GB1560232A

GB1560232A - Biphasic insulin preparations comprising crystalline insulin and monodesamido insulin

Info

Publication number: GB1560232A
Application number: GB4121/77A
Authority: GB
Original assignee: Novo Industri AS
Current assignee: Novo Nordisk AS
Priority date: 1977-02-01
Filing date: 1977-02-01
Publication date: 1980-01-30
Also published as: SE7801163L; AU503013B1; FR2378516B1; DE2803996A1; SE445887B; FR2378516A1; CA1099632A

Abstract

Injectable biphasic zinc-containing insulin preparations comprising a suspension of crystalline insulin in an aqueous medium containing dissolved monodesamidoinsulin and having a pH within the range of from 6 to 8, the crystalline insulin and the dissolved monodesamidoinsulin being derived from the same species. The injectable biphasic zinc-containing insulin preparations are prepared by bringing monodesamidoinsulin into solution in an aqueous medium containing suspended crystalline insulin, the crystalline insulin and the monodesamidoinsulin being derived from the same species.

Description

(54) IMPROVEMENTS IN OR RELATING TO BIPHASIC INSULIN PREPARATIONS COMPRISING CRYSTALLINE INSULIN AND MONODESAMIDO INSULIN (71) We, NOVO INDUSTRI A/S, a Danish Company of Novo Allé, DK-2880 Bagsvard, Denmark, do hereby declare the invention, for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following state ment:- This invention relates to injectable biphasic zinc-containing insulin preparations and to a process for preparing such preparations.

The importance of timing of the action of injected insulin is well recognised in diabetology. It is generally considered advantageous, having regard to the relative comfort of the diabetic patient, that the number of daily injections be kept at a reasonably low leveL As a consequence, over the past years endeavours have been directed towards the development of insulin preparations covering the spectrum from intermediate to prolonged durations of activity. In many cases of diabetes, a desirable insulin preparation for therapeutic use is one with a rapid onset of action combined with a protracted duration of activity.

Injectable insulin preparations having the latter properties are known. In this respect, reference is made to British Patent Specification No. 860,515 which discloses pharma ceutically stable insulin preparations consisting of a suspension of zinc containing, bovine insulin crystals in a solution having a pH within the range of 6 to 8 and containing dissolved porcine insulin, the suspended insulin and the dissolved insulin constituting the slow and quick acting insulin fractions, respectively.

In order to keep the porcine insulin in solution, particularly under storage, the suspension shows such a zinc ion content that the suspended bovine insulin crystals contain less than 0.25 milliequivalent of zinc per gram of the dried crystals at pH 7.0 of the suspension.

Under these conditions, it is not possible to substitute the suspended bovine insulin crystals by porcine insulin crystals since the latter will not remain undissolved, nor is it possible to substitute the dissolved porcine insulin by dissolved bovine insulin since the latter will not remain in solution.

It is an object of the present invention to enable the removal of, or mitigation of, the limitations referred to above, heretofore considered as being unavoidable. This object has now been attained according to the present in vention by incorporating dissolved monodesamidoinsulin into the aqueous phase of the insulin suspension.

Monodesamidoinsulin, as used herein, shall mean any chemical derivative obtained from insulin by converting a single side chain carboxamido group (of an asparaglne or a glutamine residue) of the insulin molecule into the corresponding carboxyl or carboxylate group.

It is knawn that deamidation of insulin takes place under hydrolytic conditions, particularly in add solution, thus resulting in the formation of desamidoinsulins. Since the extraction of insulin from pancreas gland tissue is usually conducted under add conditions, desamidoinsulins, particularly monodesamidoinsulin, are normal contaminants, not only of crude insulin fractions but also of insulin obtained therefrom by crystallization.

Although insulin, both of porcine and of bovine origin, contains a total of six carb oxamido groups, so that a complex mixture of desamidoinsulins could be expected, the rate of the add hydrolysis of the A-chain carboxyl terminal asparagine carboxamido group is con siderably higher than that of the remaining amido groups with the result that the desamidoinsulin fraction consists pre dominantly of monodesamido - A21 - insulin.

Thus, according to the first aspect of the present invention there is provided a stable injectable biphasic zinc-containing insulin preparation, which comprises a suspension of zinc-containing crystalline insulin in an aqueous medium containing dissolved monodesamidoinsulin and having a pH in the range of 6 to 8, the zinc-containing crystalline insulin and monodesamidoinsulin being derived from the same species.

In a specific embodiment of the present invention the crystalline insulin and monodesamidoinsulin are both of bovine origin. Thus, it has now been found that, under the conditions which prevail for making the known biphasic insulin preparations referred to above, bovine monodesamidoinsulin shows such a degree of solubility that it can replace the porcine insulin used heretofore. Even though deamidation of insulin lowers the isoelectric point, it could not be expected that the increase in solubility should be sufficiently high to enable the formation of a stable solution within a pH range of from 6 to 8. It is also surprising that the biphasic preparation possesses the stability required for practical use.

In this respect the present invention provides an important industrial progress. It is known that the amounts of bovine pancreas glands available for insulin production greatly surpass the amounts of porcine pancreas glands available for this production. By means of the present invention, it will be easter to satisfy the increasing demand for biphasic insulin preparations of the type referred to.

According to a second specific embodiment of the present invention the crystalline insulin and monodesamidoinsulin are both of porcine origin.

Thus, it has also been found that porcine monodesamidoinsulin remains in solution in the presence of zinc ions at a considerably higher concentration than does porcine insulin itself. This surprising property makes it pos sidle to make stable injectable biphasic insulin prcparations in which the suspended crystals are porcine insulin crystals remaining in suspension due to the increased zinc ion content.

It could not be expected that such a preparation would have the stability required for practical use.

The above mentioned specific embodiments of the present invention taken together open up additional ways of making injectable biphasic single species insulin preparations. It has been postulated that porcine insulin and bovine insulin contained in the known insulin preparations give rise to the formation of different insulin antibodies and that, for immuniological reasons, single species insulin preparations would possess the advantage of affording the possibility to change from one species to another. By means of the present invention, it becomes possible to make injectable biphasic insulin preparations of which the insulin constituents, both in solution and in suspension, originate fromi a single species, preferably bovine or porcine.

It is known that commercial crystalline insulin contains impurities and that these impurities can be removed to such an extent that the purified insulin emerges as a single peak in gel filtration analysis (vide e.g. Diabetes, vol. 21 (1972) pp. 657O). Herein insulin exhibiting such a degree of purity shall be termed: "Highly purified insulin". Likewise, insulin which fulfils the additional requirement of exhibiting essentially a single component when analyzed by discontinuous polyacrylamide gel electrophoresis (DISC PAGE) shall be termed: "monocomponent insulin" (vide e.g. British Patent Specification No.

1,285,023). Suspensions of crystalline insulin consisting of highly purified insulin and monocomponent insulin, respectively, are preferred embodiments of the present invention.

As mentioned previously, monodesamidoinsulin has been detected as a contaminant of crude or crystalline insulin, for example by subjecting such grades of insulin to analytical ion exchange chromatography (vide for instance: J. Biol. Chemistry, vol. 235 (196 pp. 2294 2299; Diabetes, vol. 21 (1972), p.

463). Likewise, it has been possible to identify monodesamidoinsulin, and specifically monodesamido - A21 - insulin, as a constituent of partially hydrolyzed insulin obtained under properly controlled conditions as to acid strength, temperature and duration of hydrolysis (vide e.g. J. Biol. Chemistry, vol. 237 (1962), pp. 3406 3411). However, none of these references discloses the isolation of monodesamidoinsulin.

To prepare substantial amounts of monodesamidoinsulin for the purpose of the present invention, partially hydrolyzed crude or commercial insulin, preferably prepared under properly controlled add conditions, was subjected to fractionation by ion exchange chromatography, preferably on an anion exchanger. Eluted fractions corresponding to the central part of the monodesamidoinsulin peak were collected. Recovery of the monodesamidoinsulin may be affected by precipitation, e.g. as a slightly soluble zinc complex from a solution at a pH in the range of neutrality, followed by desalting of the re-dissolved monodesamidoinsulin, e.g. by gel filtration and, finally, recovering monodesamidoinsulin from the desalted solution. Starting from monocomponent insulin, essentially pure monodesamido A21 - insulin was obtained in this fashion Alternatively, monodesamidoinsulin may be recovered during the production of monocomponent insulin as disclosed in British Patent Specification No. 1,285,024. Fractions corresponding to the monodesamidoinsulin peak may be collected and the monodesamidoinsulin contained therein recovered and used for the purpose of the present invention It is preferred that the monodesamidoinsulin used for the pur pose of this invention shows such a purity that gel filtration thereof results in a single peak and that disc polyacrylamide gel electrophoresis results in essentially a single band.

The amount of monodesamidoinsulin used depends on the degree of rapid onset of the insulin action desired. Preferably, the activity of the monodesamidoinsulin should constitute from 10 to 75, more preferably from 20 to 50, percent of the total insulin activity of the preparation, expressed in international units (i.u.) of activity.

The pH value of the monodesamidoinsulin solution should be within the range of from 6 to-8, preferably from 6.5 to 7.5.

The solubilities of insulin and monodes amidoinsulin, respectively, and hence the physical stability of the biphasic preparations of the present invention, are related to the free zinc ion concentration of the liquid phase of the preparation. Therefore, addition of auxiliary substances interfering with the avail ability of free zinc ions, such as zinc com plexing and/or zinc precipitating agents, is preferably avoided. Under such conditions the total zinc io content of a bovine insulin pre paration at pH 7 is chosen in the range of from 0.25X10-ZXA to 10-2xA, preferably from 0.45xl0-2xA to 0.85X10XA micro moles per ml, in which A indicates the total number of international units (i.u.) of insulin per ml of the preparation.

In the case of a porcine insulin preparation, the respective ranges are from 0.7X 102XA to 1.5 x 10-2 X A and 10-a X A to 1.3 X 10-2XA micromoles of zinc ion per ml.

It is to be understood that in case the pre paration contains auxiliary zinc ion binding substances and/or pH of the preparation is substantially higher than 7.0, the total zinc content of the preparation must be increased accordingly. For further guidance in this re spect, reference is made to British Patent Specification No. 840,870.

According to a further aspect of the pre, sent invention there is provided a process for preparing an injectable zinc-containing insulin preparation for clinical use, containing zinc containing crystalline insulin suspended in an aqueous medium, which process comprises bringing monodesamidoinsulin into solution in the aqueous medium, the aqueous medium hav ing a pH in the range of from 6 to 8, the zinc-containing crystalline insulin and mono desamidoinsulin being derived from the same species.

A sterile suspension of crystalline, prefer ably highly purified or monocomponent insulin, the amount of insulin being calculated to produce a final suspension of predetermined activity, e.g. 40, 80 or 100 i.u. per ml, was prepared in an aqueous solution containing a preservative, e.g. methyl paraben, a buffering agent, e.g. sodium acetate, and a physiologically acceptable salt, e.g. sodium chloride, to make the solution isotonic. The zinc content of the suspension was adjusted to the estimated value by adding a solution of a zinc salt, e.g.

zinc chloride. The pH was adjusted to a value in the range of from 6 to 8, preferably about 7, whereafter the suspension was made up to the final volume.

Separately, a sterile solution of monodes amidoinsulin having a predetermined insulin activity, e.g. 40, 80 or 100 i.u. per ml, was prepared by dissolving the calculated amount of monodesamidoinsulin in water together with preservative, buffering agent, and physiologically acceptable salt to establish isotonicity, in all instances using the same ingredients as those used for preparing the insulin suspension. Optionally, the total amount of zinc salt required for the final preparation may be divided between the insulin suspension and the monodesamidoinsulin solution. The pH was adjusted to a value in the range of from 6 to 8, preferably about 7, followed by adjustment of the final volume of the solution.

Aliquot volumes of the insulin suspension and monodesamidoinsulin solution were then combined, the volume ratio being determined by the percentage of monodesamidoinsulin activity desired in the final biphasic preparation.

The present invention is further illustrated by the following Examples which, however, are not to be construed as limiting the scope of the invention. Parts and percentages are expressed herein by weight, except where stated or apparent In the Examples, aqueous solutions and water were sterilized, the former by filtration, and subsequent handling was conducted under aseptic conditions.

Preparation of Monodesamidoinsulin.

Example 1 From crystalline porcine insulin.

Porcine insulin (0.5 g of crude insulin, crystallized from citrate buffer) was dissolved in water containing hydrochloric acid (5 ml of 0.2 N solution). Water was added to a total volume of 50 ml. The solution (pH 2.02), after being sterilized by filtration, was set aside at room temperature (250C) for 30 days.

A mixture of insulin and monodesamidoinsulin was precipitated by addition of an aqueous solution containing zinc ions (0.5 ml of M zinc acetate) followed by adjustment of pH to 5.5.

The precipitate (containing about 50 percent of monodesamidoinsulin according to DISC PAGE analysis) was dissolved in water containing EDTA (50 mg of the disodium salt), ethanol (20 ml of 60 percent v/v) and sodium chloride (0.08 g), tris(hydroxymethyl)amino- ethane being added to adjust pH to 8.4. An insoluble residue was removed by centrifugation.

The supernatant was applied to a 2.5 cmx 30 cm column containing QAE-Sephadex A-25 equilibrated with a buffer of the following composition: Tris(hydroxymethylaminoethane (121.1 g) Sodium chloride (61.36 g) Hydrochloric acid (50 ml of 6 N) Ethanol (6.24 litres of 96% v/v), filled up with water to a total volume of 10 litres. The pH of the buffer was 8.6. The word "Sephadex" is a Trade Mark Elution was conducted with the same buffer at 250C and fractions (each of 4.9 ml) were collected at an elution rate of 50 ml per hour. The O.D. of the eluate was monitored at 276 Fractions of the central part of the second main peak were pooled and the monodesamidoinsulin was precipitated by the addition of an equal volume of an aqueous solution of zinc acetate (0.01M, pH 6.6) and then recovered by centrifugation.

The precipitate, dissolved in aqueous acetic acid (5 ml of M solution) was desalted on a column (2.5 cmx40 cm) of Sephadex G 10.

Elution with aqueous acetic add (1M solution) at a rate of 0.6 ml per minute was followed by monitoring the OD. at 276 nm. Fractions corresponding to the main peak were pooled, evaporated and freeze dried, yielding monodesamidoinsulin (140 mg).

Example 2 From crystalline bovine insulin.

Bovine insulin (0.5 g of chide insulin, crystallized from cirrate buffer) was dissolved in water containing hydrochloric acid (5 ml of 0.2N solution). Water was added to a total volume of 50 mL The solution (pH 2.09), after being sterilized by filtration, was set aside at room temperature (250C) for 30 days.

Monodesamidoinsulin was recovered by a procedure identical to that of Example 1. The yield of monodesamidoinsulin was 147 mg.

Example 3 From monocomponent (MC) porcine instiln Porcine MC-insulin (40 g) was dissolved in water containing hydrochloric acid (30 ml 2N solution). Water was added to a total volume of 2000 ml. The solution (pH 2.08), after being sterilized by filtrationy was set aside at room temperature (250C) for 45 days.

Precipitation of insulin and monodesamido insulin was afforded by addition of an aqueous solution containing zinc ions (20 ml of 1M zinc acetate) followed by adjustment of pH to 5.5.

Monodesamidoinsulin was separated from other components by ion exchange chromatography on a 15 cmx40 cm column of QAE Sephadex A-25, recovered by precipitation with zinc acetate followed by desalting, by using procedures analogous to those of Example 1. The yield of porcine monodesamido A21 - insulin was 19.8 g.

Example 4 From monocomponent bovine insulin.

Bovine MC-insulin (25 g) was dissolved in water containing hydrochloric acid (25 ml of 2N solution) followed by adjustment of the volume to 2500 ml with water. The solution (pH 2.09), after being sterilized by filtration, was set aside at room temperature (250C) for 30 days.

The mixture of insulin and monodesamidoinsulin was precipitated by addition of an aqueous solution containing zinc ions (25 ml of 1M zinc acetate) followed by adjustment of pH to 5.5.

Fractionation was performed on a 10 em X 42 cm column containing QAE-Sephadex A-25 equilibrated with a buffer of a composition identical to that of Example 1. Fractions (each of 250 ml) were collected at an elution rate of 530 ml per hour. The O.D. of the eluate was monitored at 276 nm.

Fractions of the central part of the second main peak were pooled, and the monodesamidoinsulin was precipitated by addition of zinc acetate followed by desalting using the same procedure as in Example 1.

The yield of bovine monodesamido - A21 insulin was 10.8 g.

Example 5 Isolation of monodesamidoinsulin during the preparation of MC-insulin from crystal line insulin.

Monodesamidoinsulin, which emerges together with insulin in gel-filtration, is separated from the latter by anion exchange.

chromatography (cf. British Patent Specification No. 1,285,023). Fractions corresponding to the central part of the monodesamidoinsulin peak of the anion exchange chromatogram were pooled, the monodesamidoinsulin was precipitated by addition of zinc acetate and recovered in a manner analogous to those of the previous examples.

Biphasic Insulin Preparations.

Example 6 Biphasic preparation of bovine MO-insulin and dissolved bovine monodesamidoinsulin (20 percent of total activity), containing 0.18 micromoles of zinc per ml.

Al. Bovine M & insulin crystal suspension.

(a) Crystalline monocomponent bovine insulin (1.45 g) containg 0A7G of Zn and having a total activity of 40,000 i.u., was dissolved in water containing zinc chloride (602 microlitres of 1% aqueous solution, calculated as Zn++) by addition of hydrochloric acid (750 microlitres of 2N). Water was added to a total volume of 50 ml.

(b) The insulin solution so prepared was added to an aqueous solution of sodium acetate (1.36 g), sodium chloride (7.0 g) and aqueous sodium hydroxide (275 microlitres of 2N), made up to a total volume of 50 ml.

(c) The resulting mixture containing amorphous insulin (100 ml) of pH 5.48 was caused to crystallize as described in British Patent Specification No. 766,995 by seeding with an aqueous suspension (1 ml) of microcrystalline insulin having a mean diameter of about 1 micron and prepared as described in British Patent Specification No. 766,994.

A2. Preparation of MC-insulin crystal sus pension, 40 i.u. per ml.

To a solution of methyl paraben (375 mg) in water (280 ml) was added hydrochloric acid (470 microlitres of 0.2N) together with an aliquot (37.5 ml) of suspension Al. The resulting suspension, having a pH of 5.52, was left for approximately 2 hours, after which time a solution of sodium hydroxide (375 microlitres of 2N) in water (50 ml) was added slowly with stirring. Following adjustment of the total volume with water to 375 ml, the resulting suspension of bovine insulin crystals contained 40 i.u. of insulin per ml. The pH of the suspension was 7.1.

B1. Preparation of bovine monodesamido insulin solution.

Bovine monodesamido - A21 - insulin (200 mg), prepared according to Example 4 and having a total activity of 5000 i.u., was dis solved in water containing zinc chloride (150 microlitres of 1 % solution, calculated as Zn5) and hydr:chloric acid (1.35 ml of 0.2N).

The total volume was adjusted to 10 mL B2. Preparation of monodesamidoinsdin solution, 40 i.'L per ml.

To a solution of methyl paraben (130 mg) in water (75 ml) was added sodium acetate (170 mg), sodium chloride (875 mg) and aqueous sodium hydroxide solution (2.1 ml of 0.2N) followed by solution B1. The total volume was adjusted to 125 ml by addition of water, thus affording a solution of monodesamidoinsulin having a pH of 7.1 and containing 40 i.u. per ml.

C. Injectable biphasic insulin preparation.

A preparation of pH 7.1 was prepared by mixing an aliquot (80 ml) of suspension A2 with an aliquot (20 ml ) of monodesamidoinsulin solution B2. The preparation was transferred to sterilized vials.

Example 7 Biphasic preparation of highly purified bovine insulin and dissolved bovine monodes amidoinsulin (25 percent of total activity), containing 026 micromoles of zinc per mL Al. Highly purified bovine insulin crystal suspension.

The procedure was analogous to that of Example 6, except that the following amounts of materials were used: (a) Crystalline, highly purified bovine insulin (620 mg, total activity 16,000 i.u.) containing 037 percent zinc.

Aqueous zinc chloride solution (2.5 ml of 0.107 percent, calculated as Zn^) Hydrochloric acid (2.85 ml of 0.2N) Water to make up to 2 ml.

(b) Sodium acetate (544 mg) Sodium chloride (2.8 g) Aqueous sodium hydroxide (1.1 ml of 0.2N) Water to make up to 15 ml. The pH was 5.45.

(c) The aqueous suspension was seeded and then left for 24 hours to crystallize at room temperature.

A2. Preparation of insulin crystal suspension, 40 i.u. per ml.

To a solution of methyl paraben (400 mg) in water (300 ml) was added an aqueous solution of zinc acetate (103 microlitres of M solution) together with the total volume of insulin crystal suspension. Al. The resulting suspension (pH 5.56) was left for two hours, when a solution of sodium hydroxide (400 microlitres of 2N) in water (50 ml) was added slowly with stirring. The pH of the resulting suspension was adjusted to 7.03 by adding hydrochloric acid (225 microlitres of 0.2N) and the volume was made up to a total of 400 ml with water.

B1. Preparation of bovine monodesamido insulin solution.

Bovine monodesamidoinsulin, prepared according to Example 2 (240 mg, total activity 5600 i.u.), was dissolved in water containing hydrochloric acid (280 microlitres of 0.2N).

The total volume was adjusted to 20 ml.

B2. Preparation of bovine monodesamido insulin solution, 40 i.u. per ml.

To a solution of methyl paraben (140 mg) in water (105 ml) was added sodium acetate (190.4 mg), sodium chloride (980 mg) and aqueous sodium hydroxide solution (235 microlitres of 2N), followed by solution B1. The pH was adjusted to 6.99 with aqueous hydroxide solution (50 microlitres of 0.2N) and the total volume to 140 ml, thus affording a solution of bovine monodesamidoinsulin containing 40 i.u.

per ml.

C. Injectable biphasic insulin preparation A preparation of pH 7.01 was prepared by mixing suspension A2 (395 ml) with solution B2 (131.7 ml). The preparation was transferred to sterile vials.

Example 8 Biphasic preparation of porcine MGinsulin and dissolved porcine monodesamido insulin (10 percent of total activity), con taining 0.55 micromoles of zinc per ml.

Al. Porcine MC-insulin crystal suspension.

The procedure was analogous to that of Example 6, except that the following amounts of materials were used: (a) Crystalline porcine MC-insulin (1.493 g, total activity 40,000 i.u.) containing 0.37 percent zinc.

Aqueous zinc chloride solution (650 microlitres of 1.02 percent, calculated as Zone) Hydrochloric acid (710 microlitres of 2N) Water to make up to 60 ml.

(b) Sodium acetate (1.36 g) Sodium chloride (7.00 g) Aqueous sodium hydroxide (275 microlitres of 2N) Water to make up to 40 ml.

The pH was 5.49.

A2. Preparation of insulin crystal suspension, 40 i.u. per ml.

To a solution of methyl paraben (400 mg) in water (300 ml) was added a solution of aqueous zinc acetate (168 microlitres of 1M solution) together with an aliquot (40 ml) of insulin crystal suspension Al. The resulting suspension (pH 5.62) was left for two hours, when an aqueous solution of sodium hydroxide (375 microlitres of 2N) in water (50 ml) was added slowly with stirring. The pH of the resuIting suspension was adjusted to 7.02 by adding hydrochloric acid (225 microlitres of 0.2N) and the volume was made up to a total of 400 ml with water.

B. Preparation of porcine monodesamido insulin solution, 40 i.u. per ml.

Porcine monodesamido - A21 - insulin prepared according to Example 3 (86.7 mg, total activity 2000 i.u.) was dissolved in water (10 ml) to give solution B1.

To a solution of methyl paraben (50 mg) in water (35 ml) was added sodium acetate (68 mg), sodium chloride (350 mg) and aqueous sodium hydroxide solution (840 microlitres of 0.2N), followed by solution B1. The pH was adjusted to 6.98 and the total volume to 50 ml, thus affording a solution of porcine monodesamidoinsulin containing 40 i.u. per ml.

C. Injectable biphasic insulin preparation.

A preparation of pH 7.01 was prepared by mixing suspension A2 (395 ml) with solution B2 (44 ml). The preparation was transferred to sterile vials.

Example 9 Biphasic preparation of porcine MC-insulin and dissolved porcine monodesamido insulin (28 percent of total activity) con taining 0.3 micromoles of zinc per ml.

Al. The porcine insulin crystal suspension Al of Example 8 was used for this preparation.

A2. Preparation of insulin crystal suspension, 40 i.u. per ml.

To a solution of methyl paraben (400 mg) in water (300 ml) was added an aqueous solution of zinc acetate (92 microlitres of 1M solution) together with an aliquot (40 ml) of insulin crystal suspension Al of Example 8 The resulting suspension (pH 5.61) was left for two hours, when an aqueous solution of sodium hydroxide (375 microlitres of 2N) in water (50 ml) was added slowly with stirring. The pH of the resulting suspension was 7.03. The volume was made up to a total of 400 ml with water. An aliquot of this suspension (200 ml) was adjusted to pH 6.64 by addition of hydrochloric acid (200 microlitres of 0.2N).

B. Preparation of porcine monodesamido insulin solution, 40 i.u. per ml.

Porcine monodesamido - A21 - insulin prepared according to Example 3 (277.3 mg, total activity 6400 i.u.) was dissolved in water 25 ml) to give solutio

Claims

**WARNING** start of CLMS field may overlap end of DESC **.

The procedure was analogous to that of Example 6, except that the following amounts of materials were used: (a) Crystalline porcine MC-insulin (1.493 g, total activity 40,000 i.u.) containing 0.37 percent zinc.

Aqueous zinc chloride solution (650 microlitres of 1.02 percent, calculated as Zone) Hydrochloric acid (710 microlitres of 2N) Water to make up to 60 ml.

(b) Sodium acetate (1.36 g) Sodium chloride (7.00 g) Aqueous sodium hydroxide (275 microlitres of 2N) Water to make up to 40 ml.

The pH was 5.49.

(c) The aqueous suspension was seeded and then left for 24 hours to crystallize at room temperature.

A2. Preparation of insulin crystal suspension,

40 i.u. per ml.

To a solution of methyl paraben (400 mg) in water (300 ml) was added a solution of aqueous zinc acetate (168 microlitres of 1M solution) together with an aliquot (40 ml) of insulin crystal suspension Al. The resulting suspension (pH 5.62) was left for two hours, when an aqueous solution of sodium hydroxide (375 microlitres of 2N) in water (50 ml) was added slowly with stirring. The pH of the resuIting suspension was adjusted to 7.02 by adding hydrochloric acid (225 microlitres of 0.2N) and the volume was made up to a total of 400 ml with water.

B. Preparation of porcine monodesamido insulin solution, 40 i.u. per ml.

Porcine monodesamido - A21 - insulin prepared according to Example 3 (86.7 mg, total activity 2000 i.u.) was dissolved in water (10 ml) to give solution B1.

To a solution of methyl paraben (50 mg) in water (35 ml) was added sodium acetate (68 mg), sodium chloride (350 mg) and aqueous sodium hydroxide solution (840 microlitres of 0.2N), followed by solution B1. The pH was adjusted to 6.98 and the total volume to 50 ml, thus affording a solution of porcine monodesamidoinsulin containing 40 i.u. per ml.

C. Injectable biphasic insulin preparation.

A preparation of pH 7.01 was prepared by mixing suspension A2 (395 ml) with solution B2 (44 ml). The preparation was transferred to sterile vials.

Example 9 Biphasic preparation of porcine MC-insulin and dissolved porcine monodesamido insulin (28 percent of total activity) con taining 0.3 micromoles of zinc per ml.

Al. The porcine insulin crystal suspension Al of Example 8 was used for this preparation.

A2. Preparation of insulin crystal suspension,

40 i.u. per ml.

To a solution of methyl paraben (400 mg) in water (300 ml) was added an aqueous solution of zinc acetate (92 microlitres of 1M solution) together with an aliquot (40 ml) of insulin crystal suspension Al of Example 8 The resulting suspension (pH 5.61) was left for two hours, when an aqueous solution of sodium hydroxide (375 microlitres of 2N) in water (50 ml) was added slowly with stirring. The pH of the resulting suspension was 7.03. The volume was made up to a total of 400 ml with water. An aliquot of this suspension (200 ml) was adjusted to pH 6.64 by addition of hydrochloric acid (200 microlitres of 0.2N).

B. Preparation of porcine monodesamido insulin solution, 40 i.u. per ml.

Porcine monodesamido - A21 - insulin prepared according to Example 3 (277.3 mg, total activity 6400 i.u.) was dissolved in water 25 ml) to give solution B1.

To a solution of methyl paraben (160 mg) in water (130 ml) was added sodium acetate (217.6 mg), sodium chloride (1120 mg) and sodium hydroxide solution (2.688 ml of 0.2N), followed by solution B1 to give solution B. The pH was lowered from 7.30 to 6.60 with 0.2N hydrochloric acid, and the volume was made up to a total of 160 ml with water.

C. A preparation of pH 6.61 was prepared by mixing suspension A2 (pH 6.64, 195 ml) with an aliquot (75 ml) of solution B. The preparation was transferred to sterile vials.

WHAT WE CLAIM IS: 1. An injectable biphasic zinc-containing insulin preparation, which comprises a suspension of zinc-containing crystalline insulin in an aqueous medium containing dissolved monodesamidoinsulin and having a pH in the range of from 6 to 8, the zinc-containing crystalline insulin and the dissolved monodesamidoinsulin being derived from the same species.
2. An insulin preparation according to Claim 1, wherein the crystalline insulin is highly purified insulin (as hereinbefore defined).
3. An insulin preparation according to Claim 1, wherein the crystalline insulin is monocomponent insulin (as hereinbefore defined).
4. An insulin preparation according to any one of Claims 1 to 3, wherein the monodes amidoinsulin is monodesamido - A21 - insulin.
5. An insulin preparation according to any one of Claims 1 to 4, wherein the content of monodesamidoinsulin corresponds to from 10 to 75 percent of the total insulin activity of the preparation, expressed in international units of activity.
6. An insulin preparation according to Claim 5, wherein the content of monodesamidoinsulin corresponds to from 20 to 50 percent of the

total insulin activity of the preparation, expressed in international units of activity.
7. An insulin preparation according to any one of Claims 1 to 6, wherein the crystalline insulin and the monodesamidoinsulin are both of bovine origin.
8. An insulin preparation according to any one of Claims 1 to 6, wherein the crystalline insulin and the monodesamidoinsulin are both of porcine origin.
9. An insulin preparation according to Claim 7, wherein the total zinc ion content of the preparation at pH 7 is in the range of from 0.25 X 102 X A to lOx A micromoles per ml of the preparation, A being the total number of international units of insulin per ml.
10. An insulin preparation according to Claim 9, wherein the total zinc ion content of the preparation at pH 7 is in the range of from OAS X 10-2 x A to 0.85 x 10Z x A micromoles per ml of the preparation.
11. An insulin preparation according to Claim 8, wherein the total zinc ion content of the preparation at pH 7 is in the range of from 0.7x10-2xA to 1.5x10-2xA micromoles per ml of the preparation, A being the total number of international units of insulin perml.
12. An insulin preparation according to Claim 11, wherein the total zinc ion content of the preparation at pH 7 is in the range of from 10-2 XA to 1.3 X 10-2 X A micromoles per ml of the preparation.
13. A process for preparing an injectable bi phasic zinc-containing insulin preparation, con taining zinc-containing crystalline insulin sus pended in an aqueous medium, which process comprises bringing monodesamidoinsulin into solution in the aqueous medium the aqueous medium having a pH in the range of from 6 to 8, the zinc-containing crystalline insulin and monodesarnidoinsulin being derived from the same species.
14. A process according to Claim 13, where in the crystalline insulin is highly purified insulin (as hereinbefore defined).
15. A process according to Claim 13, wherein the crystalline insulin is monocom ponent insulin (as hereinbefore defined).
16. A process according to any one of Claims 13 to 15, wherein the monodesamidoinsulin is monodesamido - A21 - insulin.
17. A process according to any one of Claims 13 to 16, wherein the monodesamidoinsulin is used in an amount which corresponds to from 10 to 75, percent of the total insulin activity of the final preparation, expressed in international units of activity.
18. A process according to Claim 17, wherein the monodesamidoinsulin is used in an amount which corresponds to from 20 to 50 percent of the total insulin activity of the final preparation, expressed in international units of activity.
19. A process according to any one of Claims 13 to 17, wherein the crystalline insulin and the monodesamidoinsulin are both of bovine origin.
20. A process according to any one of Claims 13 to 17, wherein the crystalline insulin and the monodesamidoinsulin are both of porcine origin.
21. A process according to Claim 19, wherein the total zinc ion content of the preparation at pH 7 is adjusted to a content in the range of from 0.25 x lOx A to 102 x A micromoles per ml of the preparation, A being the total number of international units of insulin per mL
22. A process according to Claim 21, wherein the total zinc ion content of the preparation at pH 7 is adjusted to a content in the range of from 0.45 X 10*2 X A to 0.85 X 10-2 XA micromoles per ml of the preparation.
23. A process according to Claim 20, wherein the total zinc content of the preparation at pH 7 is adjusted to a content in the range of from 0.7x1O-2xA to 1.5 x 1=2 x A micromoles per ml of the preparation, A being the total number of international units of insulin per ml.
24. A process according to Claim 23, wherein the total zinc ion content of the preparation at pH 7 is adjusted to a content in the range of from 10-2xA to 1.3 x lOx A micromoles per ml of the preparation.
25. A process for preparing an injectable biphasic zinc-containing insulin preparation, in accordance with Claim 13 and substantially as described in foregoing Example 6.
26. A process for preparing an injectable biphasic zinc-containing insulin preparation, substantially as described in foregoing Example 7.
27. A process for preparing an injectable biphasic zinc-containing insulin preparation, substantially as described in foregoing Example 8.
28. A process for producing an injectable biphasic zinc-containing insulin preparation, substantially as described in foregoing Example 9.
29. An injectable biphasic zinc-containing insulin preparation, whenever prepared by the process of any one of Claims 13 to 28.