IE50188B1

IE50188B1 - Insulin crystal suspension and process for the preparation thereof

Info

Publication number: IE50188B1
Application number: IE1762/80A
Authority: IE
Original assignee: Hoechst Ag
Priority date: 1979-08-22
Filing date: 1980-08-21
Publication date: 1986-03-05
Also published as: EP0025868A1; ZA805152B; IL60889A0; JPS5632412A; CA1155439A; DK149347B; DE3060628D1; DK149347C; ATE1281T1; EP0025868B1; ES8205752A1; DE2933946A1; AU6162180A; AU539091B2; GR69915B; ES494260A0; DK359380A; IE801762L; IL60889A

Abstract

1. A process for the structural transformation of rhombohedrally crystallized insulin, preferably of pig origin, having a 4-Zn structure to rhombohedrons having a 2-Zn structure, which comprises maintaining the crystals having a 4-Zn structure in an aqueous medium containing less than 6% of sodium chloride, the pH of which medium is adjusted to 4.5 to 6.0 and maintained stable by addition of a buffer, at a temperature ranging from room temperature to 45 degrees C until in a sample crystals of the 4-Zn structure are no longer present.

Description

The invention provides insulin preparations containing insulin/ zinc crystals, especially those of pig insulin. These crystals are sharp-edged rhombohedrons having a 2-Zn structure. They are obtained by structural trans5 formation of rhombohedrons having a 4-Zn structure.

It is known that insulin can be crystllized in the form of rhombohedrons at a pH of from 5 to 7 with the addition of bivalent metals, preferably zine, and a buffer substance such as ammonium acetate or sodium acetate.

In some cases, an organic solvent such as acetone is added in order to prevent precipitation of amorphous insulin. It may furthermore be advantageous to influence the crystallization process by adding seed crystals. When halides (chloride or iodide), urea or formamide are present in the crystallization medium, rhombohedrons of different crystal structure, depending on the concentration of said substances, are formed. For example, in the case of a concentration of up to 6 ί of sodium chloride in the crystallization medium, rhombohedrons having a so-called 2-Zn structure are formed, and a sodium chloride content of more than 6 ? produces rhombohedrons having a so-called 4-Zn structure (M.J, Adams et al., Nature 224, 491 (1969); G. Bentley et al., Nature 26l, 166 (1976)). Both crystal formsabsorb additional zinc when the pH of the surrounding medium increases. The gross content of zinc in the crystals and the concentration of zinc in the surrounding medium, as well as size and form of the crystals determine the solubilization speed of the crystals. Thus, for example, crystals of pig insulin having a zinc content of from 0.8 to 2.5 ?, relative to the insulin weight, in a neutral solution have a delayed therapeutic action.

It is furthermore known that crystallization under the conditions which lead to rhombohedrons having a 2-Zn structure gives insufficiently shaped crystals. Often, - 3 crystals having rounded-off edges, deformed rhombohedrons or twins are formed (U.S. Patent No. 2,920,014).

On the other hand, well-shaped sharp-edged rhombohedrons are formed under the conditions giving a 4-Zn structure. It is easy to see that uniformly shaped crystals in insulin products are advantageous for therapeutic application, especially because a reproducible solubilization speed of the crystals ensures a reproducible time course of insulin action of the corresponding product.

It has been observed that insulin crystals having a 4-Zn structure are not stable under the conditions required for therapeutic application of insulin products, that is, at a sodium chloride content of up to 1.5 ί.

In these cases, the crystals suspended in a neutral medium usual for therapeutic application and containing less than 1.5 1 oi zinc are partially dissolved after some time. When on the other hand the crystals contain more zinc, their effect is not reproducible.

It is known that 4-Zn crystals of pig insulin can be transformed to 2-Zn crystals, when the are introduced into a medium being free from sodium chloride and having a pH of 6.5. However, this causes such a tension in the crystals that their exterior shape fe completely destroyed in this transformation (G. Bentley et al., J. Mol. Biol. 126, 871 (1978)).

Surprisingly, it has now been found that rhombohedral insulin crystals having a 4-Zn structure, that is, those of pig insulin as well as of bovine insulin, can be tranformed to crystals having a 2-Zn structure without perceptible deterioration of their outer shape, so that these transformed crystals can be used for preparing stable insulin products for therapeutic application. - 4 Subject of the invention is therefore a process for the structural transformation of rhombohedrally crystallized insulin, preferably of pig origin, having a 4-Zn structure to rhombohedrons having a 2-Zn structure, which comprises maintaining the crystals having a 4-Zn structure in an aqueous medium contains less than 6 2 of sodium chloride, the pH of which medium is adjusted to 4.5 to 6.0 and maintained stable by addition of a buffer, at a temperature ranging from room temperature to 45°C until in a sample crystals of the 4-Zn structure are no longer present.

The transformed crystals have sharp edges and smooth surfaces. They are as transparent as the initial 4-Zn crystals. A comparison of the X-ray spectra of the crystals before and after transformation proves the change of structure, perceptible by the change of the angles of refraction. The degree of order in the crystal, however, remains unchanged, which is proved by the ratio of height to width of the peaks.

For preparing a suspension of insulin crystals having a 2-Zn structure in accordance with the invention for therapeutic application, the starting material is crystallized insulin of 4-Zn structure which advantageously has been crystallized under sterile conditions according to known methods. For transforming the structure of the crystals, the surrounding medium is replaced by a solution which contains less than 6 2 (weight/volume) of sodium chloride, preferably from 0.5 to 2 ?, and a salt for buffering the pH, preferably sodium acetate. The solution is adjusted to a pH near the isoelectric point of insulin, that is, of from 4.5 to 6.0, preferably 5.0 to 5.8. The structural transfor35 mation is carried out at room temperature or a slightly elevated temperature of up to 45°C, preferably 37°C.

It is advantageous, but not absolutely necessary, to maintain the suspension in motion during the rearrange50188 - 5 ment reaction, for example by stirring or shaking. The structural transformation of the crystals can be observed by taking a sample and examining it, for example by X-ray structural analysis, on whether there are still crystals in the suspension having the initial 4-Zn structure. Transformation is generally complete after some hours to.several days, depending on the reaction conditions. The speed of the transformation is reduced with increasing pH and increasing concentration of sodium chloride in the medium, while it is increased with rising temperature.

Aromatic substances such as the usual preservation agents hinder the transformation. For example, addition of 0.1 ? of methylparaben inhibits the transformation of pig insulin crystals, so that these substances have to be excluded from the rearrangement reaction in accordance with the invention.

The transformation being complete, the crystal suspension is diluted with a diluting solution, so that a suspension having 40, 80 or 100 I.U./ml insulin crystals is obtained. The diluting solution contains advantageously a physiologically tolerable substance for adjusting isotonieity, for example sodium chloride; a buffer salt for buffering the pH, for example sodium acetate; and a preservation agent, for example methylparaben. It is adjusted, preferably with sodium hydroxide solution, to such a pH that after dilution the crystal suspension has a pH in the range of from 6 to 8, preferably 6.7 to 7.5. Furthermore, such an amount of zinc must be added to the diluting solution that the insulin crystals in the neutral suspension have a zinc content of from 0.9 to 2.5 ?.

The suspension of stable insulin crystals having a 2-Zn structure may be mixed with a rapidly acting neutral - 6 solution of insulin or insulin derivates, or a neutral suspension of amorphous insulin or insulin derivatives having a delayed action; the stability of the component ratio depending only on' the zinc content of the mixture.

The following examples illustrate the invention.

Example 1 A. 1.Preparation of a suspension of crystals having a 4-Zn structure Crystalline pig insulin (450,000 I.U.) containing 0.5 weight ? of zinc was dissolved in 400 ml of 0.03 N hydrochloric acid. 15 ml of a 1 ? zinc chloride solution in 0.03 N hydrochloric acid were added, and 0.03 N hydrochloric acid was added until 500 ml were obtained. The solution was sterilized by filtration and mixed with 500 ml of a solution of 70 g of sodium chloride, 14 g of sodium acetate ' 3 HgO and 10 ml of 1 N sodium hydroxide solution in water, which had been likewise sterilized by filtration. The mixture was adjusted to pH 5.4-5.5 and stirred at room temperature for 48 hours, thus causing the insulin to crystallize in the form of rhombohedrons having a 4-Zn structure.

A. 2. Structural transformation of 4-Zn to 2-Zn crystals After settling of the crystals from the clear superna30 tant, 888 ml of crystal suspension prepared according to A 1 were pumped off under sterile conditions from 1 liter of this suspension and replaced by 1013 ml of a solution sterilized by filtration which contained 50 mg of zinc chloride and 1.125 g of sodium chloride, and which was adjusted tc a pH of 5.4 to 5.5 by means of 1 N hydrochloric acid. Subsequently, the crystal suspension was stirred for 7 days at room temperature, and then for - 7 5 days at 37°C. The X-ray spectrum showed thereafter a' complete tranformation of the 4-Zn to the 2-Zn structure.

A. 3· Preparation of a neutral crystal suspension containing 40 I.U./ml To 1.125 ml of the crystal suspension prepared according to A 1 and A 2, 10.125 1 of a solution sterilized by filtration were added which contained 81 g of sodium chloride, 14.175 g of sodium acetate ' 3 HgO, 0.8-1.1 g of zinc chloride, 11.250 g of methylparaben and such an amount of 1 N sodium hydroxide solution that a pH of from 6.9 to 7.4 was adjusted in the mixture.

Example 2 Preparation of a two-phase product containing 70 % pig insulin crystals and 30 Ϊ amorphous Des-B1-phenylalanine insulin of pig origin.

To 1.125 1 of the crystal suspension prepared according to A 1 and A 2 (Example 1), 14.946 1 of a sterile suspension were added which contained 192,900 I.U. of amorphous Des-B1-phenylalanine insulin of pig origin, 119.6 6 of sodium chloride, 20.9 g of sodium acetate ‘ 3 H20, 2.362 g of zinc chloride, 16.07 g of methylparaben and such an amount of 1 H sodium hydroxide solution that a pH of 7.2-7.5 was adjusted in the mixture.

Example 3 Preparation of a two-phase product containing 75 1 of crystals of pig insulin and 25 % of dissolved Des-B1phenyl-alanine insulin of pig origin.

To 1.125 1 of the crystal suspension prepared according to A 1 and A 2 (Example 1), 13.875 1 of a sterile suspension were added which contained 150,000 I.U. of Des-B1-phenylalanine insulin of pig origin, 111.04 g of 018 8 - 8 sodium chloride, 19.43 S of sodium acetate ' 3 HgO, 292 g of zinc chloride, 15.00 g of methylparaben and such an amount of 1 N sodium hydroxide solution that a pH of 6.8-7.0 was adjusted in the mixture.

Example 4 Preparation of a two-phase product containing 75 ? of pig insulin crystals, 12.5 ί of dissolved pig insulin and 12.5 ? of dissolved Des-B1-phenylalanine insulin of pig origin.

To 1.125 1 of the crystal suspension prepared according to A 1 and A 2 (Example 1), 13.875 1 of a sterile suspension were added which contained 75,000 I.U.of pig insulin and 75,000 1.0. of Des-B1-phenylalanine insulin of pig origin, 111.04 g of sodium chloride, 19.43 g of sodium acetate ‘ 3 H20, 354 g of zinc chloride, 15.00 g of methylparaben and such an amount of 1 N sodium hydroxide solution that a pH of 6.8-7.0 was adjusted in the mixture.

Claims

1. ) A process for the structural transformation of rhombohedraflly crystallized insulin, preferably of pig origin, having a 4-Zn structure to rhorabohedrons having a 2-Zn structure, which comprises maintaining the crystals having a 4-Zn structure in an aqueous medium containing less than 6 ί of sodium chloride, the pH of which medium is adjusted to 4.5 to 6.0 and maintained stable by addition of a buffer, at a temperature ranging from room temperature to 45°C until in a sample crystals of the 4-Zn structure are no longer present.

2. ) Crystal suspension of insulin, preferably pig insulin, in an aqueous medium, having a delayed action, wherein insulin crystals in the form of rhombohedrons of 2-Zn structure are present which have been obtained by structural transformation of rhombohedrons having a 4-Zn structure.

3. ) A process for the structural transformation of rhombohedrally crystallized insulin, substantially as hereinbefore described with reference to the accompanying examples.

4. ) Insulin crystals whenever obtained by a process claimed in a preceding claim.

5. ) A crystal suspension of insulin according to Claim 2 substantially as hereinbefore described with reference to the acoanpanying examples.