CA1099632A - Biphasic insulin preparations - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

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

This invention relates to novel in~ectable biphasic zinc-containing insulin preparations and to a process for pre-paring 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 in;ections 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 pro-longed 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 i,nsulin preparations having the latter ~ I
properties are known. In this respect, reference is ~,ade to British Patent Specification No. 860,515 which discloses pharmaceutically stable insulin preparations consisting of a suspension of zinc-containing, bovine insulin crystals in a solution having a pH within thé ran~e o~ 6 to 8 and containing di.ssolved porcine insulin, the suspended insulin and the di.sso1ved insulin constituting the slow and quick acting insulin fractions, respectively. In order to keep the porcine : insulin in so1ution, particularly under storage, the suspension . shows s.uch a zinc ion content that the suspended bovine i.nsulin crystals contain less than 0.25 milliequivalent of zinc per . gram of the dried crystals at pH 7.0 of the suspension. Under : these condi.tions, it is not possible to substitute the suspended boyine insulin crystals by porcine insulin crystals since the latter will not remain undissolved, nor is it possible

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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 the limitations referred to above, heretofore con-sidered as being unavoidable. This object has now been attained according to the present invention by incorporating dissolved monodesamidoinsulin into the aqueous phase of the insulin suspension.
~ onodesamidoinsulin, as herein defined, shall mean any chemical derivative obtained from insulin by converting a single side chain carboxamido group (of an asparagine or a glutamine residue) of the insulin molecule into the corresponding carboxyl or aarbo~ylate group.
It is known that deamidation of insulin takes place under hydrolytic conditions, particularly in acid solutlon, thus resulting in the formation of desamidoinsulins. Since the extraction of insulin from pancreas gland tissue is usually conducted under acid conditions, desamidoinsulins, particularly monodesamidoinsulin, are normal contaminants, not only of crude insulin fractions but also of lnsulin obtained therefrom by crystallization.
Although insulin, both of porcine and of bovine origin, contains a total of six carboxamido groups, so that a complex mixture of desamidoinsulins could be expected, the rate of the àcid hydrolysis of the A-chain carboxyl-terminal asparagine carboxamido group is considerably higher than that of the remaining amido groups with the result that the desamidoinsulin fraction consists predominantly of monodesamido-A21-insulin.
Tllus, accordin~ to the first aspect of the present invention there is provided a stable injectable biphasic zinc-: -, 1~)9963Z
1, containing insulin preparation, which comprises a suspension ofcrystalline insulin in an aqueaus medium containing dissolved ¦ monodesamidoinsulin and having a pH within the ranse of 6 to 8, said crystalline insulin and monodesamidoinsulin being derived from the same species.
¦ In a specific ~mbodiment of the present invention thcI 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 monodesami~oinsulin shows such a degree o~ solubility that it can replace the porcine insulin used here-tofore. Even though deamidation of insulin l-owers the isoelectric point, it could not be expected that the increase in solubility . .
sould be sufficiently high to enable the formatlon 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.
I 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 pro--~ ~ ductlon. By means of the present invention, lt will be easier ~ to satisfy the increasing demand for biphasic insulin pre-~ ~, parations 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 monodesamido-insulin remains in solution in the presence of zinc ions at a considerably higher concentration than does porcine insulin ., 1~;)9963Z

itself. This surprising property makes it possible to make stable I injectable biphasic insulin preparations in which the suspended ¦ crystals are porcine insulin crystals remaining in suspensi;on ¦ due to the increased zinc ion content. It could not be expected that such a preparation would have the stability required for practical use.
I The above mentioned specific embodiments of the present ¦ - invention taken together open up additional ways of making ¦ injectable biphasic single species insuli~ preparations. It has been postulated that porcine insulin and bovine insulin contained in the known insulin preparations gLve rise to the formation of different insulin antibodies and that, for im~unological 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 biphasia insulin preparations of which the insulin constituents, both in solution and in suspension, ori-;~ ginate from a single species, preferably bovine or porcine.
¦~ ~ It is known that commercial crystalline insulin contains impuritie=~ and that these impurities can be removed to such an ~ ~ ~ extent that the purified insulin emerges as a single peak in 1~ ~ gel filtration analysis (vide e.g. Diabetes, vol. 21 (1972) ~ ~ ::
¦ ~ pp. 657-60). Hereinafter 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 electrophoreses (DISC PAGE) shall bb 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, respect-", 1(~9963Z

ively, 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 (1960), pp. 2294-2299; Diabetes, vol. 21 (1972), p. 463). Likewise, it has been possible to identify monodesamido-insulin, and specifically monodesamido-A21-insulin, as a constituent of partially hydrolyzed insulin obtained under properly controlled conditions as to acid strength, temperature, duration of hydrolysis, etc. (vide e.g. J. Biol. Chemistry, vol.
237 (1962), pp. 3406-3411). However, none of these references disclose the isolation of monodesamidoinsulin in substance.
To prepare substantial amounts of monodesamidolnsulin for the purpose of the present invention, partially hydrolyzed crude or commercial insulin, preferably prepared under properly controlled acid conditions, was subjected to fractionation by ion exchange chromatography, preferably on an anion exchanger.
Eluted fractions corresponding to the central part of the mono-desamidoinsulin peak were collected. Recovery of the monodes-~amidoinsulin may be affected by precipitation, e.g. as a slight-ly soluble zinc complex from a solution at a pH in the range of neutrality, followed by desalting of the re-dissolved mono-desamidoinsulln, 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 durlng the production of monocompon~nt lnsulln as dlsclosed i-. .'', ""', -1()99632 Britisll Patent Speclfication No. 1,285~024. Fractions correspond-ing 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 mono-desamidoinsulin used for the purpose of this invention shows such a purity that gel filtration thereof results in a single peak and that disc polyacrylamide gel electrophoresis results in essential-ly a single band.
The amount of monodesamidoinsulin used depends on the de-gree of rapid onset of the insulin a~tion desired. Usually, the activity o the monodesamidoinsulin should constitute from 10 to 75, preferably from 20 to 50, percent of the total insulin activi-ty of the preparation, expressed in international units (i.u.) of activity.
The p~-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 monodesamidoinsulin, re-spectively, and hence the physical stability of the biphasic pre-parations of the present invention, are related to the free zinc .
on concentration of the llquid phase of the preparation. There-fore, addition of auxiliary substances interfering with the avail-ability of free zinc ions, such as zinc complexing-and/or zinc precipitating agents, is preferably avoided. Under such conditions the total zinc ion content of a bovlne insulin preparation at;pH
7 is chosen in the range of from 0.25 x 10 2 x A to 10 2 x A, pre-ferably from 0.45 x 10 2 x A to 0.85 x 10 2 x A micromoles per ml, in which A indicates the total number of international units (i.u.) o~ insulin per ml of the preparation.
In the case of a porcine insulin preparation, the respective ranges are from 0.7 x 10 2 x A to 1.5 x 10 2 x A

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and 10 2 x A to 1.3 x 10 2 x A micromoles of zinc ion per ml.
It is to be understood that in case the preparation 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 respect, reference is made to British Patent Specification No. 840,870.
According to a further aspect of the present invention there is provlded a process for preparing an injectable zinc-containing insulin preparation for clinical use, containing crystalline insulin suspended in an a~ueous medium, which process comprises bringing monodesamidoinsulin into solution in the aqueous medium, said crystalline insulin and monodesamidoinsulin belng derived from the same species.
A sterile suspension of crystalline, preferably highly purified or monocomponent insulin, the amount of insulin being calculated to produce a final suspension of predetermined i , 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. sodlum acetate, and a physiolo~ically acceptable salt, e.g.~sodium chloride, to make the solution isotonic. The zinc content of the suspension was adjusted to the .
estlmated 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 monodesamidoinsulin having a predetermined lnsulin aotivity, e.g. 40, 80 or 100 i.u.
per ml, was prepared by dissolving the calculated amount of monodesamidoinsulin in water together with preservative, .
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buffering a~ent, and physiological1y 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 devided between the insulin suspension and the monodes-amidoinsulin solution. The pH was adjusted to a value in the ' ' range of from 6 to 8, preferably about 7, followed by adjust-I ment of the final volume of the solution.
Aliquot volumes of the insulin suspension and monodes-~.
amidoïnsulin solution were then combined, the volume ratio ~eing 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 ~e construed as limlting the scope of the lnventlon. 1 In the examples, aqueous solutions and water were sterilized, the former by filtration, and subsequent handling . -~; was conducted`under aseptic conditions.
:
' ~ Prepa~ation of Monodesamidoinsulin.

- Example l From crystalline porcine insulin.
. ~
Porcine insulin (O.5 g of crude insulin, crystallized ' from citrate buffer) was dissolved in water containing hydro-' chlorid acid (5 ml of 0.2 N solutionl. Water was added to a total volume of 50 ml. The solution (pH 2.021, after being sterilized by filtration, was set aside at room temperature (25C) for 30 days.
A mixture of insulin and monodesamidoinsulin was pre~
cipitated by addition of an aqueous solution contain1ng -zinc . ' _g_ ,.

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to 5.5.
The precipitate (containing about 50 percent of monodes-amidoinsulin according to DISC PAGE analysis) was dissolved in water containing EDTA (50 mg of the disodium salt), ethanol (~0 ml of 60 percent v/v) and sodium chloride (0.08 g), tris(hydroxymethyl)aminoethane being added to adjust pH to 8.4.
An insoluble residue was removed by contrifu~ation.
- The supernatant was applied to a 2.5 cm x 30 cm column ~
B containing QAE-Sephadex A-25 equilibrated with a buffer of the following composition:
Tris(hydroxymethyl)aminoethane (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.
Elution was conducted with the same buffer at 25C and fractions ~each of 4.9 mlj were collected at an elution rate of 50 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 the addition of an equal volume of an aqueous solution of zinc acetate (0.01 M, 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 cm x 40 cm) of SEPHADEX G 10. Elution with aqueous acetic acid (M solution) at a rate of 0.6 ml per minute was followed by monitoring the O.D.
a~ch~o~rK
--1()--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 crude insulin, crystallized from citrate buffer) was dissolved in water containing hydrochlorid acid (5 ml of 0.2 N 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 (25C) 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 insulin.
Porcine MC-insulin (40 g) was dissolved ln water contain-ing hydrochloric acid (30 ml 2 N solution). Water was added to , a total volume of 2000 ml. The solution (pH 2.08), after being sterilized by filtration, was set aside at room temperature (25Cj for 45 days.
Precipltation of insulin and monodesamidoinsulin was afforded by addition of an aqueous solution containing zinc ions (20 ml of M zinc acetate) folIowed by adjustment of pH to 5.5.
Monodesamidoinsulin was separated from other components by ion exchange chromatography on a 15 cm x 40 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.

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- - Example 4 From monocomponent bovine insulin.
Bovine I~C-insulin (25 g) was dissolved in water contain-ing hydrochloric acid (25 ml of 2 N 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 (25C) for 30 days.
The mixture of insulin and monodesamidoinsulin was pre-cipitated by addition of an aqueous solution containing zinc ions (25 ml of M zinc acetate) followed by adjustment of pH
to 5.5.
- Fractionation was performed on a lO cm 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 hou~. i The O.D. of the eluate was monitored at 276 nm.

Fractions of the centraI 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 monodesamldo-A21-insulin was 10.8 g.
~ ExamPle 5 :, ~
Isolation of monodesamidoinsulin during the preParation of ; ~ MC-insulin ~rom cryst~alline insulin.
Monodesamidoinsulin, which emerges togethér with insulin in gel-filtration, is separated from the latter by anion ex-change chromatography (cf. British~Patent Specification No.
1,285,023~.~Fractions corresponding to the central part of the monodesamidoinsulin peak of the anion exchange chromatogram ' ..

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were pooled, the monodesamidoinsulin was precipitated by add~tion of zinc acetate and recovered in a manner analogous to those of the previous examples.
Biphasic Insulin Preparations.
Example 6 ¦ Biphasic preparation of bovine MC-insulin and dissolved bovine monodesamidoinsulin (20 percent of total ac~ivity), containinq 0.18 micromoles of zinc per ml.
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A 1. Bovine MC-insulin crystal suspension.
(a) Crystalline monocomponent bovine insulin (1.45 g) containing 0.4~ 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++) I by addition of hydrochloric acid ~750 microlitres of 2 N).
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 2 N), made up to a total volume of 50 ml.

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~ ~ (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 a~ described in British Patent Specification No. 766,994.
A 2. Preparation of MC-insulin crystal suspension, 40 i.u.
.
per ml.

To a solution of methyl paraben (375 mg) in water (280 ml) was added hydrocbloric acid (470 microlitres of 0.2 N) . - , ..

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together wlth an aliquot (37.5 ml) o~ suspension ~ 1 The ! resulting suspension, having a pH of 5.52, was left for ¦ approximately 2 hours, after which time a solution.of sodium hydroxide (375 microlitxes of 2 N) in water t50 ml) was added ~ slowly with stirring. Following ad~.ustment of the total volume ¦ with water to 375 ml, the resulting suspension of bovine insulin l, crystals contained 40 i.u. of insulin per ml. The pH of the I ~ suspension was 7.1.
I : B 1. Preparation of bovine monodesamidoinsulin solution.
¦ Bovine monodesamido-A21-insulin (200 mg), prepared according to Example 4 and having a total activity of 5000 i.u;,.
was dissolved in water containing zinc chloride (150 microlitres . of 1~ solution, calculated as Zn++) and hydrochloric acid I . (1.35 ml of 0.2 N). The total volum~ was adjusted to 10 ml.
. B 2. Preparation of monodesamidoinsulin solution, 40 i.u.
per ml.
To a solution of methyl paraben (130 mg) in water (75 ml) : ~ was added~sodium acetate (170 mg), sodium chloride (875 mgj : ~ and aqueous sodium hydroxide solution (2.1 ml of 0.2 N) followed by solution B 1. The total.volume was adjusted to 125 ml by ~- ~ addition of water, thus affording a solution of monodesamido-~ ~ . .
insulin having a pH of 7.1 and containing 40 i.u. per ml.

:~ ~ Injectable biphasic insulin Preparation.

. ~ ~ A preparation of pH 7.1 was prepared by mixing an aliquot (80 ml) of suspension A 2 with an aliquot (20 ml of monodes-amid.oinsulin solution B 2. The preparation was transferred to . sterilized vials.
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Example 7 Biphasic preparation of hiqhIy purified ~ovi'n'e i sulin and dissolved bovine monodesamïdoinsulin ( ?5 percent of total ' acti.vity), contaïning 0.26 micr'omo'l'e's' of zinc per ml.
A 1. Highly purified bovine ins-ulin c'r'vs't'_l''sus'pens'ion.
The procedure was analogous to that of Example 6, except that the following.amounts of materials were used:
. (a), Crystalline, highly purified ~ovine insulin (620 mg, total activity 16,000 i.u.~.. containing 0.37 percent , zinc.
Aqueous zinc chloride solution (2.5 ml of 0.107 per- ' . ,' cent, calculated as Zn+~l Hydrochloric acid (2.85 ml of 0.2 N) Water to make up to 2 ml.
. (b~ Sodi.um acetate (544 mg~
Sodïum chloride (2.8 gl ~

Aqueous sodium hydroxide (1.1 ml of 0.2,N) .
, ' 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.
.: : A ~ Pre~aration of in'sulin crYstaI'sus~ension, 40 i.u. pex ml.
To a solution of meth.Yl para~en ~400 mg~ in water (300 : mll was added an aqueous solution of zinc acetate (103 micro~
li.tres of M solutionl together with the total volume of i.nsulin crystal sus.pension A 1. The resulting suspension (pH 5.56~, was .
left for two hours, when a solution of sodium hydroxide (400 . mi.crolitres of 2 Nl in wa.ter (50 mll was added 510wly with.
. stirring. The pH of the resulting suspension was adjusted to 7.03 by adding hydrochloric acid (225 microlitres of 0.2 N~

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and the volume was made up to a total of 400 ml with water.
B 1. Preparation of bovine monodesamidoinsulin solution.
Bovine monodesamidoinsulin, prepared according to ~xample 2 (240 mg, total activity 5600 i.u.), was dissolved in water containing hydrochloric acid (280 microlitres of 0.2 N).
The total volume was adjusted to 20 ml.
B 2. Preparation of bovine monodesamidoinsulin solution, 40 i.u. per ml.
To a solution of methyl paraben (140 mg) in water ~105 ml) was added sodium acetate (190.~ ~g), sodium chloride (980 mg) and aqueous sodium hydroxide solution (235 microlitres of 2 N), followed by solution B 1. The pH was adjusted to 6.99 with a~ueous sodium hydroxide solution (50 microlitres of 0.2 N) and the total volume to 140 ml, thus affording a solution o~ bovine monodesamidoinsulin containing 40 i.u. per ml.
C. Injectable biphasic insulin preparation A preparation of pH 7.01 was prepared by mixing suspension 2 (395 ml) with solution B 2 (131.7 ml). The preparation was transferred to sterile vials.
Example 8 Biphasic preparation of porcine MC-insulin and dissolved por-cine monodesamidoinsulin (10 percent of total activity), contaLning 0.55 micromoles of zinc per ml.
A 1. Porcine MC-insulin crystal suspension The procedure was analogous to that of Example 6, except that the following amounts of materials were used:
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~ -16-1~9. 9632 . , (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 Zn++) Hydrochloric acid (710 microlitres of 2 N) - Water to make up to 60 ml.
: (b) Sodium acetate (1.36 g) Sodium chloride (7.00 g) ~ ~
Aqueous sodium hydroxide~(275 microlltres of 2 N) 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.
2. Preparation of insulin crystal ~uspension, 40 i.u. Per ml ; To a solution of methyl paraben (400 mg~.i.n:wa~er (300 ml) was added a solutlon of aqueous zinc~acetate (168 mLorolitres of M solution)~together with an aiiquot (40 ml) ::of~:insulin crystal:suspension A 1. ~he resulting suspension (pH~5~.62) was left for two hours, when an aqueous solution of~sodium hydroxide~;~(375~mlorolltres of 2 Nj` in water (50 ml) was~added slowly wlth:stlrring. The pH of the resulting auspension was~adjusted to 7.02 by~adding~hydrochlorlc acid (225 microlitres of 0.2 N) and the.volume was made up to a total of 400 ml with~water.
: B.~ Preparation~of~porcine monodesamldoinsulin_so_ution, 40 i.u.
per ml.
: ~ , . Porcine monodesamido-A l-insulin prepared according to . Example 3 (86.7 mg, total activity 2000 i.u.) was dissolved in : water (10 ml) to give solution B 1.

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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 0.2 ~), followed by solution B 1. The pH was ad~usted 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 A 2 (395 ml) with solution B 2 (44 ml). The pre-paration was transferred to sterile vials.
Example 9 Biphasic preParatiOn of Porcine MC-insulin and dissolved Por-cine monodesamidoinsulin (28 Percent of total activ_ y) contain-ing 0.3 micromoles of zinc per ml.
A 1. The porcine insulin crystal suspension A 1 of Example 8 was used for this preparation.
A 2. 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 micro-litres of M solution) together with an aliquot (40 mV of insulin crystal suspension A 1 of Example 8. The resulting suspension (pH 5.61) was left for two hours, when an aqueous solution of sodium hydroxide (375 microlitres of 2 N) 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 o~ 0.2 N).

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109g632 B. Preparation of porcine monodesamldoinsu1in 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 B 1.
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 0.2 N), followed by-solution B 1 to give solution B. The pH was lowered from 7.30 to 6.60 with 0.2 N 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 ~y mixing suspension A 2 (pH 6.64, 195 ml) with an aliquot (75 ml) of solution ~.
The prepar~tion was transferred to sterile vials.

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Claims (18)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A stable injectable biphasic zinc-containing insulin preparation, which comprises 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.

2. An insulin preparation according to claim 1, wherein the crystalline insulin is highly purified insulin.

3. An insulin preparation according to claim 1, wherein the crystalline insulin is monocomponent insulin.

4. An insulin preparation according to claim 1 wherein the monodesamidoinsulin is monodesamido-A21-insulin.

5. An insulin preparation according to any one of claims 2 to 4, wherein the content of monodesamidoinsulin corresponds to from 10 to 75 percent of the total insulin activity of the preparation.

6. An insulin preparation according to claim 1, wherein the crystalline insulin and the monodesamidoinsulin are both of bovine origin.

7. An insulin preparation according to claim 1, wherein the crystalline insulin and the monodesamidoinsulin are both of porcine origin.

8. An insulin preparation according to claim 6, wherein the total zinc content of the preparation at pH 7 is from 0.25 x 10-2 x A to 10-2 x A, micromoles per ml of the preparation, A being the total number of international units of insulin per ml.

9. An insulin preparation according to claim 7, wherein the total zinc content of the preparation at pH 7 is from 0.7 x 10-2 x A to 1.5 x 10-2 x A, micromoles per ml. of the preparation, A being the total number of international units of insulin per ml.

10. A process for preparing a stable injectable biphasic zinc-containing insulin preparation, containing crystalline insulin suspended in an aqueous medium, which process comprises bringing monodesamidoinsulin into solution in an aqueous medium having a pH value in the range of from 6 to 8, said crystalline insulin and monodesamidoinsulin being derived from the same species.

11. A process according to claim 10, wherein the crystalline insulin is highly purified insulin.

12. A process according to claim 10, wherein the crystalline insulin is monocomponent insulin.

13. A process according to claim 10, wherein the mono-desamidoinsulin is monodesamido-A21-insulin.

14. A process according to any one of claims 11 to 13, 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.

15. A process according to claim 10, wherein the crystalline insulin and the monodesamidoinsulin are both of bovine origin.

16. A process according to claim 10, wherein the crystalline insulin and the monodesamidoinsulin are both of porcine origin.

17. A process according to claim 15, wherein the total zinc content of the preparation at pH 7 is adjusted to from 0.25 x 10-2 x A to 10-2 x A micromoles per ml. of the preparation, A being the total number of international units of insulin per ml.

18. A process according to claim 16, wherein the total zinc content of the preparation at pH 7 is adjusted to from 0.7 x 10-2 x A to 1.5 x 10-2 x A micromoles per ml. of the preparation, A being the total number of international units of insulin per ml.