Note: Descriptions are shown in the official language in which they were submitted.
109963Z
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
-2- ~
'' ', ' '. ` ' ' ' ' '.
.
l W96 3Z
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-
- -5-
",
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
--7--
, ' ` ' .
~W~63Z
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,
.
''' ' ' '
. .
~ g963Z
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_
,.
l~9g63Z ` ' '
ions (0.5 ml of M zinc acetate) followed by adjustment of pH
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()--
109963Z
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.
- --11--
1~9963Z
- - 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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.. . . .
' . ' ~ .~
.
~ag963z `
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.
.
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.
, : ~
~ ~ (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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. - , ..
6æ
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.
.
.. ..
. -14-
' ':
1~96~ , . . .
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~
-15-
.
iC09~63Z
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:
.. . ..
~ -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.
.
-17-
',
1(J95~63Z
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).
.
-18-
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.
' ` ' ' ' '
~ ~' ~ ' ' ' ' ' ' ' .
.
,
--19--
