DK150813B - Process for preparing stabilized insulin solutions - Google Patents

Description

in 150813

The present invention relates to a process for the preparation of stabilized insulin solutions specially adapted for use in continuous insulin delivery pump equipment.

In recent years, there has been an ever increasing effort to develop portable or implantable systems for continuous infusion of insulin. The main purpose of these efforts has been to allow the diabetic patient to administer insulin in a way that is easier to adapt to the diurnal variations in the patient's physiological insulin requirements than has been possible with conventional insulin therapy.

The mechanical portion of a continuous insulin delivery device comprises substantially such elements as an insulin reservoir, pump system, and catheter adapted to the chosen method of administration, which is usually subcutaneous, intravenous or intraperitoneal.

The pump system may be automatically activatable and may also be provided with a further manual control of the administration of insulin when there is a special need for it.

If the insulin solution is supplied from a syringe, it will also usually act as an insulin reservoir.

Spray-type devices are usually worn outside the body. However, much more complicated systems have also been developed in which the entire mechanical unit is designed for implantation, usually subcutaneously. The insulin reservoir will usually be adapted for 30 percutaneous replenishment.

The insulin's propensity to precipitate in commercially available solutions, clogging both the mechanical parts and the infusion catheter, has been found to be a major obstacle to the further development and clinical use of continuous infusion pumping equipment. In addition, efforts are being made to develop ever smaller types of insulin pumps for obvious reasons. Thereby, there is a need for more concentrated insulin solutions than those previously available, which probably further contributes to aggravating the above problems.

It is generally believed that the explanation for the precipitation phenomenon should be sought in the insulin's tendency to form insoluble fibrils, especially if insulin solutions are used at higher temperatures for a longer period. Certain facts also suggest that any kind of movement of or in the solution, including e.g. the turbulence induced by the passage of the solution through a small cross-section or narrow opening causes insulin fibrillation. It is obvious that insulin solutions are exposed to most or all such effects in any type of insulin pump equipment. The general deficiencies of the known insulin preparations in this respect are adequately documented in the literature, e.g. in a published review article by W.D. Lougheed et al. (Diabetologia vol. 19 (1980), pp. 1 - 9).

To solve this problem, it has been proposed to use acidic insulin solutions containing glutamic acid or aspartic acid, see Diabetes 3, (1981), 83, or neutral insulin preparations containing a sugar such as glucose (DS Schade et al : Satelite Symposium to the 16th European Association for the Study of Diabetes-Meeting, Greece, September 22-23, 1980, page 107). A further 25 have been proposed to use a nonionic surfactant (H. Thurow: German Publication no.

29 52 119).

However, insulin is chemically unstable in acid even under body temperature, and can react reversibly or irreversibly with carbohydrates, cf. J. Brange and S. Havelund, "Properties of Insulin in Solution", in Artificial Systems for Insulin Delivery, Assisi September 20-23, 1981, Editors: P. Brunetti et al. Raven Press, 1983. Furthermore, the aforementioned non-physiological surfactant must be considered undesirable in drugs for parenteral use.

3 150813

The present invention alleviates the above-mentioned drawbacks and relates to a method of preparing insulin solutions in which the insulin, in comparison with conventional insulin preparations, exhibits a substantially reduced propensity for precipitation under the conditions prevailing in insulin pumping equipment.

The invention is based on the surprising realization that zinc ions at certain concentrations exert a high stabilizing effect on neutral insulin solutions.

10 Zinc has been used for years as crystallization-promoting metal in the manufacture of insulin crystals. Insulin crystals used to prepare neutral insulin solutions therefore usually contain zinc, cf. eg. British Patent No. 840,870 corresponding to 15 Danish Patent No. 95007 and Danish Patent No. 116,527.

However, it is clear from the above-mentioned Danish patent no. 95007 that the insulin solution should have a content of zinc ions available for insulin of less than -3

20 13 x A x 10 milli equivalents of zinc ions per liter, where A

indicates the number of international insulin units per day. ml, corresponding to a content of zinc ions of less than approx. 1% by weight (calculated on the basis of dry insulin crystals).

In the presence of compounds which form readily soluble or complex compounds with zinc, the compositions may contain significantly more zinc if only the amount of zinc ions available to the insulin, and consequently not bound by such compounds, does not exceed the above upper limit.

If the content of zinc ions available for insulin exceeds the above upper limit of approx. 1 weight percent 2+ cents, corresponding to approx. 5 Zn / hexamer insulin, zinc-insulin complexes precipitate from the solution.

Since the vast majority of insulin in neutral solutions occurs as insulin hexamers, it is selected in this specification to express the amount of zinc per day. 6 molecules of monomeric insulin. It is of no consequence for this calculation method whether more or less amounts of other forms of insulin are present, such as dimers 2+ or hexamers. The relationship between zinc / hexamer insulin and% zinc is shown in the following equation: 5 „2+ ,,. ,. % zinc x MW. x 6

Zn / hexamer insulin = _ ms_ MW x 100 where MW stands for molecular weight1? 10 To ensure that a precipitation of zinc-insulin complexes is avoided, the zinc insulin content of commercial insulin preparations has always been well below the upper limit mentioned above, and a study of all known, commercial, neutral insulin preparations by applicants has shown that The zinc ion concentration in known compositions is approx. 2 - 3.5 Zn / hexamer insulin. Also, the compositions prepared in the above-mentioned Danish Patent No. 95007 have a zinc content substantially below the upper limit for precipitation of zinc-insulin complexes, the highest content of zinc being 20 of 0.7% zinc based on dry matter insulin, which corresponds to 2+ approx. . 3.7 Zn / hexamer.

However, it can be seen from FIG. 1, that insulin preparations having a zinc ion content of 2 and even up to 4 2+

Zn / hexamer insulin is unstable under conditions that mimic conditions of continuous insulin administration because insulin precipitates within a few days. Therefore, the insulin compositions described in the above-mentioned Danish Patent No. 95007 are unsuitable for continuous administration as they are within the unstable range of FIG. This is further confirmed by experiments in which the applicants have shown that a preparation prepared as described in Example 2 of Danish Patent No. 95007 and containing 0.7% zinc has a stability factor of only 2.5, which means that the preparation is too unstable to be used for continuous administration.

Therefore, it is a surprising realization that an increase of zinc ions to certain specific concentrations is just below the upper limit of zinc insulin precipitation of approx. Zn / hexamer insulin exerts a stabilizing effect on insulin solutions under the conditions described above for continuous insulin administration.

The present invention relates to a process for preparing an insulin solution having a pH between about 7 and ca. 8, suitable for use in continuous insulin administration by being physically stable under conditions of such administration, and optionally comprising a preservative, an isotonic solution, a buffer and a complexing agent, and this method is characterized by For example, in said insulin solution, a substantially ionized zinc salt is incorporated in a concentration corresponding to a content of zinc ions available for insulin of more than 4 Zn / hexamer insulin but below the limit of precipitation of zinc-insulin complexes.

By the term "zinc ions available for insulin" is meant zinc ions which are not bound to other compounds which form complex compounds with zinc, e.g. citrate, glycine or phosphate.

The upper limit of the zinc ion content is the amount of zinc causing precipitation of zinc insulin complexes, 2+ i.e. ca. 5 Zn / hexamer insulin, as mentioned above.

According to a preferred embodiment of the present invention, the content of zinc ions available for insulin does not exceed 4.8 Zn / hexamer insulin, and the content of zinc ions available for insulin preferably ranges from about 1 to about 5 inches. 4.2 - approx. 4.5 Zn / hexamer insulin.

Preferred zinc salts are zinc acetate and zinc chloride.

According to a further preferred embodiment of the invention, phenol is used as a preservative and glycerol as an agent to render the solution isotonic.

As mentioned earlier, the content of zinc 35 refers to zinc ions available for insulin. In the presence of zinc complexing agents, some of the zinc is bound to the complexing agent. However, since the content of 6 150 813 zinc ions available for insulin must still be above 4 Zn / hexamer insulin, more zinc must be added. The total content of zinc in insulin preparations comprising a zinc complexing agent is therefore more or less above the limits mentioned above, depending on the amount of complexing agent used and its complex constant.

It is often preferred to use a zinc complexing agent, thereby ensuring a greater operable range for the total zinc content based on the insulin content. For example, any of the equipment used for reservoir, pump system, hoses, etc., binds or releases zinc, the presence of a zinc complexing agent will ensure that the content of zinc ions available for insulin will be within the scope of the present invention.

When the insulin solution contains a zinc complexing agent, it has been found that the solution to be physically stable under the conditions of continuous insulin administration must exhibit a total zinc ion content of at least 5 2+ 20 Zn / hexamer insulin, and in a preferred embodiment of the present invention. set the total zinc content 2+ content to at least 6 Zn / hexamer insulin.

The zinc complexing agents used in the novel insulin solutions should be soluble and physiologically acceptable.

Suitable zinc complexing agents are amino acids, e.g. glycine, alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, proline, tryptophan, asparagine, glutamic acid and histidine, and oligopeptides such as diglycine.

Another group of suitable zinc complexing agents are carboxylic acids such as acetic acid, or hydroxycarboxylic acids such as citric acid, 3-hydroxybutyric acid and lactic acid.

Preferred zinc complexing agents are glycine 35 and citric acid. Also preferred are diglycine and histidine.

The smallest total zinc ion concentration to achieve a significant stabilizing effect, i.e. a stability factor (as defined in the detailed part of the specification) in the range of 5-10 depends on the complexing agent used. However, a stability factor over 10 is preferred.

If a weak complexing agent is used, e.g. glycine 10 mM, a clear stabilization is achieved at a total zinc content of approx. 5 Zn / hexamer insulin, cf. Table I and Figs. II, and the preferred range for the 2+ total zinc content is from approx. 5 to approx. 8 Zn / hexamer 10 insulin.

Table I -2 40 IU swine sulxn per ml in a 10 molar solution of glycine with the zinc concentration varied, prepared analogously to Example 7.

2+

Zinc Concentration - Zn Per * Stability Traction Hexamer Factor (M) Insulin 20 _________ 1.0 10 ~ 4 2.4 1 1.8 10 ~ 4 4.4 4 2.0 10 "4 4.9 7 2.2 10" 4 5.4 45 25 2.6 10 ~ 4 6.4 49 3.4 10 "4 8.4 8 3.8 10" 4 9.4 10 4.0 10 ~ 4 9.9 15 30 * Total content of zinc ions.

2+

The relationship between total Zn / hexamer insulin, molar zinc concentration and international insulin units 35 can be calculated as follows: (Zn) = X · 0.99 · 10 Ymol / liter, where 8 150813 (Zn) is the molar zinc concentration expressed in mol / liter , X = international insulin units per day. ml. and 2+ Y = Zn / hexamer insulin 5 If a stronger zinc complex is used, the agents, e.g. citric acid, a total zinc content 2+ content of approx. 40 to 200 Zn / hexamer insulin to achieve significant stabilization, cf. the following Table II.

When citric acid is used as a complexing agent, the preferred total zinc content at pH 7.4 in millimoles can be expressed as 0.004 xA + kxB, where A is international units of insulin per day. ml and B is the millimolar citrate concentration, k is in the range of 0.5 - 1.0, with a k value of approx. 0.7 - 0.8 is preferred.

Table II

40 IU of swine insulin per day. ml of varying zinc and citrate concentrations, prepared analogously to Example 8.

é I »I — I— i —11 I | - I

2+ 20 zinc concen- Zn pr. * Stabilization Hexamer Citrate Functional (M) Insulin (M) Tor 25 1.0 10 "4 2 10 ~ 2 1 0.6 10-2 150 10 ~ 2 6 0.8 10-2 200 10-2 10 0.9 10 ~ 2 225 10-2 10 0.95 10-2 238 10-2 19 30 1.0 10 ~ 4 2 2.0 10-3 1 1.6 10 “3 40 2.0 10-3 6 1.8 10-3 45 2.0 10 ~ 3 30 2.0 10-3 50 2.0 10-3 19

- I

35 * Total content of zinc ions.

When a zinc complexing agent is used, the upper limit of the total zinc content is the amount of zinc which causes a precipitation of the zinc insulin complex, i.e. when the content of zinc ions not bound by the 2+ 5 complexing agent exceeds approx. 5 Zn / hexamer insulin.

The stability test described below can be used as a means to ensure that with the complexing agents used, the preferred 10 range of 4.2 - 4.5 Zn / hexamer insulin is provided for the insulin. The total zinc content of insulin solutions containing complexing agents can be expected to be between 5 and 10 Zn / hexamer insulin when weak complexing agents are used, and can be expected to range from approx. 10 to approx. 200 Zn / hexamer insulin when strong complexing agents are used.

In addition to the stability test, the upper limit of the total zinc content, i.e. the zinc content which causes a precipitation of zinc-insulin complexes is used to determine the amount of total zinc in an insulin solution containing complexing agent, whereby a substantial increase in stability is obtained.

It has been found that the total amount of zinc ions in an insulin solution containing complexing agent can be expected to be from ca. 50 to approx. 90% of the amount of zinc ions which would precipitate zinc-insulin complexes.

The total amount of zinc ions is preferably from ca. 60 to approx. 90% and more preferably from approx. 75 to approx.

30 90% of the amount of zinc ions which would precipitate zinc-insulin complexes.

Neutral insulin preparations containing complexing agents or buffers forming zinc complexes are known from the specification of the above-mentioned Danish Patent No. 116,527 and from the specification of Danish Patent Application No. 1851/80. However, the highest zinc content described is 1% Zn based on the weight of dry insulin corresponding to 2+ to about 10%. 5.5 Zn / hexamer insulin. Applicants have reproduced Examples 2 and 6 of Danish Patent Specification No. 116,527, cf. The following Examples 9 and 10. It can be seen from this that the known compositions exhibit a stability factor of only 3.5 and 1. It can therefore be indirectly concluded that the zinc content of these preparations is available for insulin, i.e. zinc, which is not bound to the complexing 2+ agent, is below 4 Zn / hexamer insulin. This is because the known compositions both contain complexing agents, namely phosphate buffer and citrate buffer, respectively.

Therefore, part of the zinc will be bound to the complexing agent and is therefore not available for the insulin, and the known compositions are, as shown, unsuitable for continuous insulin administration, having too low physical stability for such administration. The other examples in Danish Patent No. 116,527 describe compositions having a zinc content of between 0.1 and 0.5% zinc (total zinc). Therefore, these compositions are outside the scope of the present invention.

The concentration of insulin in the solution readily prepared according to the present invention is preferably in the range of from 5 to 750 I.U. and especially from 40 - 500 I.U. per. ml. The insulin solutions may e.g. is prepared by dissolving crystalline zinc insulin, e.g. a highly purified form of insulin, such as "monocomponent" insulin (cf. British Patent No. 1,285,023) in water in the presence of an acid, e.g. hydrochloric acid. An aqueous solution of the preservative, e.g. phenol or an alkyl phenol such as cresol or methyl parahydroxybenzoate are separately prepared and may also contain, if desired, an osmotic pressure control agent such as glycerol, preferably in an amount intended to render the final solution isotonic, and if desired a zinc complexing agent such as citrate or glycine. This solution is then added to the acidic insulin solution followed by the addition of a base, e.g. sodium hydroxide solution, for setting to a neutral pH. In the present application, neutral pH is understood to mean a pH in the range of from about 1150813 to approx. 7 - approx. 8. The calculated amount of the zinc salt, e.g. zinc acetate, the buffer (if desired) e.g. TRIS and the zinc complexing agent can be added at this stage, after which the pH is adjusted. Alternatively, the zinc salt may be incorporated into the acidic insulin solution prior to its neutralization. The resulting solution is finally filled to the desired volume with water, sterilized by filtration and then transferred to sterile vials.

10 Stability Test

The insulin solutions thus prepared are subjected to a stability test under forced conditions as follows:

Vials with a volume of 12.5 ml each containing a 10 ml sample of the solution and fitted with a rubber cap are placed vertically on a shaking platform (Type 01 T623TBSH02, purchased from HETO, Birkerød, Denmark), which is fully immersed in a water bath maintained at a temperature of 41 ° C ± 0.1 ° C. The platform is subjected to a horizontal shaking motion with a frequency of 100 rpm and an amplitude of 50 mm.

The opalescence of the samples is measured at regular time intervals on a Fischer DRT 1000 nephelometer equipped with an ampoule adapter. The fibrillation time is defined as the 25 time that elapses until the specimen develops a turbidity of 10 nephelometric turbidity units (NTU).

Each test is performed with test samples and control samples without added zinc salt (4-5 ampoules of each) treated side by side. The control samples correspond to the test samples except 2 + 30 except that the control samples contain approx. 2 Zn / hexamer insulin, the zinc content of the control samples being derived from the crystallization of the insulin. The stability factor is calculated as the ratio of the average fibrillation times for test specimens and control specimens, respectively: The invention will be explained in further detail with reference to the drawing, in which

FIG. 1 shows the stability factor at 40 ° C as a function of zinc ion concentration (100 I.U. swine insulin per ml, prepared according to Example 1), and

FIG. 2 shows the stability factor at 41 ° C as a function of the total content of zinc ions in the presence of a weak complexing agent (40 I.U. swine insulin per ml in 10 mM glycine prepared analogously to Example 7).

It can be seen from FIG. 1, the stability of the insulin solution is drastically increased when the zinc ion content is above 4 2+ 10 Zn / hexamer insulin.

In the presence of a weak complexing agent, a substantial increase in stability is obtained at a higher 2+ zinc ion content, e.g. above 5 Zn / hexamer insulin because some of the zinc is bound to the complexing agent and consequently 15 is not available for the insulin.

Further details of the practice of the present invention will be apparent from the following examples.

In the examples, aqueous solutions and water 20 were first sterilized by filtration and the subsequent operations were performed under aseptic conditions.

Example 1 100 I.U. swine insulin per ml containing 4.2 2+ 25 Zn / hexamer insulin.

Crystalline monocomponent pig insulin (264 mg) with a zinc content of 0.4% and a total activity of 10,000 30 I.U. was dissolved in water (50 ml) containing hydrochloric acid (325 μΐ normal) and then an aqueous solution (25 ml) containing 1.6 g glycerol and 200 mg phenol was added. The pH of the solution was adjusted to 7.5 using a sodium hydroxide solution. Zinc acetate (1.56 ml of 10 mM solution) was added and the pH was re-adjusted to 7.5 and the total volume was filled up to 100 ml with water. The resulting solution was sterilized by filtration and then aseptically transferred to ampoules (10 ml).

Stability factor:> 15.

5

Example 2,500 I.U. swine insulin per ml containing 4.2 2+

Zn / hexamer insulin.

10

Crystalline monocomponent porcine insulin (20.75 g) containing 0.4% zinc and having a total activity of 550,000 I.U. was dissolved in 550 ml of water containing hydrochloric acid (10 ml of normal).

To a sample of this solution (50 ml) was added zinc acetate (78 μΐ molar solution) followed by the addition of 25 ml of an aqueous solution containing 1.6 g of glycerol and 200 mg of phenol. The pH of the solution was adjusted to 7.4 by means of sodium hydroxide solution and the total volume was adjusted to 100 ml with water. The resulting solution was sterilized by filtration and then aseptically transferred to ampoules (10 ml).

Stability factor: 5.

25

Example 3 100 I.U. human insulin per ml containing 4.3 2+

Zn / hexamer insulin.

30 ___________________

Crystalline monocomponent human insulin (1.67 g) containing 0.4% zinc and having a total activity of 45,000 I.U. was dissolved in 225 ml of water containing hydrochloric acid (1.46 35 ml of normal). To a sample of this solution (25 ml) was added zinc acetate (100 μΐ 0.1 molar solution) to which 10 ml of an aqueous solution containing 0.8 g glycerol and 0.1 g phenol were added. . The pH of the solution was adjusted to 7.4 by means of sodium hydroxide solution and the volume was adjusted to 50 ml with water. The resulting solution was sterilized by filtration and then aseptically transferred to ampoules (10 ml).

Stability factor: 26.

Example 4 10 100 I.U. swine insulin per ml containing 4.5 2+

Zn / hexamer insulin.

The procedure was analogous to that described in Example 1 except that 1.89 ml of a 10 mM zinc acetate 2+ solution was added to provide 4.5 Zn / hexamer insulin.

Stability factor:> 33.

Example 5 40 I.U. human insulin per ml containing 4.2 2+ 25 Zn / hexamer insulin.

Crystalline monocomponent human insulin (741 mg) containing 0.4% zinc and having a total activity of 20,000 30 I.U. was dissolved in 60 ml of water containing hydrochloric acid (640 μΐ normal) and zinc acetate (3.96 ml 0.01 M). 400 ml of an aqueous solution containing 1 g of phenol and 8 g of glycerol were added, the pH was adjusted to 7.45 by means of a sodium hydroxide solution and the volume was filled up to 35 500 ml with water. The solution was sterilized by filtration and then aseptically transferred to ampoules (10 ml).

15 150813

Stability factor: 34,

Example 6 5 100 I.U. human insulin per ml containing 4.2 2+ -2 Zn / hexamer insulin in 10 molar TRIS.

Crystalline monocomponent human insulin (1852 mg) containing 0.4% zinc and having a total activity of 50,000 I.U. was dissolved in 60 ml of water containing hydrochloric acid (1600 μΐ normal) and zinc acetate (99 μΐ molar). 400 ml of an aqueous solution containing 1 g of phenol and 8 g of glycerol were added, then the pH was adjusted to 7.45 with TRIS 15 (606 mg) and sodium hydroxide solution and the volume was adjusted to 500 ml with water. The resulting solution was sterilized by filtration and then aseptically transferred to ampoules (10 ml).

Stability factor: 16.

Example 7 40 I.U. swine insulin per ml in 2.6 * 10 ^ and 10 2 2 + 25 molar solution of zinc and glycine, respectively. Zn / hexamer insulin = 6.4.

Crystalline monocomponent porcine insulin (1.49 g) containing 0.4% zinc and having a total activity of 40,000 I.U. was dissolved in 120 ml of water containing hydrochloric acid (1280 μΐ normal) followed by the addition of 730 ml of an aqueous solution containing 2 g of phenol, 8 g of glycerol and 75.1 mg of glycine. The pH was adjusted to 7.5 using 35 sodium hydroxide solution. Zinc acetate (160 μΐ molar) was added, the pH was adjusted to 7.5 and the volume was adjusted to 1000 ml with water. The solution was sterilized by filtration and then aseptically transferred to ampoules (10 ml).

Stability factor: 49.

5

Example 8 -3 -3 40 I.U. pork loin per. ml in 1.8'10 and 2 * 10 2+ molar solution of zinc and citrate respectively. Zn / hexamer 10 insulin = 45

Crystalline monocomponent porcine insulin (746 mg) containing 0.4% zinc and having a total activity of 20,000 15 I.U. was dissolved in 60 ml of water containing hydrochloric acid (640 μΐ normal). 400 ml of an aqueous solution containing 1 g phenol and 8 g glycerol were added and the pH was adjusted to 7.5 with sodium hydroxide solution. Citric acid (210 mg monohydrate) and zinc acetate (850 μΐ molar) were added and the pH was adjusted to 7.5. The solution was sterilized by filtration and then aseptically transferred to ampoules (10 ml).

Stability factor: 30.

25

Example 9 (Comparison) 40 I.U. bovine insulin pr. ml in 13.3 mM sodium phosphate 2+ phate solution. (Total zinc 1% 5.5 Zn / hexamer insulin).

30 ________________

Example 2 of Danish Patent No. 116,527 was reproduced. The zinc content was adjusted to approx. 5.5 2+

Zn / hexamer insulin by the addition of zinc acetate and the pH-35 was adjusted to 6.91. The solution was sterilized by filtration and then aseptically transferred to ampoules (10 ml).

17 150813

Stability Factor: 3.5 Example 10 (Comparison) 5 40 I.U. bovine insulin pr. ml in 10 mM citrate solution 2+. (Total zinc 1% Λ / about 5.5 Zn / hexamer insulin).

Example 6 of Danish Patent No. 116,527 was reproduced. The zinc content was adjusted to approx. 5.5 2+

Zn / hexamer insulin by the addition of zinc acetate and the pH was adjusted to 7.5. The solution was sterilized by filtration and then aseptically transferred to ampoules (10 ml).

15

Stability factor: 1

Claims (4)

150813 - 18 - 5 PATENT REQUIREMENTS A process for preparing an insulin solution having a pH between about 7 and ca. 8 suitable for use in continuous insulin administration by being physically stable under conditions of such administration and optionally comprising a preservative, isotonic agent, buffer and complexing agent, characterized in that in said insulin solution is incorporated a substantially ionized zinc salt at a concentration corresponding to a content of zinc ions available for insulin of more than 4 Zn / hexamer insulin, but below the limit of precipitation of zinc-insulin complexes. 20 Process according to claim 1, characterized in that a substantially ionized zinc salt is incorporated in a concentration corresponding to a content of zinc ions available to the insulin of more than 4 Zn / he-2+ 25 xamer insulin and less than approx. 4.8 Zn / hexamer insulin. Process according to claim 1, characterized in that a substantially ionized zinc salt is incorporated in a concentration corresponding to a content of 30 zinc ions available for the insulin of from approx. 4.2 to about 2+ 4.5 Zn / hexamer insulin. Process according to any one of the preceding claims, characterized in that the zinc salt 35 used is zinc acetate or zinc chloride.