CA1094549A - Therapeutic insulin preparation and a process for the production of a stable insulin preparation with protracted effect - Google Patents
Therapeutic insulin preparation and a process for the production of a stable insulin preparation with protracted effectInfo
- Publication number
- CA1094549A CA1094549A CA243,587A CA243587A CA1094549A CA 1094549 A CA1094549 A CA 1094549A CA 243587 A CA243587 A CA 243587A CA 1094549 A CA1094549 A CA 1094549A
- Authority
- CA
- Canada
- Prior art keywords
- insulin
- process according
- basic
- aqueous solution
- protamine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
- C07K14/625—Extraction from natural sources
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
ABSTRACT OF THE DISCLOSURE
Novel stable insulin preparation with protracted action and low antigenicity is obtained by reacting insulin in stabilized monomer or loosely aggregated form with an organic compound having basic groups, preferably a basic polypeptide or a cleavage product of a basic polypeptide, such as protamine.
Novel stable insulin preparation with protracted action and low antigenicity is obtained by reacting insulin in stabilized monomer or loosely aggregated form with an organic compound having basic groups, preferably a basic polypeptide or a cleavage product of a basic polypeptide, such as protamine.
Description
~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-
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-
2'~
lO~S~'lL9 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.
., , ~O~S~9 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-
lO~S~'lL9 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.
., , ~O~S~9 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-
-3-~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.
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.
-4-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.
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
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.
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.
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.
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.
~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.
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.
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 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~
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.
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.
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.
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.
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 -8a-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 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~
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.
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.
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-10~l5~9 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 lO~S'19 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 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.
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 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 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.
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.
- 18 ~
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.
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.
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.
~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.
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.
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 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~
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.
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.
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.
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.
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 -8a-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 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~
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.
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.
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-10~l5~9 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 lO~S'19 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 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.
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 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 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.
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.
- 18 ~
Claims (20)
1. In a process for producing a stable insulin prepara-tion with protracted action, wherein insulin is purified and then is reacted with an organic base containing amino groups in an aqueous medium maintained at a pH of about 6 to about 9, and the insulin preparation is isolated, the improvement comprising utilizing for said reaction with said organic base a medium containing a protein depolymerizing or protein dissociating stabilizer to maintain said purified insulin in a dissolved monomeric or loosely aggregated form during said reaction, thereby producing a stable insulin preparation with protracted action and reduced or no antigenicity.
2. A process according to claim 19 characterized in that the insulin used is bovine insulin.
3. A process according to claim 1, characterized in that the organic compound comprising amino groups is a basic polypeptide or a cleavage product of a basic polypeptide.
4. A process according to claim 3, characterized in that the basic polypeptide is a protamine.
5. A process according to claim 1, characterized in that the medium is a buffered aqueous solution of urea.
6. A process according to claim 1, characterized in that the medium is a buffered aqueous solution containing about 7 mol urea, and that the reaction product is precipita-ted by dilution of the reaction mixture.
7. A process for the production of a stable insulin preparation with protracted action and low antigenicity, characterized in that a solution of insulin containing a stabilizer is subjected to ion exchange by elution with an eluent containing a protein dissociating or protein depoly-merizing stabilizer that maintains the insulin in stabilized monomer or loosely aggregated form, whereupon a fraction containing insulin 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.
8. A process according to claim 7, characterized in that the insulin used is bovine insulin.
9. A process according to claim 7, characterized in that the eluent is a buffered aqueous solution of urea.
10. A process according to claim 7, characterized in that the eluent is a buffered aqueous solution containing 7 mol urea, and the reaction product is precipitated by dilution of the reaction mixture.
11. A process according to claim 7, characterized in that the basic polypeptide is selected from the group consisting of polyarginine, somatostatin, protamine and globin.
12. A process according to claim 7, characterized in that the basic polypeptide is protamine.
13. Directly prepared low antigenic insulin bound to an organic base containing amino groups and exhibiting no detectable antibody formation, when tested by standard procedures comprising injection into rabbits with adjuvants, whenever prepared by the process of claim 1 or an obvious chemical equivalent thereof.
14. Directly prepared low antigenic insulin bound to a basic polypeptide or a cleavage product of a basic poly-peptide whenever prepared by the process of claim 3 or claim 7 or an obvious chemical equivalent thereof.
CLAIMS SUPPORTED BY THE SUPPLEMENTARY DISCLOSURE
CLAIMS SUPPORTED BY THE SUPPLEMENTARY DISCLOSURE
15. A process according to claim 1, wherein the medium is a buffered aqueous solution containing a compound selected from the group consisting of a lower alkanol, dialkylforma-mide, acetamide, N-alkyl-acetamide and acetonitrile.
16. A process according to claim 7, wherein the eluent is a buffered aqueous solution of a compound selected from the group consisting of a lower alkanol, dialkyl-formamide, acetamide, N-alkyl-acetamide and acetonitrile.
17. A process according to claim 16, wherein the eluent is an aqueous buffered solution containing 50-70%
lower alkanol.
lower alkanol.
18. A process according to claim 17, wherein the lower alkanol is ethanol.
19. Directly prepared low antigenic insulin bound to a basic amine exhibiting no detectable antibody formation when tested by (1) injection into rabbits first with Freund's complete adjuvant then injection with Freund's incomplete adjuvant and (2) measuring [insulin-antibody complexing by the method] of Ortved-Andersen et al [Acta Endocr.(Kbh.) 69, 195-208, 1972], whenever prepared by the process of claims 1, 15 or 16 or an obvious chemical equivalent thereof.
20. Directly prepared low antigenic insulin bound to a basic amine exhibiting no detectable antibody formation when tested by (1) injection into rabbits with Freund's incomplete adjuvant and (2) measuring insulin antibody formation by treating the serum of said injected rabbits with I125 insulin and measuring any of bound I125 insulin by precipitation with PEG 6000, whenever prepared by the process of claims 1, 15 or 16 or an obvious chemical equivalent thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK8375AA DK140801B (en) | 1975-01-15 | 1975-01-15 | Process for the preparation of a stable long-acting insulin preparation. |
| DK83/75 | 1975-01-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1094549A true CA1094549A (en) | 1981-01-27 |
Family
ID=8089535
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA243,587A Expired CA1094549A (en) | 1975-01-15 | 1976-01-12 | Therapeutic insulin preparation and a process for the production of a stable insulin preparation with protracted effect |
Country Status (36)
| Country | Link |
|---|---|
| JP (1) | JPS5946939B2 (en) |
| AR (1) | AR213088A1 (en) |
| AT (1) | AT362492B (en) |
| BE (1) | BE837600A (en) |
| BR (1) | BR7600206A (en) |
| CA (1) | CA1094549A (en) |
| CH (1) | CH631625A5 (en) |
| CS (1) | CS208147B2 (en) |
| DD (1) | DD124379A5 (en) |
| DE (1) | DE2600971C2 (en) |
| DK (1) | DK140801B (en) |
| EG (1) | EG11988A (en) |
| ES (1) | ES444558A1 (en) |
| FI (1) | FI66751C (en) |
| FR (1) | FR2297634A1 (en) |
| GB (1) | GB1524431A (en) |
| GR (1) | GR58608B (en) |
| HU (1) | HU175142B (en) |
| IE (1) | IE42395B1 (en) |
| IL (1) | IL48845A (en) |
| IN (1) | IN143279B (en) |
| IT (1) | IT1054211B (en) |
| KE (1) | KE2919A (en) |
| LU (1) | LU74175A1 (en) |
| MX (1) | MX4052E (en) |
| MY (1) | MY7900169A (en) |
| NL (1) | NL166464C (en) |
| NO (1) | NO146698C (en) |
| NZ (1) | NZ179733A (en) |
| OA (1) | OA05209A (en) |
| PL (1) | PL99927B1 (en) |
| PT (1) | PT64696B (en) |
| RO (1) | RO71576A (en) |
| SE (1) | SE437219B (en) |
| YU (1) | YU8376A (en) |
| ZA (1) | ZA7658B (en) |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9545487B2 (en) | 2012-04-13 | 2017-01-17 | Boehringer Ingelheim International Gmbh | Dispenser with encoding means |
| US9682202B2 (en) | 2009-05-18 | 2017-06-20 | Boehringer Ingelheim International Gmbh | Adapter, inhalation device, and atomizer |
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| US9757750B2 (en) | 2011-04-01 | 2017-09-12 | Boehringer Ingelheim International Gmbh | Medicinal device with container |
| US9827384B2 (en) | 2011-05-23 | 2017-11-28 | Boehringer Ingelheim International Gmbh | Nebulizer |
| US9943654B2 (en) | 2010-06-24 | 2018-04-17 | Boehringer Ingelheim International Gmbh | Nebulizer |
| US10004857B2 (en) | 2013-08-09 | 2018-06-26 | Boehringer Ingelheim International Gmbh | Nebulizer |
| US10011906B2 (en) | 2009-03-31 | 2018-07-03 | Beohringer Ingelheim International Gmbh | Method for coating a surface of a component |
| US10016568B2 (en) | 2009-11-25 | 2018-07-10 | Boehringer Ingelheim International Gmbh | Nebulizer |
| US10099022B2 (en) | 2014-05-07 | 2018-10-16 | Boehringer Ingelheim International Gmbh | Nebulizer |
| US10124129B2 (en) | 2008-01-02 | 2018-11-13 | Boehringer Ingelheim International Gmbh | Dispensing device, storage device and method for dispensing a formulation |
| US10124125B2 (en) | 2009-11-25 | 2018-11-13 | Boehringer Ingelheim International Gmbh | Nebulizer |
| US10195374B2 (en) | 2014-05-07 | 2019-02-05 | Boehringer Ingelheim International Gmbh | Container, nebulizer and use |
| US10722666B2 (en) | 2014-05-07 | 2020-07-28 | Boehringer Ingelheim International Gmbh | Nebulizer with axially movable and lockable container and indicator |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK374579A (en) * | 1979-09-07 | 1981-03-08 | Nordisk Insulinlab | PROCEDURE FOR THE PREPARATION OF AN INJUCABLE INSULIN PREPARATION |
| US4459226A (en) * | 1982-02-26 | 1984-07-10 | Eli Lilly And Company | Process for recovering insulin |
| US4801575A (en) * | 1986-07-30 | 1989-01-31 | The Regents Of The University Of California | Chimeric peptides for neuropeptide delivery through the blood-brain barrier |
| US4878892A (en) * | 1987-02-10 | 1989-11-07 | Drug Delivery Systems Inc. | Electrolytic transdermal delivery of polypeptides |
| AU609769B2 (en) * | 1987-02-10 | 1991-05-09 | Drug Delivery Systems Inc. | Electrolytic transdermal delivery of proteins |
| US6221367B1 (en) | 1992-06-15 | 2001-04-24 | Emisphere Technologies, Inc. | Active agent transport systems |
| US5629020A (en) | 1994-04-22 | 1997-05-13 | Emisphere Technologies, Inc. | Modified amino acids for drug delivery |
| US5714167A (en) | 1992-06-15 | 1998-02-03 | Emisphere Technologies, Inc. | Active agent transport systems |
| US5447728A (en) | 1992-06-15 | 1995-09-05 | Emisphere Technologies, Inc. | Desferrioxamine oral delivery system |
| US5643957A (en) | 1993-04-22 | 1997-07-01 | Emisphere Technologies, Inc. | Compounds and compositions for delivering active agents |
| US6051258A (en) | 1995-06-07 | 2000-04-18 | Emisphere Technologies, Inc. | Proteinoid emulsions and methods for preparation and use thereof |
| US5750147A (en) | 1995-06-07 | 1998-05-12 | Emisphere Technologies, Inc. | Method of solubilizing and encapsulating itraconazole |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1285023A (en) * | 1968-08-09 | 1972-08-09 | Novo Terapeutisk Labor As | Improvements in or relating to injectable insulin preparations |
| US3758683A (en) * | 1971-04-30 | 1973-09-11 | R Jackson | Insulin product |
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1975
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1976
- 1976-01-06 ZA ZA00760058A patent/ZA7658B/en unknown
- 1976-01-07 GR GR49734A patent/GR58608B/en unknown
- 1976-01-07 IN IN41/CAL/76A patent/IN143279B/en unknown
- 1976-01-12 FI FI760058A patent/FI66751C/en not_active IP Right Cessation
- 1976-01-12 CA CA243,587A patent/CA1094549A/en not_active Expired
- 1976-01-12 NZ NZ179733A patent/NZ179733A/en unknown
- 1976-01-13 DE DE2600971A patent/DE2600971C2/en not_active Expired
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- 1976-01-13 LU LU74175A patent/LU74175A1/xx unknown
- 1976-01-13 IE IE62/76A patent/IE42395B1/en unknown
- 1976-01-13 IT IT19221/76A patent/IT1054211B/en active
- 1976-01-14 AT AT21376A patent/AT362492B/en not_active IP Right Cessation
- 1976-01-14 OA OA55703A patent/OA05209A/en unknown
- 1976-01-14 PT PT64696A patent/PT64696B/en unknown
- 1976-01-14 RO RO7684491A patent/RO71576A/en unknown
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- 1976-01-14 NO NO760118A patent/NO146698C/en unknown
- 1976-01-14 BR BR7600206A patent/BR7600206A/en unknown
- 1976-01-14 PL PL1976186526A patent/PL99927B1/en unknown
- 1976-01-14 DD DD190805A patent/DD124379A5/xx unknown
- 1976-01-14 FR FR7600854A patent/FR2297634A1/en active Granted
- 1976-01-14 GB GB1309/76A patent/GB1524431A/en not_active Expired
- 1976-01-14 MX MX764822U patent/MX4052E/en unknown
- 1976-01-14 NL NL7600338.A patent/NL166464C/en not_active IP Right Cessation
- 1976-01-15 CS CS76270A patent/CS208147B2/en unknown
- 1976-01-15 HU HU76NO198A patent/HU175142B/en unknown
- 1976-01-15 ES ES444558A patent/ES444558A1/en not_active Expired
- 1976-01-15 BE BE163542A patent/BE837600A/en not_active IP Right Cessation
- 1976-01-15 IL IL48845A patent/IL48845A/en unknown
- 1976-01-15 CH CH47076A patent/CH631625A5/en not_active IP Right Cessation
- 1976-01-16 JP JP51003293A patent/JPS5946939B2/en not_active Expired
- 1976-01-17 EG EG16/76A patent/EG11988A/en active
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1978
- 1978-01-14 AR AR261924A patent/AR213088A1/en active
-
1979
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- 1979-12-30 MY MY169/79A patent/MY7900169A/en unknown
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| US10011906B2 (en) | 2009-03-31 | 2018-07-03 | Beohringer Ingelheim International Gmbh | Method for coating a surface of a component |
| US9682202B2 (en) | 2009-05-18 | 2017-06-20 | Boehringer Ingelheim International Gmbh | Adapter, inhalation device, and atomizer |
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