin and its analogs in crystalline form. This applies in particular to insulin glargine.
WO2015084694 describes a method for crystallization of insulin glargine from a basic solution (at pH higher by at least 1 than the isoelectric point of an insulin analog) containing a crystals stabilizing agent (phenolic compound) and an organic solvent miscible with water (such as ethanol,
methanol, acetone or isopropanol), wherein the crystallization is initiated by the addition of a zinc salt.
WO2014122653 describes a method for crystallization of insulin glargine from a 30% solution of isopropanol using pH 7 and zinc ions to initiate it.
The known methods of obtaining crystalline insulin glargine are associated with the problem of the content of zinc at the levels from 0.2 to 0.6% in the active substance obtained by these methods, which necessitates taking into account its content when preparing the ready form of the drug. The object of the invention
The object of the invention is to provide a crystalline form of zinc-free insulin glargine produced without the use of phenol at the crystallization stage, meeting the pharmacopoeial requirements for this active substance, stable for at least 12 months within the pharmacopoeial requirements of this active and readily soluble substance.
Unexpectedly, the purpose set forth above has been achieved in the present invention.
The essence of the invention
The invention relates to a solid form of zinc—free insulin glargine, preferably a crystalline form of zinc-free insulin glargine .
Another object of the invention is a method for obtaining a crystalline insulin glargine, characterized in that a crystallization mixture is prepared comprising an aqueous solution containing isopropanol, sodium citrate and insulin glargine, and then, by a gradual dilution of the crystallization mixture, crystallization process is initiated and continued, after which the resulting crystalline form of insulin glargine is separated, preferably in a zinc-free form.
The method of the invention consists in preparing a mixture in which insulin glargine is dissolved and then
initiating the crystallization by diluting the mixture with water.
Preferably, the mixture under initial conditions is an aqueous solution of the following components:
- insulin glargine at a concentration of 5 to 20 mg/ml,
- isopropanol at a concentration of 20 to 40%,
- sodium citrate at a concentration of 0.1 to 0.5M.
Preferably, pH in the range of 6 to 9 is used.
Preferably, the crystallization is initiated by gradual addition of water.
Preferably, the dilution is carried out until the organic solvent concentration is below 70% of its concentration value at the time of initiation of the crystallization, preferably not higher than 50% of the initial concentration.
Preferably, the crystallization is carried out in the temperature range of 10 °C to 20 °C.
Detailed description of the invention
For the purpose of this description, the following terms are understood as follows.
Zinc-free insulin glargine - a crystalline form of insulin glargine, for which the zinc content determined by available analytical method is below the sensitivity of this method, in particular less than 0.01% by mass.
Initiation of crystallization - the moment of beginning of the crystallization process observed as a clouding of the crystallization mixture. This clouding occurs at a specific composition and at the specific pH and temperature values achieved when diluting the crystallization mixture by adding water .
The process of crystallization - a process of gradual dilution of the crystallization mixture to achieve the established composition, pH and temperature of the mixture, aimed at obtaining insulin glargine in a crystalline form.
Determination of the zinc content in the resulting crystalline insulin glargine preparation was performed by atomic absorption spectroscopy in accordance with the European Pharmacopoeia (2.2.23 Method I) . The zinc content has been determined in % by mass, where the detection limit (LOD) for this method is 0.01%.
The crystallization mixture is obtained by mixing, at a specified temperature, an aqueous protein solution with an organic solvent and salt. After adjusting pH to the desired value, the mixture is ready to initiate the crystallization.
The order in which the ingredients are added is of no great significance, but it is most preferred that organic solvent is added first to the protein solution.
The initiation of crystallization and the crystallization process itself take place by diluting the mixture with water, resulting in a reduction in the concentration of organic solvent and salt.
The following parameters have the most important effects on the course of the method according to the invention: insulin glargine concentration, pH, organic solvent concentration, salt concentration, temperature of the mixture, but the parameters, such as the intensity of mixing during the crystallization process and the dilution rate with water, may also influence the quality of the obtained results.
The insulin glargine concentration in the crystallization mixture should be between 5 and 20 mg/ml.
Isopropanol at a concentration of 20 to 40% was used as the organic solvent.
Sodium citrate at a concentration of 0.1 to 0.5 M was used as the salt.
The crystallization temperature affects the moment of crystallization initiation and its efficiency and should be between 10 and 20 °C.
The pH value should be between 6.0 and 9.0. Too high pH value, especially above 9, can significantly accelerate the degradation of insulin glargine.
It is preferred that the crystallization mixture before the dilution is clear - this prevents the precipitation of protein from the crystallization mixture in an amorphous form, which may no longer transform into the crystalline form, reducing efficiency and/or making it difficult to filter the crystals .
The process of mixing can also influence the course of the crystallization process. The crystalline form is formed both with and without mixing. However, the crystallization is preferably carried out with stirring. Mixing speeds up making the composition of the mixture uniform throughout the entire volume during the addition of water, which results in the crystalline material obtained being more homogeneous and its crystallization proceeds faster.
The duration of crystallization depends on the dilution rate and the time needed to achieve a balance between the crystal form and the dissolved protein. The slower the rate of dilution of organic solvent and salt, the bigger the crystals are obtained. Therefore, it is preferred to slowly add water to the mixture .
Examples
For a better understanding of its essence, the invention has been further explained by the following examples.
Example 1.
To an aqueous solution containing protein at a concentration of approx. 8 mg/ml, isopropanol at concentration of 20% and 0.5 M sodium citrate, with pH 8.5 and temperature ca . 20 °C, water was added in portions to a final protein concentration of approx. 4 mg/ml. In this way, insulin glargine was obtained in a crystalline form. The obtained crystals are shown in Fig. 1, which presents their microscope
image obtained with a Nikon Eclipse 80i microscope with a Nikon Plan Apo 100x/1.4 objective.
Example 2.
To an aqueous solution containing protein at a concentration of approx. 16 mg/ml, isopropanol at concentration of 20% and 0.1M sodium citrate, with pH 6.5 and temperature of about 10 °C, water was added in portions to a final protein concentration of approx. 8 mg/ml. In this way, insulin glargine was obtained in a crystalline form. The obtained crystals are shown in Fig. 2, which presents their microscope image obtained with a Nikon Eclipse 80i microscope with a Nikon Plan Apo 100x/1.4 objective.
Example 3.
The crystals of Example 2 were filtered, washed with water and dried in a vacuum. The obtained precipitate was weighed in an amount of 77 mg and 3 ml of water with pH of about 3 was added. The precipitate dissolved to give a clear solution with a concentration of approx. 25 mg/ml.
Claims
1. Solid form of zinc-free insulin glargine.
2. The form of insulin glargine according to claim 1, characterized in that it has a crystalline form.
3. A method for obtaining a crystalline form of insulin glargine, characterized in that a crystallization mixture is prepared comprising an aqueous solution containing isopropanol, sodium citrate and insulin glargine, and then, by a gradual dilution of the crystallization mixture, crystallization process is initiated and continued, after which the resulting crystalline form of insulin glargine is separated, preferably in a zinc-free form.
4. The method according to claim 3, characterized in that the mixture under initial conditions is an aqueous solution of the following components:
- insulin glargine at a concentration of 5 to 20 mg/ml,
- isopropanol at a concentration of 20 to 40%,
- sodium citrate at a concentration of 0.1 to 0.5 .
5. The method according to claim 3, characterized in that pH is from 6 to 9.
6. The method according to claim 3, characterized in that the crystallization is initiated by gradual addition of water.
7. The method according to claim 6, characterized in that the dilution is carried out until the organic solvent concentration is below 70% of its concentration value at the time of initiation of the crystallization, preferably not higher than 50% of the initial concentration.
8. The method according to claim 3, characterized in that the crystallization is carried out in the temperature range of 10 °C to 20 °C.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PLP.426224 | 2018-07-06 | ||
| PL426224A PL238016B1 (en) | 2018-07-06 | 2018-07-06 | Crystal, zinc-free form of insulin glargine and method of its preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2020009594A1 true WO2020009594A1 (en) | 2020-01-09 |
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ID=69060575
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/PL2019/050038 WO2020009594A1 (en) | 2018-07-06 | 2019-07-06 | A crystalline, zinc-free form of insulin glargine and the method of its preparation |
Country Status (2)
| Country | Link |
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| PL (1) | PL238016B1 (en) |
| WO (1) | WO2020009594A1 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998042749A1 (en) * | 1997-03-20 | 1998-10-01 | Novo Nordisk A/S | Zinc free insulin crystals for use in pulmonary compositions |
| US20050085621A1 (en) * | 2002-10-29 | 2005-04-21 | Aventis Pharma Deutschland Gmbh | Crystals of insulin analogs and processes for their preparation |
| EP2708550A1 (en) * | 2011-05-09 | 2014-03-19 | Gan & Lee Pharmaceuticals | Preparation method for insulin glargine crystal |
| WO2015084694A2 (en) * | 2013-12-04 | 2015-06-11 | Merck Sharp & Dohme Corp. | Method for preparing crystalline insulin |
| CN105585628A (en) * | 2016-01-28 | 2016-05-18 | 通化东宝药业股份有限公司 | Preparation method of insulin glargine and insulin glargine prepared by same |
-
2018
- 2018-07-06 PL PL426224A patent/PL238016B1/en unknown
-
2019
- 2019-07-06 WO PCT/PL2019/050038 patent/WO2020009594A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998042749A1 (en) * | 1997-03-20 | 1998-10-01 | Novo Nordisk A/S | Zinc free insulin crystals for use in pulmonary compositions |
| US20050085621A1 (en) * | 2002-10-29 | 2005-04-21 | Aventis Pharma Deutschland Gmbh | Crystals of insulin analogs and processes for their preparation |
| EP2708550A1 (en) * | 2011-05-09 | 2014-03-19 | Gan & Lee Pharmaceuticals | Preparation method for insulin glargine crystal |
| WO2015084694A2 (en) * | 2013-12-04 | 2015-06-11 | Merck Sharp & Dohme Corp. | Method for preparing crystalline insulin |
| CN105585628A (en) * | 2016-01-28 | 2016-05-18 | 通化东宝药业股份有限公司 | Preparation method of insulin glargine and insulin glargine prepared by same |
Also Published As
| Publication number | Publication date |
|---|---|
| PL238016B1 (en) | 2021-06-28 |
| PL426224A1 (en) | 2020-01-13 |
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