WO2020009595A1

WO2020009595A1 - A crystalline form of insulin glargine with a stoichiometric content of zinc and the method of its preparation

Info

Publication number: WO2020009595A1
Application number: PCT/PL2019/050039
Authority: WO
Inventors: Radosław DARŁAK; Jacek Pstrzoch; Paweł ŚWIECA; Konrad SOŁTYSEK; Agnieszka LEDER
Original assignee: Bioton S.A.
Priority date: 2018-07-06
Filing date: 2019-07-06
Publication date: 2020-01-09
Also published as: PL239258B1; PL426225A1

Abstract

A method of obtaining a new crystalline form of insulin glargine with a zinc content corresponding to a molar ratio of zinc to insulin of 1:3 is disclosed, with should find applications in making pharmaceutical preparations intended for the treatment of diabetes.

Description

A crystalline form of insulin glargine with a stoichiometric content of zinc and the method of its preparation

Field of the invention

The invention relates to a method for obtaining a crystalline form of insulin glargine with a zinc content corresponding to a molar ratio of zinc to insulin of 1:3, 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.

Known methods involve the use of phenol or its derivatives during the crystallization of insulin glargine, which necessitates removal of the phenolic compound residues from the obtained crystals. Another problem is the strict control of the level of zinc in the obtained crystalline form of insulin glargine, so that the produced preparation meets pharmacopoeial standards.

The purpose of the invention

The basic object of the invention is to provide a crystalline form of insulin glargine in the absence of phenolic compounds, meeting the pharmacopoeia purity reguirements specified for this active substance, stable for at least 12 months within the pharmacopoeial requirements and readily soluble.

It is also an object of the invention to provide a method for the preparation of a crystalline form of insulin glargine that allows obtaining this form of active substance with a stoichiometrically determined zinc content and falling within the pharmacopoeial standards.

It is a particular object of the invention to provide this form of insulin glargine in the form of crystals with edge lengths of up to 10 pm.

It is also a particular object of the invention to provide a crystalline form of insulin in the form of crystals of uniform size and shape and stability that facilitates their further processing, such as sifting, fractionation and drying. The crystals obtained should not crack too easily during these operations in order not to interfere with their course, e.g. by clogging the sieve.

Unexpectedly, the purpose set forth above has been achieved in the present invention.

The essence of the invention

The present invention relates to a crystalline form of insulin glargine with a zinc content corresponding to a molar ratio of zinc to insulin glargine of 1:3. Preferably, it is in the form of crystals with edge lengths up to 10 pm.

A further object of the invention is a process for obtaining a crystalline form of insulin glargine characterized in that it comprises the use of a crystallization mixture constituting an aqueous solution with pH of 6.5 to 7.5 and composed of:

- insulin glargine at a concentration of 5 to 11 mg/ml,

- an organic solvent selected from isopropanol or 1-propanol at a concentration of 15 to 35% by weight,

- sodium citrate at a concentration of 10 to 30 mM,

- zinc (II) compound at a concentration of 2 to 8 mM,

wherein a crystallization mixture is preferably obtained, the crystallization step is initiated, at the end of which the crystalline form of insulin glargine is separated, wherein the zinc content in the obtained crystalline form of insulin glargine corresponds to a molar ratio of zinc to insulin glargine of 1:3.

Preferably, the crystallization mixture comprises a zinc salt, preferably zinc acetate.

Preferably, the duration of the crystallization step is from 30 minutes to 12 hours, preferably at least 3 hours.

In a first preferred variant, the crystallization is carried out in a temperature gradient, and the crystallization step is initiated by lowering the initial temperature of the crystallization mixture to the initiation temperature of the crystallization, wherein the initial temperature is above 28 °C. Particularly preferably, at the beginning of the crystallization step the mixture is maintained at the initiation temperature for a period of time in the range of 30 to 60 min. It is particularly preferred that the temperature of the crystallization mixture is lowered during the crystallization step, preferably to a final temperature in the range of 10 °C to 18 °C, especially around 12 °C.

In a second preferred variant, the crystallization is carried out at a constant temperature ranging from 10 °C to 27 °C, and the crystallization step is initiated by obtaining a crystallization mixture by mixing two solutions:

(a) solution RA containing insulin glargine and an organic solvent, and

b) solution RB containing a zinc (II) compound and sodium citrate .

Detailed description of the invention

The method according to the invention consists in preparing a mixture in which insulin glargine is initially dissolved, and then, by changing the temperature or composition of the mixture, its solubility is lowered thereby initiating the crystallization process.

The presented method of crystallization makes it possible to obtain insulin glargine substance in a crystalline form, without the use of phenol and its derivatives . The crystallization mixture contains water, recombinant insulin glargine, zinc compound, organic solvent and sodium citrate.

In a preferred embodiment, the method of the invention consists in preparing a solution of insulin glargine in water with the addition of an organic solvent, a zinc compound and sodium citrate, and then initiating the crystallization by changing the temperature. In a further preferred embodiment, the method of the invention consists in the preparation of two process solutions: a solution RA containing insulin glargine, water and an organic solvent and an solution RB containing water, sodium citrate and a zinc compound. Crystallization is initiated by addition of RB solution to the RA solution at a constant temperature.

The efficiency of both preferred embodiments of the method according to the invention oscillates around 90% .

For the purpose of this description, the following terms are understood as follows.

Initial conditions - fixed composition, pH and temperature of the mixture before initiation of crystallization.

Initiation of crystallization - change of the initial conditions of the mixture leading to the start of the crystallization process.

Final conditions - fixed composition, pH and temperature of the mixture to which they are brought to during crystallization. They are characterized by lower solubility of the protein than in the initial conditions.

The process of crystallization - the process of gradual transformation of the mixture from initial to final conditions aimed at obtaining insulin glargine in a crystalline form.

Initial temperature - temperature of the mixture in initial conditions.

Initialization temperature - temperature at which the crystallization of insulin glargine starts due to the decrease of its solubility in the mixture.

Final temperature - temperature of the mixture at which crystallization ends. In the process according to the first preferred embodiment of the invention, the crystallization is carried out in a temperature gradient. The crystallization mixture is obtained by mixing the protein solution with an organic solvent, sodium citrate and a zinc compound. The order in which the components are added is of no great significance, but it is most preferred that the solvent is added first to the protein solution. After pH correction to the set point, the crystallization is initiated by lowering the temperature of the mixture .

In the process according to the second preferred embodiment of the invention, the crystallization is carried out at a constant temperature and the initiation of the crystallization takes place by mixing two solutions, i.e. a protein and an organic solvent solution (solution RA) and a solution containing zinc ions and citrate (solution RB) . The crystallization is carried out at a constant temperature, preferably in the range of 10 °C to 27 °C.

In both preferred embodiments, important parameters of the method of the invention include: insulin glargine concentration, pH, organic solvent concentration, sodium citrate concentration, temperature of individual process steps, and Zn2+ ion concentration. The obtained results may also be influenced by such parameters as the mixing method during the crystallization process and the time of crystallization .

The insulin glargine concentration in the crystallization mixture should be between 5 and 11 mg/ml. The concentration of insulin glargine in the crystallization mixture determines the time required for obtaining protein crystals with a yield of 90% and higher. An increase in the protein concentration in the given range results in a shortening of the crystallization time by approx. 50%. The pH value should be between 6.5 and 7.5. The pH value of the crystallization mixture of about 7 allows complete conversion of the amorphous form of insulin glargine to a crystalline form. It has been found that lowering the pH to 6 or raising it to 8 results in a mixture of both the crystalline and the amorphous forms.

The concentration of the organic solvent should be between 15 and 35%. The organic solvents used are: isopropanol or 1- propanol or a mixture thereof. The content of solvent in the crystallization mixture has an effect on the solubility of insulin glargine in solution. It has been found that the content of the organic solvent in the crystallization mixture of less than 15% or more than 35% results in a mixture of crystalline and amorphous forms of insulin glargine.

The concentration of sodium citrate should be between 10 and 30 mM. It was found that the presence of sodium citrate affects the solubility of the protein and the stability of the crystalline form. Too low salt concentration results in a mixture of both crystalline and amorphous forms. Too high concentration of salt causes deformation and overgrowth of the crystals as well as a decrease in the efficiency of the process. The role of sodium citrate molecules is also the buffering of the crystallization mixture. In case when an organic solvent will be present next to the salt in the mixture, it should be remembered that incorrect choice of salt concentration relative to the solvent concentration may cause separation of the mixture, which results in a mixture of crystalline and amorphous forms divided between the two phases .

The initial temperature (i.e. the temperature of the initial mixture in which the insulin glargine is located) should not be lower than the crystallization initiation temperature. Preferably, the starting temperature is above the crystallization initiation temperature - so that the solution is clear. This prevents the protein from being precipitated in an amorphous form which may no longer go into the crystalline form, reducing efficiency and/or making it difficult to filter the crystals. However, too high temperature (especially above 40 °C) should be avoided, which may contribute to the denaturation of the protein or significantly accelerate its degradation. In the first embodiment, the initial temperature is above 28 °C. In a second embodiment of the invention, in which all steps are carried out at substantially the same temperature, the initial temperature, the initiation of crystallization and the final temperature should be constant, and their value should be in the range of 10 °C to 27 °C.

In a variant of embodiment of the invention carried out with a temperature gradient, the initiation of the crystallization takes place at a temperature lower than the initial temperature. The initiation temperature is the temperature at which an excess of insulin glargine in relation to its solubility begins to crystallize - this is manifested by the opalescence and then the clouding of the mixture. The time of maintaining the crystallization mixture at the initiation temperature translates into the quality of the crystals. It is preferred to keep the mixture at the initiation temperature for a period of time in the range of 30 to 60 min. This has a positive effect on the homogeneity of the crystal forms obtained and the size of the crystals. The temperature of the crystallization mixture is then lowered until the final temperature is reached.

The final temperature (i.e. the temperature of the mixture after completion of crystallization) should not be higher than the initial temperature. In the case of crystallization carried out by lowering the temperature, the final temperature has an effect on the efficiency of the process - and is limited only by the possibility of freezing the mixture. The lower the temperature, the lower the solubility and thus the higher the efficiency. Preferably, the final temperature of the crystallization is in the range of 10-18 °C.

The concentration of zinc ions in the crystallization mixture should be between 2 and 8 mM. Crystallization does not take place without its participation in the ranges of protein, isopropanol and salt concentration determined in accordance with the invention. It was found that under these conditions zinc is responsible for stabilization of the crystalline forms, and its content in the crystals is close to the theoretical, stoichiometric content of zinc (0.36%) (i.e. for 1 Zn molecule there are 3 insulin molecules). Determination of the zinc content in the obtained crystalline insulin glargine preparation was performed by atomic absorption spectrometry (ASA) according to the procedure described in the European Pharmacopeia (Ph Eur . 9.0-2.2.23 Method) and determined in % by weight with a detection limit (LOD) of 0.01%.

The process of mixing can also influence the course of the crystallization process. The initiation of the crystallization may be accompanied by intensive mixing, while the crystallization process has a favorable course with gentle mixing .

In an embodiment of the invention carried out in a temperature gradient, mixing is used to make the entire amorphous form transformed into a crystalline form. It also shortens the crystallization time to <6h. Mixing increases the efficiency of heat transfer between the crystallization mixture and the cooling medium and/or shortens the mixing time of the mixture components when changing its composition.

In a variant of the invention carried out at a substantially constant temperature, mixing shortens the time needed to obtain a homogeneous reaction mixture and shortens the crystallization time. It has been found that, regardless of the embodiment, too vigorous mixing leads to relatively small crystals, and too mild extends the crystallization time in relation to those mixed properly.

In one preferred embodiment, the desired homogeneity and shape of the crystals is obtained for a crystallization time in the range of 30 to 60 minutes. Crystals are obtained that have a regular shape and high durability to prevent them from cracking in subsequent processing steps. Thanks to this, they can be easily filtered, because they do not contain too large crystals with irregular shapes, which usually break during sifting and the resulting fraction clogs the sieve.

The method of the invention yields insulin glargine in the form of crystals with edge lengths up to 10 pm.

Examples

For a better understanding of its essence, the invention has been further explained by the following examples. The attached figures show microscopic images of the obtained crystals. A Nikon Eclipse 80i microscope with a Nikon Plan Apo 100x/1.4 lens was used.

Example 1.

To prepare the solution RA, 11.25 ml of isopropanol was added to 25 ml of the 15 g/L insulin glargine solution. The resulting mixture was stirred for 1 minute. To prepare the solution RB, 0.3 ml of a 1M zinc acetate solution was added to 0.375 ml of 1M sodium citrate solution. The resulting mixture was stirred for 1 minute and then its pH was adjusted to 7.0. The solution RB was added to the solution RA and the pH of the mixture was adjusted to 7.0. The obtained crystallization mixture was stirred vigorously for 10 min and then allowed to crystallize at ambient temperature. The obtained insulin crystals were filtered. The yield of the crystallization process after 12 hours was 90%. The obtained crystals are shown in Fig. 1.

Example 2.

To prepare the solution RA, 7.5 ml of n-propanol was added to 25 ml of the 15 g/L insulin glargine solution. The resulting mixture was stirred for 1 minute. To prepare the solution RB, 0.125 ml of a 1M sodium citrate solution and 0.15 ml of zinc acetate were added to 4.725 ml of water. The solution RB was added to the solution RA and the pH of the mixture was adjusted to 7.0. The obtained crystallization mixture was stirred vigorously for 10 min at 10 °C and then left to crystallize at this temperature. The efficiency of the crystallization process after more than 12 hours was 90%. The obtained crystals are shown in Fig. 2.

Example 3.

100 ml of a crystallization mixture was prepared containing 5 g/L glargine insulin, 30 ml isopropanol, 0.8 ml 1 zinc acetate solution and 2 ml sodium citrate solution, with pH 7.0 and temperature of 29 °C. The mixture was cooled to 27 °C with vigorous stirring. After reaching the crystallization initiation temperature, the stirring was stopped and the temperature reached was maintained for 60 minutes. Then, gentle mixing was started and cooling of the mixture to 12 °C was continued for 150 minutes. The obtained crystals are shown in Fig. 3.

Claims

1. A crystalline form of insulin glargine with a zinc content corresponding to a molar ratio of zinc to insulin glargine of

1:3.

2. The form of insulin glargine according to claim 1, characterized in that it is in the form of crystals with edge lengths up to 1Ό pm.

3. A method for obtaining a crystalline form of insulin glargine, characterized in that it comprises the use of a crystallization mixture constituting an aqueous solution with a pH of 6.5 to 7.5 and composed of:

- insulin glargine at a concentration of 5 to 11 mg/ml,

- sodium citrate at a concentration of 10 to 30 mM,

- zinc (II) compound at a concentration of 2 to 8 mM.

4. The method according to claim 3, characterized in that the crystallization mixture comprises a zinc salt, preferably zinc acetate .

5. The method according to claim 3, characterized in that the duration of the crystallization step is from 30 minutes to 12 hours, preferably at least 3 hours.

6. The method according to claim 3, characterized in that the duration of the crystallization step is from 30 minutes to 60 minutes .

7. The method according to claim 3, characterized in that the crystallization is carried out in a temperature gradient and the crystallization step is initiated by lowering the initial temperature of the crystallization mixture to the initiation temperature of the crystallization, wherein the initial temperature is above 28 °C.

8. The method according to claim 7, characterized in that at the beginning of the crystallization step the mixture is maintained at the initiation temperature for a period of time in the range of 30 to 60 min.

9. The method according to claim 7 or 8, characterized in that the temperature of the crystallization mixture is reduced during the crystallization step, preferably to a final temperature having a value in the range of 10 °C to 18 °C, particularly preferably around 12 °C.

10. The method according to claim 3, characterized in that the crystallization is carried out at a constant temperature in the range of 10 °C to 27 °C, and the crystallization step is initiated by obtaining a crystallization mixture by mixing two solutions :

(a) a solution RA containing insulin glargine and an organic solvent, and

b) a solution RB containing a zinc (II) compound and sodium citrate.