Note: Descriptions are shown in the official language in which they were submitted.
~O~'lS~
This invention relates to a therapeutic insulin
preparation and a process for producing a stable insulin
preparation with protracted effect and low antigenicity by
reacting insulin with an organic compound comprising basic
groups.
In the treatment of diabetes mellitus insulin
preparations derived from swine or ox pancreas are generally
used. Thus approximately 30% of the world consumption of insu-
lin is based on porcine insulin and approximately 70% on bovine
insulin. Insulin from other animals has been suggested, for
instance sheep insulin, but so far it has not attained any major
commercial significance.
Insulin therapy previously involved several
inconveniences which manifested themselves inter alia as allergy
and lipodystrophy~
It has long been known that the conventional
insulin treatment for most patients resulted in the formation of
insulin antibodies, which might lead to increased insulin
requirements, in that unknown amounts of insulin may be bonded
to the antibodies and during the continuance of that bonding the
insulin will be ineffective as regulator of the blood sugar.
The primary cause of the antibody formation and
the consequent high insulin consumption was long supposed to be
the presence of various impurities in normal commercial insulin.
As examples of such impurities may be mentioned
insulin dimer, proinsulin, intermediary insulin tthe stage
between proinsulin and insulin), arginine insulin, ethylester
insulin, mono-desamido insulin and didesamido insulin. The
first three of these compounds are known to be highly antigenic.
I~ s also known that th~ fourth or the fifth or both of these
are highly antigenic, while the sixth and the seventh compound
and the insulin molecule itself are not or but slightly anti-
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genic. Cf. Schlichtcrull, Monocompone~t Insulin and itsClinical Implications, 16th March, 1973.
It is known to remove the said impurities by gel
filtration and/or ion exchange, whereby it is possible to obtain
a highly purified insulin containing substantially only a single
component. In recent years insulin preparations which have been
freed of at any rate some of the said impurities have been
marketed, and in many cases they have resulted in reduced anti-
body formation in diabetics.
It has been found that quick-acting preparations of
both porcine and bovine insulin can be produced so pure that
they will not, or at any rate only to a very small degree, cause
insulin antibody formation, cf. Schlichtcrull, Monocomponent
Insulin and its Clinical Implications, 16th March 1973. And it
has proved to be possible to produce preparations with protract-
ed action and having substantially reduced antigenicity if the
production is based on highly purified porcine insulin, cf. T.
Deckert et al. Diabetologia, vol. 10, pp~703-8, 1974. But it
has also been found that even if bovine insulin is produced in a
purity that with the present analytical methods must be regard-
ed as just as high as the highly purified porcine insulin that
can be made today and so pure that, as stated abGve, it will not,
or at any rate only to a very low degree, cause antibody formation
when used in quick-acting preparations, protracted action low-
antigenic preparations based on highly purified bovine insulin
have not yet been marketed. This is a serious drawback because
protracted-action preparations based on bovine insulin are
preferred internationally.
The formation o~ antibody in the live organism
against a chemical compound such as insulin may be due to such
chemical differences in the primary structure, c~. Arguilla E.R.
.,
,
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et al: Immunochemistry of Insulin, Handbook of Physiology, Ch. 9,
p. 160, 1972, that the chemical compound has a steric structure
which is incompatible with and repelled by the organism, cf.
Arquilla, EoR~ et al: Immunology Conformation and Biological
Activity of Insulin, Diabetes, vol. 18, p. 194, 1969, or that
the chemical compound has such total dimensions (aggregation)
that it is incompatible with and repelled by the organism, cf.
Arquilla, E oR~ et al: Immunochemistry of Insulin, Handbook of
Physiology, Ch. 9, pO 160, 1972.
Furthermore it is possible to cause antibody
formation by administering together with the chemical compound
a component capable of activating the immunity mechanism.
Several tests tend to indicate that the physical
state of insulin plays an essential part in the antibody forma-
tion. (Kumar et al: Horn. Metab. Res. 6, (1974) 185-177, and
Piers et al: Neth J. Med. 17 ~1974) pp. 234-238).
The reason why the quick-acting highly purified
insulin preparations do not, or only to a limited degree, cause
antibody formation probably is that in those cases the insulins,
besides being highly purified, are present in monomer form or
form but loose aggregates, whereas in the previously produced
and marketed protracted-action highly purified insulin prepara-
tions the insulin appeared in an aggregated form and with
properties that resulted in antibody formation. This problem is
very pronounced in respect of bovine insulin, which is probably
due to the fact that bovine insulin is more inclined to form
aggregates than porcine insulin.
Bovine insulin without additives is known to have a
wider isoelectric precipitation zone or interval than porcine
insulin. It is also known that this wide isoelectric precipita-
tion zone may be restricted to the same width as known for por-
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~0~5~1~
cine insulin by the addition of certain substances such as phenol
or m-cresol, cf. U.K. Patent No. 1,222,100. The said wider iso-
electric precipitation zone of bovine insulin is assumed to be
due to the fact that bovine insulin aggregates at pH values close
to neutral.
The invention is based on the recognition that it
is possible to stabilize highly purified insulin so that it
will not during production or in protracted-action prepara-
tions during storage or in use form aggrégates that might
cause antibody formation.
This object has been accomplished according to the
invention, which is specific in that the reaction is carried
out with a highly purified insulin in stabilized monomer or
loosely aggregated form.
In accordance with the invention, in a process for
the production of a stable insulin preparation with protracted
action and low antigenicity by reacting insulin with an orga-
nic base containing amino groups to prepare a stabilized insu-
lin, there is provided an improvement which comprises carrying
out the reaction in a medium for the reaction which contains a
protein dissociating or protein depolymerizing stabilizer to
maintain the insulin in a dissolved monomeric or loosely aggre-
gated form during the reaction.
In the process according to the invention the highly
purified insulin in the form of stabilized monomer or as a
loose aggregate is reacted with an organic compound comprising
basic groups, such as amino groups or substituted amino groups.
A preferred compound comprising basic groups is a polypeptide,
for instance polyarginine, somatostatine, protamine or globin,
or a cleavage product of such a basic polypeptide. me pre-
ferred depolymerizing stabilizer is urea.
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lO~tS~l~
According to the invention the reaction is expediently
carried out in the presence of m-cresol or glucose. M-cresol
is well known to act as a preservative and glucose is an iso- -
tonic diluent.
The invention may be employed in connection with
insulin from many different animal species, such as swine, ox
or sheep, but it is in particular significant in connection
with bovine insulin.
A preferred embodiment of the invention is specific
in that a solution of insulin containing a stabilizer is sub-
jected to ion exchange by elution with an eluent containing a
stabilizer which maintains the insulin in stabilized monomer or
loosely aggregated form, whereafter a fraction containing in-
sulin freed of impurities is added to a solution containing a
basic polypeptide or a cleavage product of a basic polypeptide
and the precipitated insulin complex is isolated. This proce-
dure provides effective security against aggregation, in that
the highly purified insulin is not first isolated as such with
the conseq1lent risk of aggregation. Besides, the said process
is very simple in that the protracted-action insulin prepara-
tion is precipitated directly in a stable form.
The invention will be illustrated in detail by the
following examples.
EXAMPLE 1
250 mg. of recrystallized bovine insulin was dissolved
in 5.2 ml of stabilized buffer solution consisting of 7 M (mo-
lar) of deionized urea and 0.02 M of tris having a pH of 8.1.
~he solution was mixed with 5.2 ml of 7 M of urea. The pH of
the mixture was adjusted to 8.1. A column of a diameter of
5 cm~rwas packed with a layer 2.1 cm high DEAR cellulose (What-
~09~5~9
mann DE 52*) and equilibrated with a buffer solution of theabove composition. The insulin solution was introduced in the
column and elution performed at a rate of 75 ml per hour ac-
cording to the following schedule:
2.5 hours with a buffer of the composition
defined above,
3 hours with a buffer of the composition
- defined above to which had been added 0.0045
mole of ~dium chloride per liter,
12 hours with a buffer of the composition
defined above to which had been added 0.011
mole of sodium chloride per liter.
me eluate was divided into fractions. me highly
purified fractions were collected and the content of insulin
determined. In a 7 M urea solution of the same volume as the
mixture of the highly purified fractions protamine was dis-
solved in the amount necessary for obtaining the isophanic
ratio of highly purified insulin to protamine.
The insulin solution was added slowly and dropwise
to the protamine solution while stirring and, possibly after
dilution to a urea concentration of lM, a protamine-insulin
complex in amorphous state will be precipitated.
The precipitate was isolated by centrifuging and
presented only one band practically when 300 ~g is subjected
to isoelectric focusing in polyacrylamide gel and using 2% of
ampholine (pH 3-10) in 6M of deionized urea.
EXAMPLE 2
me procedure of Example 1 was repeated, and in the
protamine solution was introduced zinc chloride corresponding
to 0.5% Zn ions calculated on the amount of insulin and 0.3% m-
cresol based on the volume of the mixture. me protamine-insu-
lin complex was then precipitated in crystalline state.
* Trade Mark
,~
,~, "
~0~3~51~1C9
The precipitate was isolated by centrifuging and
presented practically only one band when 300 ~ g was subjected
to isoelectric focusing in polyacrylamide gel using 2% of am-
pholine (pH 3-10) in 6 M deionized urea.
EXAMPLE 3
me procedure of Example 1 was repeated, but as
starting material was used recrystallized bovine insulin
purified by gel filtration on Sephadex G-50*.
me prepared product was of the same purity as the
product described in Example 1.
EXAMPLE 4
~he procedures of Examples 1 and 2 were repeated, but
as starting material was used porcine insulin prepared by
salting-out an aqueous crude extract formed in the production
of insulin by addition of salt to obtain 3.5 M at pH 8.5. me
obtained salt cake was d~salted in conventional manner prior to
the ion exchange.
me prepared product was of the same purity as the
product described in Example 1.
EXAMPLE 5
The procedure o~ Example 4 was repeated and the ob-
tained salt cake was subjected to gel filtration on Sephadex
G-50*.
me product was of the same purity as the product
described in Example 1.
EXAM LE 6
m e procedures of Examples 1-3 were repeated, but
porcine insulin was used instead of bovine insulin.
me formed protamine-porcine insulin complex was as
pure as the complex prepared in Example 1
* Trade Mark
f --7
~94S't9
EXAMPLE 7
The procedure of Example 1 was repeated but instead
of protamine was used poly-L-arginine, degree of polymerization
295. me precipitate was isolated in the manner described in
Example 1~
EXAMPLE 8
me procedure of Example 1 was repeated but to the
pooled highly purified insulin ~ractions was added a 0.2%
aqueous solution of bis-(4-amino-2-methyl-quinolyl-6) urea-
hydrochloride in an amount equivalent to 10% by weight of theinsulin amount present in said fractions. Thereby, possibly
after dilution to a urea concentration of lM with 0.025 M so-
dium phosphate buffer, pH 7.3, an insulin complex was precipi-
tated, which was maintained some time and then isolated by cen-
trifuging.
The prepared product was of the same purity as the
product described in Example 1.
EXAMPLE 9
The procedure of Example 8 was repeated, but as
starting material was used recrystallized bovine insulin
purified by gel filtration on Sephadex G-50*. The prepared
product was of the same purity as the product described in
Example 1.
EXAMPLE 10
The procedures of Examples 8 and 9 were repeated but
porcine insulin was used instead of bovine insulin.
The formed porcine insulin complex was as pure as the
complex prepared in Example 1.
EXAMPLE 11
The procedure of Example 8 was repeated, but as
* Trade Mark
io9~l5~9
ctarting material was used porcine insulin, produced by salt-
ing-out an aqueous crude extract formed in the production of
insulin by addition of salt to obtain 3.5 M at pH 8.5. The
obtained salt cake was desalted in conventional manner prior
to the ion exchange.
The prepared product was of the same purity as the
product described in Example l.
EXAMPLE 12
me procedure of Example ll was repeated and the ob-
tained salt cake was subjected to gel filtration on Sephadex
G-50*.
me product was of the same purity as the product
described in Example l.
* Trade Mark
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lb9l~5'l9
SUPPLEMENTARY DISCLOSURE
Applicant has carried out tests under the invention
using other protein depolymerizing or protein dissociating
stabilizers such as a lower alkanol, dialkylformamide, aceta-
mide, N-alkyl-a~etamide and acetonitrile.
In the drawings which illustrate the invention:
Figure 1 shows results of the insulin binding cap-
acity of conventional NHP bovine insulin,
Figure 2 shows results of the insulin binding cap-
acity of purified NHP bovine insulin;
Figure 3 shows results of the insulin binding cap-
acity of a new insulin prepared according to the invention,
Figure 4 shows results of /0 bound 125I-insulin in
a conventional NHP bovine insulin;
Figure 5 shows results of % bound 125I-insulin
in a purified NHP bovine insulin, and
Figure 6 shows results of % bound 125I-insulin
in a new NHP bovine insulin prepared according to the in-
vention.
These tests which further illustrate the invention
are given below by way of examples.
1~9~5~1L9
EXAM LE 13
A column of 2.6 x 80 cm was packed with DEAE-
Sephadex A-25TM which was previously swelled and equilibrated
in a bllffer consisting of 0.15 M ammonium hydroxide in 7 M
dimethyl-formamide adjusted to a pH of 9.0 with 5 N hydro-
chloric acid, 300 mg bovine insulin purified by gel filtra-
tion on Sephadex G-50* were dissolved in 40 ml of the above
buffer and introduced in the column, whereupon an elution
was performed for 2 hours with the above buffer at a rate
of 200 ml per hour. Then the elution was continued, and at
the same rate a linear gradient was impressed, said gradient
being produced from 2700 ml of the above buffer and 2100 ml
buffer consisting o~ 0.25 M ammonium hydroxide in 7 M dime-
thylformamide adjusted to pH e 9.0 with 5 N hydrochloric acid.
The eluate was divided into fractions. The highly
purified fractions were collected, and the insulin content
was determined. The pH was adjusted to 7.9 with 5 N hydro-
chloric acid, and m-cresol was added until the solution
contained 0.2% of said m-cresol. When the total zinc content
of the solution had ~een adjusted to 0.5% by weight of the
insulin content, the amount of protamine sulphate necessary
to obtain the isophanic ratio was added in the form of a 1%
aqueous solution, and the mixture was stirred carefully.
After a standing period of 15 minutes at 20C 6 volume
1/75 sodium phosphate buffer was added, p~ = 7.3, containing
0.~/O m-cresol, and after a short stirring it was left to
stand at 20~C for 16 hoursO Hereby the amorphously precipi-
tated protamine-insulin-complex was crys~allized.
By replacing the above liquid by a known carrier
medium the precipitate was converted into an injectable
insulin preparation~
-- 10 --
V
5 ~ 9
WnAen a precipitate of 300 pg was isoelectrically
focussed in polyacrylamide gel using 2% ampholine (pH =
3-10) in 6 M deionized urea, said precipitate showed
essentially only one band.
EXAMPLE 14
A column with a diameter of 2.6 cm and a height of
4.4 cm was packed with QAE-Sephadex A-25TM which was previous-
ly swelled and equilibrated in a buffer consisting of 0.1 M
tris-(hydroxymethyl)-amino methane in 60 vol.% ethanol
adjusted to pH = 7.35 with 5 N hydrochloric acid. ~50 g
bovine insulin~ which had been purified by gel filtration on
Sephadex G-SOTM , were dissolved in 10 ml 0.1 M tris in 60%
ethanol adjusted to pH = 8.5 with hydrochloric acid, and
after filtering the solution was introduced in the column.
Then the column was eluted with the equilibration buffer at
a rate of 10 ml per hour. The eluate was divided into
fractions, the highly purified fractions were collected, and
the insulin content was determined. Then m-cresol was added
until the solution contained 0.2% of said m-cresol, where-
upon the total zinc content was adjusted to 0.5% by weight
of the insulin content. Then the amount of protamine sul-
phate necessary to obtain the isophanic ratio was added in
the form of a 1% aqueous solution, and the mixture was stirred
carefully. After a standing period of 15 minutes at 20C 9
volume 1/75 sodium phosphate buffer was added, pH = 7.3,
containing 0.2% m-cresol) and after a short stirring it was
left to stand at 20C for 16 hours. Hereby the amorphously
precipitated protamine-insulin-complex was crystallized.
By replacing the a~ove liquid by a known carrier
medium the precipitate was converted into an injectabl~
insulin preparation.
~'
lO9~S~
When a precipitate of 300 ~g was isoelectrically
focussed in polyacrylamide gel using 2% ampholine (pH - 3-10)
in 6 M deionized urea, said precipitate showed essentially
only one band.
EXAMPLE 15
In a refrigerated room at 5C a column with a
diameter of 2.6 cm and a height of 11 cm was packed with
QAE Sephadex A-25TM which was previously swelled and equilibra-
ted in a buffer consisting of 0.5 M tris (hydroxymethyl)-
amino methane in 8 M acetamide adjusted to pH = 8.0 with
glacial acetic acid.
300 mg bovine insulin, which had been purified ~y
gel filtration on Sephadex G-50TM, were dissolved in 30 ml
of the above buffer and introduced in the column, whereupon
an elution was performed for one hour at a rate of 80 ml per
hour. Then the elution was continued, and at the same rate
a linear gradient was impressed, said gradient being prepared
from 2000 ml of the above buffer and 2000 ml of the same
buffer containing furthermore 0.15 M sodium chloride.
2G The eluate was divided into fractions. The highly
purified fractions were collected and the insulin content
was determined. After admi~ture of m-cresol until a content
of 0.2% was obtain~d, the total zinc content of the solution
was adjusted to 0.5% by weight of the insulin content with
an 0.15 M zinc chloride solution, and then the amoun`t of pro-
tamine sulphate necessary to obtain the isophanic ratio was
added in the form of a 1% aqueous solution~ The mixture was
stirred carefully, and after a standing period of 15 minutes
7 volume 1/75 M sodium phosphate buffer was added, pH = 7.3,
containing 0.2% m-cresol, whereupon it was left to stand for
another 16 hours at 20C. Hereby the amorphously precipi-
- 12 -
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tated protamine-insulin-complex was crystallized.
By replacing the above liquid by a known carrier
medium the precipitate was converted into an injectable
insulin preparation.
When a precipitate of 300 ,ug was isoelectrically
focussed in polyacrylamide using a 2% ampholine (ph = 3-10)
in 6 M deionized urea, said precipitate showed essentially
only one band.
EXA~PLE 16
In a refrigerated room at 5C a column with a
diameter of 2.6 cm and a height of 12 cm was packed with
QAE Sephadex A-25TM which was previously swelled and equili-
brated in a buffer consisting of 0.05 M tris ~hydroxymethyl)-
amino methane in 7 M acetonitrile adjusted to pH = 8.2 with
5 ~ hydrochloric acid.
300 mg bovine insulin, which had been purified by
gel filtration on Sephadex G-50 M, were dissolved in 30 ml
of the above buffer, the pH being adjusted to 9.3 with 2 N
NaOH and then to 8.2 with 2 N hydrochloric acid and intro-
duced in the column, whereupon an elution was performed forone hour with the above buffer at a rate of 75 ml per hour.
Then the elution was continued, and at the same rate a
linear gradient was impressed, said gradient being produced
from 1800 ml of the above buffer, and 1800 ml of the same
buffer containing furthermore 0.15 M sodium chloride.
The eluate was divided into fractions, The highly
purified fractions were collected, and the insulin content
was determined. After admixture of m-cresol until a content
of 0.2% was obtained, the total zinc content of the solution
was adjusted to 0.5% by weight of the insulin content with
an 0.15 M zinc chloride solution, and then the amount of
- 13 -
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protamine sulphate necessary to obtain the isophanic ratio
was added in the form of a 1% aqueous solution. The mixture
was stirred carefully, and after a standing period of 15
minutes 6 volume 1/75 sodium phosphate buffer was adde~,
pH = 7.3, containing 0.2~/~ m-cresol, whereupon it was left
to stand for another 16 hours at 20C. Hereby the amorphously
precipitated protamine-insulin-complex was crystallized~
By replacing the above liquid by a known carrier
medium the product was converted into an injectable insulin
preparation.
When a precipitate of 300 ,ug was isoelectrically
focussed in polyacrylamide ~el using 2% ampholine (ph = 3-10)
in 6 M deionized urea, said precipitate showed essentially
only one band.
EXAMPLE 17
Example 15 was repeated, however, the buffer now
consisted of 0.05 M tris (hydroxymethyl)-amino methane in
5 M aqueous N- methylacetamide adjusted to pH = 8.2 with 5 N
hydrochloric acid, and only 4 volume phosphate buffer were
admixed with the insulin solution at the crystallization.
EXAMPLE 18
The procedure of Example 16 was repeated, the
collected highly purified insulin fractions being introduced
in a col~mn packed with Sephadex G-25 which was swelled in a
- medium consisting of 1/60 M disodium-mono-hydrogen-phosphate
in 1 M acetamide pH 7.65~ so that the volume introduced did
not exceed 20% of the total volume of the column. The column
was then eluted with the above medium at a rate of 20 ml per
hour per cm , and the insulin containing eluate fractions
were collected. After determination-of the insulin content
the total zinc content of the solution was ad~usted to 0.5%
r ~ 14
.1 ji~l
lO~?~S~19
by weight of the insulin content with 0.15 M zinc chloride
in 1 M acetamide, and then were added 1/4 volume 1/100 M
hydrochloric acid containing 1.5% m-cresol, and the amount of
protamine sulphate necessary to obtain the isophanic ratio.
Having been left to stand for 16 hours at 20C the precipi-
tated protamine-insulin-complex was crystallized. The pro-
duct obtained thereby was of the same purity as the product
described in Example 16.
sy replacing the above liquid by a known carrier
medium the precipitate can be used to prepare injectable
insulin preparations.
To illustrate the changed immunogenic properties
of the protamine-insulin-complex isolated according to the
invention comparative tests have been conducted on rabbits,
which are the animals normally used for examination of the
antigenic properties of insulin and insulin-like components.
In the comparative tests the following preparations
were used:
1. Conventional NPH bovine insulin prepared in conven-
tional manner from recrystallized bovine insulin
containing the impurities mentioned in the specifi-
cation.
2. Purified NPH bovine insulin prepared in conventional
manner from purified crystalline bovine insulin but
freed of the impurities mentioned in the specifica-
tion by column chromatography.
3. Purified NPH bovine insulin prepared according to
the invention as described in Example 2, New NPH
Bovine Insulin.
., . _ .
The rabbits were injected subcutaneously every 10th
day with a constant dose of 20 i.u. of the insulin preparation
to be tested. It was not possible to show any essential
- 15 -
lO~L15~1L9
antibody fol~ation after the injection of conventional NPH
bovine insulin without adjuvants, and with a view to compari-
son it was therefore necessary to adopt an immunization pro-
cedure normally used in the preparation of antibodies, that
is to inject the insulin preparations of the 1st injection
e~lllsified in Freund~s ~omplete Adjuvant and in subsequent
injections emulsified in Freund's Incomplete Adjuvant.
The insulin antibody formation was examined at
intervals of 20 days using the method described by Ortved-
Andersen et al. (Acta Endocr. (Kbh.) 69, 195-208, 1972).
The obtained results are illustrated in Figs. 1, 2
and 3.
It will be seen from the results that it was pos-
sible to show formation of antibody to conventional NPH
bovine insulin and reduced formation of antibody to purified
NPH bovine insulin, whereas it was practically impossible to
show any formation of antibody to the new NPH bovine insulin
prepared according to the invention. It should be noted that
preparations 2 and 3 were purified by the same chromatography
purification, the only difference being that preparation 2
was prepared by first isolating the insulin and then reacting
it with protamine in known manner, whereas preparation 3 was
prepared by conducting the reaction in a urea-containing eluate
without prior isolation of the insulin.
A further series of tests were made using a milder
immunization procedure from which the initial stimulation
of the immunity instrument with killed bacteria had been ex-
cluded, i.e. the injected preparations were in all cases
emulsified in Freund's Incomplete Adjuvant. The rabbits
were injected every 10th day with a constant dose of 20 i.u.
The insulin antibody formation was examined at
intervals of 10 days using a newly developed method described
- 16 -
10~ 9
below.
The obtained r~ults are illustrated in Figs. 4, 5
and 6.
It will be seen from the results that it was not
possible to show any formation of antibody to the new NPH
bovine insulin prepared according to the invention.
P_ Method for Antibody Determination
100 ~1 rabbit serum, 100 ,ul 125I-insulin, about
2/uU/ml, lOO~ul insulin solution, 250~uU/ml and 700 ~1 phos-
phate buffer, 0.04 M pH 7.4 with 0.15 M NaCl and 0.5 %
human albumin was incubated for 48 hours at 4C. 500,ul PEG
6000 was added, 360 g/l mixed on rotamixer and the sample
was left to stand for one hour at 20C.
~ fter centrifuging for 10 minutes at 3000 r/min.the supernatant was decanted off and scrapped, 125I in pre-
cipitate was counted.
In each test is included a sample with an excess of
guinea pig antibovine insulin, max.bonding, and a sample with
rabbit serum from non-immunized rabbits, 0-sample. The re-
sults were calculated as follows:
O/~ = To . 100
T -T
wherein T = counting number for unknown,
To = counting number of 0-sample,
TM = counting number for max.bonding
Literature
1) Hunter, R.: Standardization of the chloramine T method
of protein iodination.
Proc.Soc.Exp.Biol.Med. 133 (1970) 989~
2) Desbuquois, B & Aurbach, G.D.: Use of polyethylene glycol
to separate free and antibody-bound peptide hormones in
radioimmunoassays.
- 17 -
J.Clim.Endocrinol. 33 (1971) 732.
3) Gennaro, W.D. & Van Normone, J.D.: Quantitation of free,
total and antibody-bound insulin in insulin-treated diabetics.
Clin.Chem. 21/7 (1975) 873.
4) Vinik, A.I., Jaffe, B.I., Seftel, H.C., Distiller, L.A.
& Jackson, W.P.V.: Clinical aspects of monocomponent insul-
ins in the treatment of diabetes.
S.A. Medical Journal, April (1976) 587.
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