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.