CA1162868A - Process for preparing insulin esters - Google Patents
Process for preparing insulin estersInfo
- Publication number
- CA1162868A CA1162868A CA000370483A CA370483A CA1162868A CA 1162868 A CA1162868 A CA 1162868A CA 000370483 A CA000370483 A CA 000370483A CA 370483 A CA370483 A CA 370483A CA 1162868 A CA1162868 A CA 1162868A
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- insulin
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- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Endocrinology (AREA)
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- General Health & Medical Sciences (AREA)
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- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Forklifts And Lifting Vehicles (AREA)
Abstract
ABSTRACT
Insulin and insulin-like compounds from certain species can in the presence of an L- threonine ester, water, a water miscible organic solvent, and trypsin be transpeptidized into a threonineB30 ester of human insulin in which the protecting group(s) can be removed whereby human insulin is formed.
Insulin and insulin-like compounds from certain species can in the presence of an L- threonine ester, water, a water miscible organic solvent, and trypsin be transpeptidized into a threonineB30 ester of human insulin in which the protecting group(s) can be removed whereby human insulin is formed.
Description
~L~62~
This invention relates to the conversion o insulin and insulin-like compounds into human insulin.
In the treatment of diabetes mellitus insulin preparations derived from porcine or bovlne insulin have generally been used. Bovine, poxcine, and human insulins exhibit minor differences with respect to their amino acid composition, the difference between human and porcine insulin being confined to a single amino acid in that the B30 amino acid of human insul'in is threonine whereas that of porcine insUlinisalanine However, it could be argued that the ideal insulin preparation for treatment of human beings would be an insul'in having exactly the same chemical structure as that of human insulin.
For the production of natural human insulin - the necessary amount of human pancreas glands i~ not ; available.
'Synthetic human insulin has been prepared ~n a small scale at great expense, vide Helv.Chi~.Acta 57, 2617, and 60, 27. Semisynthetic human insulin has been prepared from porcine insulin by tedious path-ways, vide Hoppe-Seyler's Z.~hysiol.Chem. 357, 759, and Nature 280, 412. However, the present invention relates to a process which can be used for preparing human insulin on an industrial scaleO ~
.,. . '''~
'
This invention relates to the conversion o insulin and insulin-like compounds into human insulin.
In the treatment of diabetes mellitus insulin preparations derived from porcine or bovlne insulin have generally been used. Bovine, poxcine, and human insulins exhibit minor differences with respect to their amino acid composition, the difference between human and porcine insulin being confined to a single amino acid in that the B30 amino acid of human insul'in is threonine whereas that of porcine insUlinisalanine However, it could be argued that the ideal insulin preparation for treatment of human beings would be an insul'in having exactly the same chemical structure as that of human insulin.
For the production of natural human insulin - the necessary amount of human pancreas glands i~ not ; available.
'Synthetic human insulin has been prepared ~n a small scale at great expense, vide Helv.Chi~.Acta 57, 2617, and 60, 27. Semisynthetic human insulin has been prepared from porcine insulin by tedious path-ways, vide Hoppe-Seyler's Z.~hysiol.Chem. 357, 759, and Nature 280, 412. However, the present invention relates to a process which can be used for preparing human insulin on an industrial scaleO ~
.,. . '''~
'
2 i~28~
US p~tent specification ~o. 3,276,961 purports to relate to a process for preparin~ semisynthetic human insu-lin. l1cwever, the ~ield of human insulin is poor because the process is performed in water, under which conditions trypsin causes a splitting of the Arg - Gl~ bond, vide J. Biol.Chem. 236, 743.
A known semisynthetic process for preparing human insulin comprises the following three steps:
First, porcine insulin is converted into porcine des-(AlaB30)-insulin by treatment with carboxypeptidase A, vide Hoppe-S~yler's A.Physiol.Chem. 3S9, 799. In the second step porcine des-(t~laB3C)-insulin is subjec ted to a trypsin-catalysed coupling with Thr-OBut, whereby human insulin ThrB30-tert-butyl ester is formed.
Finally, said ester is treated~with triflouroacetic acid yielding human insulin, vide Nature 280, 412. The first step, however, results in a partial removal of Asn , yielding des-(~la ~AsnA21)-insulinO This derivative gives, after the two subsequent reactions, rise to a contamination of des-(AsnA21)-insulin in the semisyntetic human insulin prepared, a contamination which cannot easily be removed with known preparative methods.
Des-(AsnA2~ sulin L~ossesses low biological activity ~about 5%), vide Amer.J.Med. 40, 750.
One object of this invention is to provide a process which converts a non-human insulir.
in~o human insulin.
.
A second object of this invention is to provide a process which converts porcine insulin and certain impurities therein into human insulin via a threonineB30 ester o~ human insulin.
Further objects and the advantages of this invention will be apparent in this description.
~L6~
This invention is based upon the discovery that the amino acid or peptide chain bound to the carbo-nyl group of LysB29 in the insulin compound can be inter-changed with a threonine ester. Said interchange is herein referred to as a transpeptidation.
The term "insulin compounds" as used herein encompasses insulins and insulin-like compounds con~
taining the human des~ThrB30)insulin moiety, the B30 amino acid of-the insulin being alanine (in insulin ~rom, e.g., hog, dog, and fin and sperm whale) or serine (rabbit~. The term "insulin-like compounds" as used herein encompasses proinsulin derived from any of the above species and primates, together with intermediates from the conversion of proinsulin into insulin. As examples of such intermediates can be mentioned split proinsulin, desdipeptide proinsulins, desnonapeptide pro-insulin, and diarginine insulins, vide R. Chance: In Proceedings of the Seventh Congress of IDF, Buenos Aires 1970, 292 - 305, Editors: R.R. Rodrig~es & J.Y.-Owen, Excerpta Medica, Amsterdam.
The process according to this invention comprises transpeptidizing an insulin compound or a salt or complex thereof with an L-threonine ester or a salt thereof in a mixture of water, a water miscible organic solvent, and trypsin, the content of water in the reaction mixture being less than about 50 per cent (volume/volume) and the reaction temperature being below about 50 C;
~. ~
Although trypsin is best known for its proteolytic properties, workers in the art have recognized that trypsin is capable of catalyzing the coupling of des-(AI.aB3a) -insulin and a threonine~tert-butyl ester, vide Nature 280, 41~.
In the present process the trypsin is used to catalyze transpeptidation.
!
The transpeptidation can be performed by dissolving 1~ the insulin compound, 2) an L-threonine ester, and 31 trypsin in a mixture of water and at least one wa~er miscible organic solvent, optionally in the ~resence of an acid.
Preferred water miscible orqanic solvents are polar solvents. As specific examples of such solvents can be mentioned methanol, ethanol, 2-propanol/ 1,2-ethan-~ diol, acetone, di.oxane, tetrahydrofuran, N,N-dimethylfor-mamide, formamide, N,N-dimethylacetamide, N-methylpyrro-lidone, hexamethylphosphortriamide, and acetonitrile.
Depending on which water miscible organic solvent is used, on the chosen reaction temperature, and on the presence oE an acid in the reaction mixture, the content of water in the reaction mixture should be less than about 50 per cent (v/v), preferably less than about 40 per cent (v/v), and more than 10 per cent (v/v).
On advantage of decreasing the amount of water in the reaction mixture is that the formation of byproducts is thereby decreased. Similarly, by increasing the amount of acid in the reaction mixture it is possible to decrease the byproduct formation. The increase in yield is positive correlated to a high percentage of organic solvent.
The type of trypsin used is not material to the practice of this invention. Trypsin is a well characterized enzyme which is commercially available in high purity, notably of bovine and porcine source.
Furthermore, trypsin of microbial origin may be used.
Moreover, the trypsin form, e.g., crystalline trypsin (soluble form), immobilized trypsin or even trypsin derivatives (so long as the trypsin activity is retained) is not material to practice of this invention. The term trypsin as employed herein is intended to include trypsins from all sources and all forms of trypsin that retain the transpeptidating activity, including proteases with trypsin-like specificity, e.g. Acromobacter lyticus protease, vide Agric.Biol.Chem. 42, 1443.
1~
62~8 As examples of active trypsin derivatives can be mentioned acetylated trypsin, succ:inylated trypsin, glutaraldehyde treated trypsin, and immo~ilized trypsin derivatives.
If an immobilized trypsin is used it is suspended in the medium.
Organic or inorganic acids such as hydrochloric acid, forric aci~, acetic aci~, propionic acid, and butyric acid, or bases such as pyridine, TRIS, N-methylmorpholine, and N-ethylmorpholine may be added to bring about a suitab]e buffer system. Oryanic acids are preferred. However, the reaction can be conducted without such additions. The amount of acid added is usually less than about 10 equivalents per equivalent of the L-threonine ester. Preferably, the amount of acid is between 0.5 and 5 equivalents per equivalent of the L-threonine ester. Ions, which stabilize trypsin such as calcium ions, may be added.
The process may be performed at a tempera-ture in the range between 50 C and the freezing point of the reaction mixture.
Enzymatic reactions with trypsin are usually performed at - about 37 C in order to give a sufficient reaction rate, However, in order to avoid inactivation of trypsin - it is advantageous to perform the process according to the present invention at a temperature below ambient. In practice reaction temperatures above about 0 C are preferred.
. .. .. .
7 1~2~6~
The reaction time is usually between several hours and several days, depending upon the reaction tempera-ture, upon the amount of trypsin added, and upon other reaction conditions.
The weight ratio between trypsin and the insu-lin compound in the react:ion mixture is normally above about 1:200, preferably ahove about 1:50, and below about 1 : 1 .
The L-threonine esters contemplated for practice of this invention can be depicted by the general formula Thr(R5)-OR4 II
wherein R represents a carboxyl protecting group, and R represents hydrogen or a hydroxyl protecting group, or a salt thereof.
The threonineB30 esters of human insulin resulting from the transpeptidation can be depicted by the general formula ; (Thr(R5)-oR4)B3o-h-In III
wherein h-In represents the human des-(Thr 30~.insulin moiety, and R4 and R5 are as defined above.
... .
~1~ii2~
Applicable L-threonine esters of formula II
are those in which R~ is a carboxyl protectinq group which can be removed from the threonineB30 ester of ~uman insulin -(formula III) under conditions which do not cause substantia irreversible alteration in the insulin molecule. As examples of such carboxyl pxotecting groups can be mentioned lower aLkyl, e.g., methyl, ethyl, and tert-butyl, substituted benzyl such as p-methoxybenzyl and 2,4,6-trimethylbenzyl and diphenyl-methyl, and groups of the general formula - CH2CH2SO2R6, where~n R represents lower al~yl such as methyl, ethyl, propyl, and n-butyl. Cuitable hydroxyl Drotecting grouPs R5 are thoSe which can be removed from the threonineB30 ester of human insulin (formula III) under conditions which do not cause substantial irreversible alteration in the insulin molecule. As an example of such a group (R5) can be mentioned tert-butyl.
Lower al~yl groups contain less than 7 carbon atoms, preferably less than S carbon atoms.
Further protection groups commonly used are described by Wunsch Methoden der Organischen Chemie (Houben-Weyl), Vol. XV/l, editor: Eugen Muller, Georg Thieme Verlag, Stuttgart 1974.
~2~
Some L-threonine esters (formula II) are known compounds and the remaining L-threonine esters (formula II) can be prepared in analogy with the preparation of known compounds.
The L-threonine esters of formula II may be the free bases or suitable organic or inorganic salts thereof preferably acetates, propionates, butyrates, and hydrohalides such as hydrochlorides.
It is desirable to use the reactants, i.e. the insulin compound and the L-threonine ester (formula II), in high concentrations. The molar ratio between the L-threonine ester and the insulin compound is preferably above about 5:1, most preferably from about 5:1 to about 1000:1, and optimally from about 20:1 to about 250:1.
It is desirable that the concentration of L-threonine ester (formula II) in the reaction mixture exeed 0.1 molar.
Human insulin can be obtain from the threonine esters of human insulin (formula III) by removal of the protecting group R and any protecting group R by known methods known ~ se. In case R4 is methyl, ethyl, or a group -C~H2S02R6, wherein R6 is as defined above, the said protecting group can be - removed at gentle basic conditions in an aqueous medium, preferably at a pH value of about 8 - 121 e.q. at about 9.5. As the base can be used ammonia, triethylamine, or hydroxides of alkali metals such as sodium hydroxide.
In case R~ is tert-butyl, substituted benzyl such as p-metho~ybenzyl or 2,4,6--trimethylbenzyl or diphenyl-methyl the said group can be removed by acidolysis, preferably with trifluoroacetic acid. The trifluoro-acetic acid may be non-aqueous or may contain some water, - or it may be diluted with an organic solvent such as dichloromethane. In case R5 is tert-butyl said group can be removed by acidolysis, vide above.
Preferred threonineB30 esters of human insulin of the formula III are compounds, wherein R5 is hydrogen, and these are prepared from L-threonine esters of the formula II, wherein R5 is hydrogen.
The transpeptidation of insulin compounds ~into threonine esters of human insulin can be described in more detail on the basis of the following formula assigned to the insulin compounds:
- Rl-tl-----A-----21) (l___B~__2g)-R2 (I) wherein . ............................ ~
~L~L6%53~
A---21) (l---B---29)~
represents the human des(ThrB30)insulin moiety wherein GlyAl is connected to the substituent designated ~ and LysB29 is connected to the substituent designated R2, R2 represents an amino acid or a peptide chain containing not more than 36 amino acids, and Rl repre-sents hydrogen or a ~roup of the general formula R -X-, wherein X represents arginine or lysine, and R3 represents a peptide chain containing not more than 35 amino acids, or R2 together with R3 represent a peptide chain containing not more than 35 amino acids, with the proviso that the number of amino acids present in Rl plus R2 is less than 37.
Thus, the transpeptidation of this invention converts any of the above insulin compounds into threonineB30 esters of human insulin (formula III), which then can be deblocked to form human insulin.
:
8~
A further advantage of this inventiol- is that insulin-lik~ compounds present in crude insulin and present in some commercial insulin preparations and covered by the formula I by the transpeptidation of this invention are convert~d into threonineB30 esters of human insulin, which then can be deblocked to form human insulin.
Examples of insulin-like compounds of formula I
appear from the following:
Porcine diar~inine insulin ;Rl is hydro~en, and R is -Ala-Arg-Arg), porcine proinsulin(R3 toaether.with R2 is -Ala-Arg-Arg-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala--Val-Glu-Leu-Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu-~ila--Leu-Glu-Gly-Pro-Pro-Gln-Lys-, wherein the terminal alanyl is connected to LysB29~ do~.proinsulin ~3 together with R2 is -Ala-Arg-Arg-Asp-Val-~lu-Leu-Ala-Gly-Ala-Pro-Gly--Glu-Gly-Gly-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ala-Leu-Gln--Lys-, wherein the terminal alanyl is connected to LysB29~, porcine split proinsulin (R is Ala-Leu-Glu-Gly-Pro-Pro-Gln-Lys-, and R is -Ala--Arg-Ar~-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala-Val-Glu-Leu--Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu), porcine desdipeptide proinsulin (Rl is hydro~en, and R2 is -Ala-Arg-Arg-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala-Val--Glu-Leu-Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu-Ala--Leu-Glu-Gly-Pro-Pro-Gln), human proinsulin (R to~ather with R is -Thr-Arg-Arg-Glu-Ala-Glu-Asp~Leu~Gln-Val-Gly--Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu--Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-, wherein the terminal threonyl is connected to LysB29), and monkey proinsulin (R3 toqether with R2 is -Thr-Ar~-Ar~--Glu-Ala-Glu-As~-Pro-Gln-Val-Glv-Gln-Val-Glu-Leu-Gly--Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu--Glu-Gly-Ser-Leu-Gln-Lys-~ wherein the ter~inal threonyl is connected to ~ysB29.
2B~
: 13 Hence, in all these impurities covered by formula I
the R substituent designated R3-X- is exchanged with hydrogen.
A preferred embodiment of the present invention comprises reacting crude porcine insulin containing insulin-like compounds or a salt or complex thereof with an L-threonine ester (formula II) or a salt thereof under the above conditions whereafter the protecting group R4 and any protecting group R5 is removed. By this process the porcine insulin together with insulin-like compounds therein is converted into human insulin.
As examples of a complex or a salt of an insulin compound (formula I) can be mentioned a zinc complex or zinc salt.
When selecting the reaction conditions according to the above explanation and considering the results obtained in the following examples it is possible to obtain a yield of threonineB30 ester of human insulin which is higher than 60 per cent, and even higher than 80 per cent, and under certain preferred conditions higher than 90 per cent.
3Li~2~
The process according to the present invention has, therefore, the followincJ advantagcs over the prior art:
a) The enzymatic hydrolysis to remove Alas30 e.g., with carboxypeptidase A, ls omitted.
b) The isolation of an intermediate compound, such as porcine des-(AlaB30)-insulin, is unnecessary.
c) Contamination with des(Asn l)-insulin deriv~tives is avoided.
d) Proinsulin and other insulin-like im-purities present in crude insulin are - via the threonineB30 ester of human insulin -converted into human insulin by the process of this invention, whereby the yield is increased.
. .
el Antigenic insulin-like compounds, vide British Patent No. 1,285,023, are converted into numan ir.sulin.
A preferred procedure for preparing human insulin is as follows:
1) The starting material used for the transpeptidation is crude porcine insulin, e.g., crystalline insulin obtained by the use o~ a citrate buffer , vi~e Patent Specification No. U.S.A.
2,626,228.
2) If there is any trypsin activity left after the transpeptidation, it is pre~erred to remove it, e.g., under conditions where trypsin is inactive, e.g., in acid medium below pH 3. Trypsin can be removed by separation according to molecular weight, e.g., by gel filtration on "Sephadex G-50~ or "~io-gel P-3U~
in lM acetic acid, vide Nature 280, 412.
* Trade Mark B
US p~tent specification ~o. 3,276,961 purports to relate to a process for preparin~ semisynthetic human insu-lin. l1cwever, the ~ield of human insulin is poor because the process is performed in water, under which conditions trypsin causes a splitting of the Arg - Gl~ bond, vide J. Biol.Chem. 236, 743.
A known semisynthetic process for preparing human insulin comprises the following three steps:
First, porcine insulin is converted into porcine des-(AlaB30)-insulin by treatment with carboxypeptidase A, vide Hoppe-S~yler's A.Physiol.Chem. 3S9, 799. In the second step porcine des-(t~laB3C)-insulin is subjec ted to a trypsin-catalysed coupling with Thr-OBut, whereby human insulin ThrB30-tert-butyl ester is formed.
Finally, said ester is treated~with triflouroacetic acid yielding human insulin, vide Nature 280, 412. The first step, however, results in a partial removal of Asn , yielding des-(~la ~AsnA21)-insulinO This derivative gives, after the two subsequent reactions, rise to a contamination of des-(AsnA21)-insulin in the semisyntetic human insulin prepared, a contamination which cannot easily be removed with known preparative methods.
Des-(AsnA2~ sulin L~ossesses low biological activity ~about 5%), vide Amer.J.Med. 40, 750.
One object of this invention is to provide a process which converts a non-human insulir.
in~o human insulin.
.
A second object of this invention is to provide a process which converts porcine insulin and certain impurities therein into human insulin via a threonineB30 ester o~ human insulin.
Further objects and the advantages of this invention will be apparent in this description.
~L6~
This invention is based upon the discovery that the amino acid or peptide chain bound to the carbo-nyl group of LysB29 in the insulin compound can be inter-changed with a threonine ester. Said interchange is herein referred to as a transpeptidation.
The term "insulin compounds" as used herein encompasses insulins and insulin-like compounds con~
taining the human des~ThrB30)insulin moiety, the B30 amino acid of-the insulin being alanine (in insulin ~rom, e.g., hog, dog, and fin and sperm whale) or serine (rabbit~. The term "insulin-like compounds" as used herein encompasses proinsulin derived from any of the above species and primates, together with intermediates from the conversion of proinsulin into insulin. As examples of such intermediates can be mentioned split proinsulin, desdipeptide proinsulins, desnonapeptide pro-insulin, and diarginine insulins, vide R. Chance: In Proceedings of the Seventh Congress of IDF, Buenos Aires 1970, 292 - 305, Editors: R.R. Rodrig~es & J.Y.-Owen, Excerpta Medica, Amsterdam.
The process according to this invention comprises transpeptidizing an insulin compound or a salt or complex thereof with an L-threonine ester or a salt thereof in a mixture of water, a water miscible organic solvent, and trypsin, the content of water in the reaction mixture being less than about 50 per cent (volume/volume) and the reaction temperature being below about 50 C;
~. ~
Although trypsin is best known for its proteolytic properties, workers in the art have recognized that trypsin is capable of catalyzing the coupling of des-(AI.aB3a) -insulin and a threonine~tert-butyl ester, vide Nature 280, 41~.
In the present process the trypsin is used to catalyze transpeptidation.
!
The transpeptidation can be performed by dissolving 1~ the insulin compound, 2) an L-threonine ester, and 31 trypsin in a mixture of water and at least one wa~er miscible organic solvent, optionally in the ~resence of an acid.
Preferred water miscible orqanic solvents are polar solvents. As specific examples of such solvents can be mentioned methanol, ethanol, 2-propanol/ 1,2-ethan-~ diol, acetone, di.oxane, tetrahydrofuran, N,N-dimethylfor-mamide, formamide, N,N-dimethylacetamide, N-methylpyrro-lidone, hexamethylphosphortriamide, and acetonitrile.
Depending on which water miscible organic solvent is used, on the chosen reaction temperature, and on the presence oE an acid in the reaction mixture, the content of water in the reaction mixture should be less than about 50 per cent (v/v), preferably less than about 40 per cent (v/v), and more than 10 per cent (v/v).
On advantage of decreasing the amount of water in the reaction mixture is that the formation of byproducts is thereby decreased. Similarly, by increasing the amount of acid in the reaction mixture it is possible to decrease the byproduct formation. The increase in yield is positive correlated to a high percentage of organic solvent.
The type of trypsin used is not material to the practice of this invention. Trypsin is a well characterized enzyme which is commercially available in high purity, notably of bovine and porcine source.
Furthermore, trypsin of microbial origin may be used.
Moreover, the trypsin form, e.g., crystalline trypsin (soluble form), immobilized trypsin or even trypsin derivatives (so long as the trypsin activity is retained) is not material to practice of this invention. The term trypsin as employed herein is intended to include trypsins from all sources and all forms of trypsin that retain the transpeptidating activity, including proteases with trypsin-like specificity, e.g. Acromobacter lyticus protease, vide Agric.Biol.Chem. 42, 1443.
1~
62~8 As examples of active trypsin derivatives can be mentioned acetylated trypsin, succ:inylated trypsin, glutaraldehyde treated trypsin, and immo~ilized trypsin derivatives.
If an immobilized trypsin is used it is suspended in the medium.
Organic or inorganic acids such as hydrochloric acid, forric aci~, acetic aci~, propionic acid, and butyric acid, or bases such as pyridine, TRIS, N-methylmorpholine, and N-ethylmorpholine may be added to bring about a suitab]e buffer system. Oryanic acids are preferred. However, the reaction can be conducted without such additions. The amount of acid added is usually less than about 10 equivalents per equivalent of the L-threonine ester. Preferably, the amount of acid is between 0.5 and 5 equivalents per equivalent of the L-threonine ester. Ions, which stabilize trypsin such as calcium ions, may be added.
The process may be performed at a tempera-ture in the range between 50 C and the freezing point of the reaction mixture.
Enzymatic reactions with trypsin are usually performed at - about 37 C in order to give a sufficient reaction rate, However, in order to avoid inactivation of trypsin - it is advantageous to perform the process according to the present invention at a temperature below ambient. In practice reaction temperatures above about 0 C are preferred.
. .. .. .
7 1~2~6~
The reaction time is usually between several hours and several days, depending upon the reaction tempera-ture, upon the amount of trypsin added, and upon other reaction conditions.
The weight ratio between trypsin and the insu-lin compound in the react:ion mixture is normally above about 1:200, preferably ahove about 1:50, and below about 1 : 1 .
The L-threonine esters contemplated for practice of this invention can be depicted by the general formula Thr(R5)-OR4 II
wherein R represents a carboxyl protecting group, and R represents hydrogen or a hydroxyl protecting group, or a salt thereof.
The threonineB30 esters of human insulin resulting from the transpeptidation can be depicted by the general formula ; (Thr(R5)-oR4)B3o-h-In III
wherein h-In represents the human des-(Thr 30~.insulin moiety, and R4 and R5 are as defined above.
... .
~1~ii2~
Applicable L-threonine esters of formula II
are those in which R~ is a carboxyl protectinq group which can be removed from the threonineB30 ester of ~uman insulin -(formula III) under conditions which do not cause substantia irreversible alteration in the insulin molecule. As examples of such carboxyl pxotecting groups can be mentioned lower aLkyl, e.g., methyl, ethyl, and tert-butyl, substituted benzyl such as p-methoxybenzyl and 2,4,6-trimethylbenzyl and diphenyl-methyl, and groups of the general formula - CH2CH2SO2R6, where~n R represents lower al~yl such as methyl, ethyl, propyl, and n-butyl. Cuitable hydroxyl Drotecting grouPs R5 are thoSe which can be removed from the threonineB30 ester of human insulin (formula III) under conditions which do not cause substantial irreversible alteration in the insulin molecule. As an example of such a group (R5) can be mentioned tert-butyl.
Lower al~yl groups contain less than 7 carbon atoms, preferably less than S carbon atoms.
Further protection groups commonly used are described by Wunsch Methoden der Organischen Chemie (Houben-Weyl), Vol. XV/l, editor: Eugen Muller, Georg Thieme Verlag, Stuttgart 1974.
~2~
Some L-threonine esters (formula II) are known compounds and the remaining L-threonine esters (formula II) can be prepared in analogy with the preparation of known compounds.
The L-threonine esters of formula II may be the free bases or suitable organic or inorganic salts thereof preferably acetates, propionates, butyrates, and hydrohalides such as hydrochlorides.
It is desirable to use the reactants, i.e. the insulin compound and the L-threonine ester (formula II), in high concentrations. The molar ratio between the L-threonine ester and the insulin compound is preferably above about 5:1, most preferably from about 5:1 to about 1000:1, and optimally from about 20:1 to about 250:1.
It is desirable that the concentration of L-threonine ester (formula II) in the reaction mixture exeed 0.1 molar.
Human insulin can be obtain from the threonine esters of human insulin (formula III) by removal of the protecting group R and any protecting group R by known methods known ~ se. In case R4 is methyl, ethyl, or a group -C~H2S02R6, wherein R6 is as defined above, the said protecting group can be - removed at gentle basic conditions in an aqueous medium, preferably at a pH value of about 8 - 121 e.q. at about 9.5. As the base can be used ammonia, triethylamine, or hydroxides of alkali metals such as sodium hydroxide.
In case R~ is tert-butyl, substituted benzyl such as p-metho~ybenzyl or 2,4,6--trimethylbenzyl or diphenyl-methyl the said group can be removed by acidolysis, preferably with trifluoroacetic acid. The trifluoro-acetic acid may be non-aqueous or may contain some water, - or it may be diluted with an organic solvent such as dichloromethane. In case R5 is tert-butyl said group can be removed by acidolysis, vide above.
Preferred threonineB30 esters of human insulin of the formula III are compounds, wherein R5 is hydrogen, and these are prepared from L-threonine esters of the formula II, wherein R5 is hydrogen.
The transpeptidation of insulin compounds ~into threonine esters of human insulin can be described in more detail on the basis of the following formula assigned to the insulin compounds:
- Rl-tl-----A-----21) (l___B~__2g)-R2 (I) wherein . ............................ ~
~L~L6%53~
A---21) (l---B---29)~
represents the human des(ThrB30)insulin moiety wherein GlyAl is connected to the substituent designated ~ and LysB29 is connected to the substituent designated R2, R2 represents an amino acid or a peptide chain containing not more than 36 amino acids, and Rl repre-sents hydrogen or a ~roup of the general formula R -X-, wherein X represents arginine or lysine, and R3 represents a peptide chain containing not more than 35 amino acids, or R2 together with R3 represent a peptide chain containing not more than 35 amino acids, with the proviso that the number of amino acids present in Rl plus R2 is less than 37.
Thus, the transpeptidation of this invention converts any of the above insulin compounds into threonineB30 esters of human insulin (formula III), which then can be deblocked to form human insulin.
:
8~
A further advantage of this inventiol- is that insulin-lik~ compounds present in crude insulin and present in some commercial insulin preparations and covered by the formula I by the transpeptidation of this invention are convert~d into threonineB30 esters of human insulin, which then can be deblocked to form human insulin.
Examples of insulin-like compounds of formula I
appear from the following:
Porcine diar~inine insulin ;Rl is hydro~en, and R is -Ala-Arg-Arg), porcine proinsulin(R3 toaether.with R2 is -Ala-Arg-Arg-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala--Val-Glu-Leu-Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu-~ila--Leu-Glu-Gly-Pro-Pro-Gln-Lys-, wherein the terminal alanyl is connected to LysB29~ do~.proinsulin ~3 together with R2 is -Ala-Arg-Arg-Asp-Val-~lu-Leu-Ala-Gly-Ala-Pro-Gly--Glu-Gly-Gly-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ala-Leu-Gln--Lys-, wherein the terminal alanyl is connected to LysB29~, porcine split proinsulin (R is Ala-Leu-Glu-Gly-Pro-Pro-Gln-Lys-, and R is -Ala--Arg-Ar~-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala-Val-Glu-Leu--Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu), porcine desdipeptide proinsulin (Rl is hydro~en, and R2 is -Ala-Arg-Arg-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala-Val--Glu-Leu-Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu-Ala--Leu-Glu-Gly-Pro-Pro-Gln), human proinsulin (R to~ather with R is -Thr-Arg-Arg-Glu-Ala-Glu-Asp~Leu~Gln-Val-Gly--Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu--Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-, wherein the terminal threonyl is connected to LysB29), and monkey proinsulin (R3 toqether with R2 is -Thr-Ar~-Ar~--Glu-Ala-Glu-As~-Pro-Gln-Val-Glv-Gln-Val-Glu-Leu-Gly--Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu--Glu-Gly-Ser-Leu-Gln-Lys-~ wherein the ter~inal threonyl is connected to ~ysB29.
2B~
: 13 Hence, in all these impurities covered by formula I
the R substituent designated R3-X- is exchanged with hydrogen.
A preferred embodiment of the present invention comprises reacting crude porcine insulin containing insulin-like compounds or a salt or complex thereof with an L-threonine ester (formula II) or a salt thereof under the above conditions whereafter the protecting group R4 and any protecting group R5 is removed. By this process the porcine insulin together with insulin-like compounds therein is converted into human insulin.
As examples of a complex or a salt of an insulin compound (formula I) can be mentioned a zinc complex or zinc salt.
When selecting the reaction conditions according to the above explanation and considering the results obtained in the following examples it is possible to obtain a yield of threonineB30 ester of human insulin which is higher than 60 per cent, and even higher than 80 per cent, and under certain preferred conditions higher than 90 per cent.
3Li~2~
The process according to the present invention has, therefore, the followincJ advantagcs over the prior art:
a) The enzymatic hydrolysis to remove Alas30 e.g., with carboxypeptidase A, ls omitted.
b) The isolation of an intermediate compound, such as porcine des-(AlaB30)-insulin, is unnecessary.
c) Contamination with des(Asn l)-insulin deriv~tives is avoided.
d) Proinsulin and other insulin-like im-purities present in crude insulin are - via the threonineB30 ester of human insulin -converted into human insulin by the process of this invention, whereby the yield is increased.
. .
el Antigenic insulin-like compounds, vide British Patent No. 1,285,023, are converted into numan ir.sulin.
A preferred procedure for preparing human insulin is as follows:
1) The starting material used for the transpeptidation is crude porcine insulin, e.g., crystalline insulin obtained by the use o~ a citrate buffer , vi~e Patent Specification No. U.S.A.
2,626,228.
2) If there is any trypsin activity left after the transpeptidation, it is pre~erred to remove it, e.g., under conditions where trypsin is inactive, e.g., in acid medium below pH 3. Trypsin can be removed by separation according to molecular weight, e.g., by gel filtration on "Sephadex G-50~ or "~io-gel P-3U~
in lM acetic acid, vide Nature 280, 412.
* Trade Mark B
3~ Other impurities such as unreacted porcine insulin may be removed by the use o anion and/or cation exchange chromato-graphy, vide Examples 1 and 2.
4) Thereafter, thle threonineB30 ester of human insulin is debloc~ed and human insulin is isolated, e.g., crystalli~ed,in a manmer known ~ se.
By this process human insulin of an accep-table pharmaceutical purity can be obtained and be further ~purified, if desired.
Furthermore, the present invention relates to novel threonineB30 esters of human insulin wherein the ester moiety is different from tert-butyl and methyl.
Abbreviations- used are in accordance with the rules approved (1974) by the IUPAC-IUB Commission on Biochemical Nomenclature, vide Collected Tentative Rules & Recommendations of the Commission on Bio-chemical Nomenclature IUPAC-IUB, 2nd ed., Maryland 1975.
~L~62~
Analytical tests:
The conversion of porcine in.sulin and porcine proinsulin into human insulin esters can be demonstrated by DISC PAGE electrophoresis in 7.5% polyacrylamide gel in a buffer consisting of 0.375 M Tris, 0.06 M HCl, and 8 M
urea. The pH of the buffer is 8.7. ESters of the formula III migrate with a speed of 75~ of that of porcine insulin. Porcine proinsulin migrating with 55% of the speed of porcine insulin is by the process according to the present invention converted into the same product.
Identification of the conversion product as compounds of the formula III is due to the following criteria:
(a) The electrophoretic migration of the human insulin esters of formula III in relation to porcine insulin corresponds to the loss of one negative charge.
(b) The amino acid composition of the stained protein bonds in the gel representing compounds of the formula III is identical with that of human insulin, i.e. 3 mol threonine and l mol alanine per mol insulin, and the composition of porcine insulin is 2 mol threonine and 2 mol alanine per mol insulin. The technique for analyzing amino acid compositions of protein bonds in bolyacrylamide gels has been described in Eur.J.Biochem. 25, 147.
(c) The proof that the incorporated threonine is placed as C-terminal amino acid in the B chain, is proved by oxidative sulfitolysis of the S-S bridges o~
insulin in 6 M guanidinium hydrochloride followed by separation of ~ and B chains by ion exchange chromatography on "SP Sephadex". DigeStion of the B
chain S-sulfonate with carboxypeptidase A liberates .~
!~
only the C-terminal amino acid. The technique has been described by Markussen in Proceedings of the Symposium on Proinsulin, Insulin and C-peptide, Tokushima, 12 - 14 July, 1978, (Editor: saba, Kaneko & Y~niahara) Int.Congress Series No. 468, Excerpta Medica, Amsterdam-Oxford. The analysis is performed after the ester group has been split from compounds of the formula III.
Those three analyses prove unambiguously that the conversion into human insulin has taken place.
The conversion of poxcine insulin and porcine proinsulin into human insulin esters can be followed quantitatively by E~PLC (high pressure liquid chromato-graphy) on reverse phase. ~ 4 x 300 mm~/u Bondapak C18 column" (Waters Ass.) was used and the elution was performed with a buffer comprising 0.2 M ammonium sulphate (adjusted to a pH value of 3.5 with sulphuric acid) and containing 26 - 50% acetonitrile. The optimal acetonitrile concentration depends on which ester of the formula III one desires to separate from porcine insulin. In case R4 is methyl, and R5 is hydrogen, separation is achieved in 26~ (v/v) of acetonitrile. Porcine insulin and (Thr-0~4e)B30-h-In (Me is methyl) elute after 4.5 and 5.9 column volumes, respectivily, as well separated symmetrical peaks.
Before the application on the HPLC column the proteins in the reaction mixture were precipitated by addition of 10 volumes of acetone. The precipitate was isolated by centrifugation, dried in vacuo, and dissolved in 0.02 M sulph~ric acid.
The process for preparing human insulin esters and human insulin is illustrated by the following examples which, however, are not to be construed as limiting. The examples illustrate some preferred em-bodiments of the process according to the invention.
.
.... . ........ . . . . . . ....... ... .... . .. _ .... _ .. _ _ _ _ __ _ _ _ . . _.. .... . ... ... .
Example 1 200 mg crude porcine insulin, crystallized once, was dissolved in 1.8 ml 3.33 M acetic acid. 2 ml of a 2 M solution of Thr-OMe (Me is methyl) in N,N-di~.ethyl-acetamide, and 20 mg of trypsin dissolved in 0.2 ~nl of water were added. After storage for 18 hours at 37C
the proteins were precipitated by the addition OI 40 ml of acetone, and the precipitate was isolated by centri--fugation. The supernatent was discarded. Analysis of the precipitate by ~IPLC using 26~ acetonitrile (vide Analytical tests) showed a 60% conversion of porcine insulin into(Thr-~e)B30-h-In . The precipitate was dis-solved in 8 ml freshly deionized 8 M urea, the pH value was adjusted to 8.0 with 1 M ammonia, and the solution was applied to a 2.5 x 25 cm column packed with "PAE A-25 Sephadex",-equilibrated with a 0.1 M ~nium chloride buf~er, which contained 60% (v/v) ethanol, the pH value of which was adjusted to 8.0 with ammonia. Elution ~7as carried out with the same buffer, and fractions of 15 ml were collected.
(Thr-CMe) -h-In was found in the fractions Nos. 26 - 46, and unreacted porcine insulin in the fractions Nos.
90 - 120. The fractions Nos. 26 - 46 were pooled, the ethanol evaporated _ vacuo, and the (Thr-OMe)B30--h-In was crystallized in a citrate buffer as described by Schlichtkrull et al., Handbuch der inneren Medizin, 7/2A, 96, Berlin, Heidelberg, New York 1975. The yield was 95 mg of crystals having the same rhombic shape as porcine insulin crystallized in the same manner. The amino acid composition was found to be identical with that of human insulin. Further analytical tests descri-bed in the above section: "Analytical Tests", prooved that the resulting product was ~Thr-OMe)B30-h-In.
~xample 2 100 mg porcine insulin fulfilling the puri-ty requirements stated in British Patent No. 1,285,023 was dissolved in 0.9 ml 3.33 M acetic acid and, there-.
. . ~ .
. ~ ,___,,~ _, ___,.. ... . _ . .. . . . . .. ... _ .. ~ ......... ... _ _ .. _.. _. _ .. __ ... __.. _ .. _ . ___.__ ___._.___._.. _____.____ .. ____.___._.. --- -- -- - ~a~ ~ ~ -~2~
after, L ml of a 2 M solution of threonine methyl ester in N,N-dimethylfon~ude and 12 mg TFCK (tosylphenylaL~L~hlorcmeth ketone) treated trypsin dissolved in 0.1 ml water were added. After an incubation for 24 hours at 37C the reaction was stopped by the addition of 4 ml 1 M
phosphoric acid. The(Thr-oMe)B3o-h~In obtained was separated from non-reacted porcine insulin by lon ex-change chromatography on a 2.5 x 25 cm column of ~SP-Sephadex~ with an eluent comprising 0.09 ~ sodium chloride and 0.02 M sodium dihydrogen phosphate(pH value of the buffer: 5.5) in 60~ ethanol. Frac~ions containing (Thr-o~e)B30-h-In were collected, the ethanol was removed Ln vacuo, and the product was crystallized as described in Example 1. The yield was 50 mg of ~Thr-OMe3B30-h-In.
Example 3 100 mg porcine proinsuiin was dissolved in 0.9 ml of 3.33 M acetic acid and converted into 5Thr-OMe)B30-h--In and purified as described for porcine insulin in Example 1. The conversion of proinsulin into (Tpr-OMe) --h-In was found to be 73% by HPLC analysis of the acetone precipitate. The yield of crystalline (Thr-O~e)B30--h-In was 54 mg.
Example 4 100 mg porcine insulin was dissolved in 0.9 ml of 2.77 M:acetic acid in water and reacted analogously ~ the process described in Example 2. After completion o~ the reaction the proteins were precipitated by the addition of 10 volumes of acetone. Analysis by DISC PAGE showed a conversion into (Thr-OMe)B30-h-In of 70%.
Example 5 1~0 mg porclne insulin was dissolved in 0.9 ml of ~, 1~6~
3.33 ~. acetic acid and 1 ml 2 M Thr-OMe in N-methylpyrro-lidone was added. The reaction was performed in a mar.ner ~loyous ~o that described in Example 4 and the conversion into (Thr-OMe)B30-h-In was 20%.
Example 6 100 mg porcine insulin was dissolved in 0.9 ml of 2.77M acetic acid in~water and 1 ml 2 M Thr-OMe in HMPA (hexamethylphosphortriamide) was added. The reaction was performed in a m~n~er analogous to'that described in Example 4. The convers~on into (Thr-OMe)B30-h-In was 80~.
Example 7 100 mg porcine insulin was dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-dimethyl-acetamide was added. The reaction was performed in a manner ana,logous to that described in Ex,ample 4. The conversion into (Thr~OMe)B30-h-In was 80%
Example 8 100 mg porcine insulin was dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-dimethylacetamide was added. Thereafter, 200 U trypsin activity (measured against the substrate BAEE) immobi-lized on 1 g of glass beads was added and after incubation at 37C during 24 hours the trypsin bound to the glass was filtred off. After completion of the reaction the proteins were precipitated by the addition of 10 volumes of acetone. Analysis by DISC PAGE showed a conversion into (Thr-OMe) 3,-h-In of 40~.
Example 9 100 mg porcine insulin was dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-dimethyl-; ~ . ., ~ -,, acetamide was added. Thereafter, 300 U trypsin (activity measured with the substrate BAEE) immobilized on 200 mg CNBr activated "Sephadex G-150" was added.
~fter incubation at 37C during 24 hours the trypsin bound to "S~phadex" was filtred off. After completion of the reaction the proteins were precipitated by the addition of 10 volumes of acetone. Analysis by DISC PAGE
showed a conversion into (Thr-OMe) 30-h-In of 70~.
Example 10 The process described in Example 7 was repeted, provided that the ester used was 2 M Thr-OBut (But is tert-butyl) in N,N-dimethylacetamide. The conversion into (Thr oBu!t)B30 h In was 80%
Example 11 The process described in Example 8 was repeted, provided that the ester used ~as 2 M Thr-OBut in N,N-di-methylacetamide. The convers~on into (Thr-OBut)B30-h-In was 30%.
Example 12 The process described in Example 9 was repeted, provided that the ester used was 2 M Thr-OBut in N,N-di-methylacetamide. The convers~on into (Thr-OBut) 3 -h-In was 70~.
Example 13 100 mg porcine proinsulin was dissolved in 0.9 ml :of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-dimethyl-acetamide was added. The reaction was perfonm~d in a manner analogous to that described in Example 4. The convers'ion into (Thr OMe)B30 h In was 80~
s ~.~
6~3 Example 14 100 mg porcine proinsulin was dissolved in 0.9ml of 3.33M ace~c acid and 1 ml 2 M Thr-OMe in N,N-dimethyl-acetamide was added. Themixture was treated with immobi-lized trypsin analogically to the ~rocess described in Example B. The convexs\~on into ~Thr-OMe) -h-In was 40~.
Example 15 100 mg porcine proinsulin was dissolved in 0.9ml O~ 3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-di-methylacetamide was added. The mixture was treated analogically to the process described in Example 9 with immobilized trypsin. The convers~on into (Thr-OMe)R30-h-In was 70%.
Example 16 100 mg porcine proinsulin was dissol~ed in 0.9 ml of 3.33 M acetic acid and 1 ml 2 M Thr-OBut in N,N-di-methylacetamide was added. The reaction was performed in a manner anahogous to that described in Example 4.
The conversion into (Thr-OBut)B30-h-In was 80%
.
Example 17 100 mg porcine proinsulin was dissolved in 0.9 ml Of 3.33 M acetic acid and 1 ml 2 M Thr-OBu in N,N-di-methylacetamide was added. The mixture was treated with trypsin immobilized to glass beads analogically to process described in Example 8. The conver~ion into (Thr-OBut)B3 -h-In was 40%.
Example 18 100 mg poxcine proinsulin was dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml 2 M Thr-OBut in N,N-di-methylacetamide was added. The mixture was treated ~Z8~
wit,. trypsin immob~lized to CNBr activated "Sephadex G-150" analogically with the process described in Example 9. The conversion into ~Thr-OBu )B30-h-In was 70%.
Example 19 100 mg porcine insulin was dissolved in 0.5 mlof 6 M acetic acid and 1 ml 1 M Thr-OTmb (Tmb is 2,4,6-tri~
methylbenzyl) in N,N-dimethylac~tamide was added.
Furthermore, 0.5 ml N,N-dimethylacetamide and 5 mg TPCK
treated trypsin in 0.1 ml water were added. The reaction mixture was stored at 32C for 44 hours. After completion of the reaction the proteins were precipitated by the addition of 10 volumes of acetone. Analysis by DISC
PAGE showed a conversion into (Thr-OTmb)B30-h-In of 50%.
Example 20 100 mg porcine insulin was dissolved in 0.9 ml of 3 M acetic acid and 1 ml 2 M Thr-OMe in dioxane was added.
The reaction was performed in a manner analogous to that descri~ed in Example 4 and ~he conversion i~to (Thr-OMe)B30_ -h-In was 10%.
Example 21 100 mg porcine insulin was dissolved in 0.9 ml of 3 M acetic acid and 1 ml 2 M Thr-OMe in acetonitrile was added. The reaction was performed in a manner analogous to that described in Example 4 and the conversion into (Th~-OMe)B30--h-In was 10~.
'~' ''I
24 ~ i~
Example 22 250 mg of crystallin~(Thr-oMe)B3o-h-In was dis-persed in 25 ml of water and dissolved by the addition of 1 N sodium hydroxide solution to a pH value of 10Ø The pH value was kept constant at 10.0 for 24 hours at 25 C.
The human insulin formed was crystallized by the addition of 2 g of sodium chloride, 350 mg of sodium acetate tri-hydrate and 2.5 mg of zinc acetate dihydrate followed by the addition of 1 N hydrochloric acid to obtain a pH value of 5.52. After storage for 24 hours at 4 C the rhombohedral crystals were isolated by centrifugation, washed with 3 ml of water, isolated by centrifugation, and dried in vacuo. Yield: 220 mg of human insulin.
Example 23 10Q mg (Thr-OTmb~B30-h-In was dissolved in 1 ml of ice cold trifluoroacetic acid and the solution was stored for 2 hours at 0 C. The human insulin formed was precipitated by the addition of 10 ml of tetrahydro-furan and 0.97 ml o~ 1.03 M hydrochloric acid in tetra-hydrofuran. The precipitate formed was isolated by centri-fugation, washed with 10 ml of tetrahydrofuran, isolated by centrifugation, and dried in vacuo. The precipitate was dissolved in 10 ml of water and the pH value of the solution was adjusted to 2.5 with 1 N sodium ~ydroxide solution. The human insulin was precipitated by the addition of 1.5 g of sodium chloride and isolated by centrifugation. The precipitate was dissolved in 10 ml of water, and the human insulin was precipitated by the addition of 0.8 g of sodium chloride, 3.7 mg of zinc acetate dih~drate and a.l4 g of sodium acetate trihydrate followed by the addition of 1 N
sodium hydroxide solution to obtain a p~ value of 5.52.
After storage for 24 hours at 4 C, the precipitate was isolated by centrifugation, washed with 0.9 ml of water, isolated by centrifugation and dried in vacuo. Yield:
90 mg of human insulin.
2~
Example 24 100 mg of ~orcine insulin was dissolved in 0.5 ml of 10 M acetic acid and 1.3 ml of 1.54 M Thr-OMe in N,N-dimethylacetamide was added. The mixture was cooled to 12C. 10 mg of trypsin dissolved in 0.2 ml of 0.05 M
calcium acetate was added. After 48 hours at 12C the proteins were precipitated by addition of 20 ml of acetone. The conversion of porcine insulin into (Thr-OMe) -h-In was 97% by HPLC.
Example 25 20 mg of porcine insulin was dissolved in a mix-ture of 0.08 ml of 10 M acetic acid and 0.14 ml of water.
0.2 ml of 2 M Thr-OMe in N,N-dimethylacetamide was added and the mixture was cooled to -10C. 2 mg of trypsin dis-solved in 0.025 ml of 0.05 M calcium acetate was added.
After 72 hours at -10C the proteins were precipitated by addi~ion of 5 ml of~acetone. The conversion of porcine insulin into (Thr-OMe) -h-In was 64% by HPLC.
Example 26 20 mg of porcine insulin was dispensed in 0.1 ml of water. Addition of 0.6 ml of 2 M Thr-O~e in N,N-dimethylacetamide caused the insulin to go into solution.
The mixtu~e was cooled to 7C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added. After 24 hours at 7C the proteins were precipitated by addition of 5 ml of acetone. The conversion of porcine insulin into (Thr-OMe)B30-h-In was 62~ by HPLC.
.1 ~
25a ~162~
Example 27 20 mg of porcine insulin was dissolved in 0.135 ml of 4.45M propionic acid. 0.24 ml of 1.67 M Thr-OMe in N,N-dimethylacetamide was added. 2 mg of trypsin in 0.025 ml of ~.., ~L6;2 ~
o.05 M calcium acetate was added and the mixture was kept at 37C for 24 hours. The proteins were precipitated by addition of 10 vol~nes of 2-propanol. The conversion of porcine insulin into (Thr-OMe)B30-h-In was 75~ by HPLC.
Example 28 20 mg of porcine insulin was dispersed in 0.1 ml of water. 0~4 ml 2 M T~Me in N,N-dimethylace~de was added follcwed by 0.04 ml of 10 N hydro~hLoric acid caused the insulLn to go in~4 solution. 2mg ~f trypsin dissolved in 0.025 ml of 0.05 M
calcium acetate was added and the mixture was ~ept at 37C
for 4 hours. Analysis by HPLC showed a 46% conversion of porcine insulin into (Thr-OMe)B30-h-In.
Example 29 20 mg of porcine insulin was dissolved in 0.175 ml of 0.57 M acetic acid. 0.2 ml of 2 M Thr-OMe in N,N-dimethylacetamide was added followed by the addition of 0.025 ml of 0.05 M calcium acetate. 10 mg of a crude preparation of AchromObacter lyticus protease was added and the mixture was kept for 22 hours at 37C. The proteins were precipi-tated by the addition of 10 volumes of acetone. The conver-sion of porcine insulin into (Thr-OMe)B30-h-In waS 12% ~EP~C.
.
Example 30 20 mg of porcine insulin was dispersed in 0.1 ml of 0.5 M acetic acid. Addition of 0.2 ml of 0.1 M Thr-OMe in N,N-dlmethylacetamide dissolved the insulin. The mix-ture was cooled to 12C and 2 mg of trypsin in 0.025 ml of 0.05 M caLcium acetate was added. After 24 hours a~ 12C
the analysis by HPLC showed a 42% conversion of porcine insulin into (Thr-OMe)B30-h-In ~.
. ~;
~z~
Example 31 2 mg of rabbit insulin was dissolved in 0.135 ml of 4.45 M acetic acid. 0.24 ml of 1.67 M Thr-OMe in N,N-dimethylacetamide was added followed by the addition of 1.25 mg trypsin in 0.025 ml of 0.05 M calcium acetate.
The mixture was kept at 37C for 4 hours. Analysis by HPLC showed an 88% conversion of rabbit insulin into (Th~OMe)B30-h-In. Rabbit insulin elutes before porcine insulin from the HPLC column, the ratio between the elu-tion volumes of rabbit insulin to (Thr-OMe)B30-h-In being 0.72.
Example 32 2 mg of porcine diarginine insulin (Arg 31-ArgB32-insulin) was dissolved in 0.135 ml of 4.45 M acetic acid.
0.24 ml of 1.67 M Thr-OMe in N,N-dimethylacetamide was added followed by the addition of 1.25 mg of trypsin in 0.025 ml of 0.05 M calcium acetate. The mixture was kept at 37C for 4 hours. Analysis by HPLC showed a 91% conver-sion of diarginine insulin into (Thr-OMe) -h-In. Diarginine insulin elutes before procine insulin from the HPLC column, the ratio between the elution volumes of diarginine insulin to (Thr-OMe)B30-h-In being 0.50.
Example 33 2 mg of porcine intermediates (i.e. a mixture of des-dipeptide (Lys62-Arg63)proinsulin and desdipeptide(Arg31-Arg32)proinsulin) was reacted analogously to the reaction described in Example 32. Analysis by HPLC showed 88% (Thr-OMe)B30-h-In .
Z8~i~
Example 34 20 mg of porcine insulin was dissolved in 0.1 ml of 2 M acetic acid. 0.2 ml of 2 M Thr-OMe in N,N-dimethyla-cetamide was added and the mixture was cooled to -18C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added and the mixture was kept for 120 hours at -18C.
Analysis by HPLC showed that the conversion into (Thr-OMe)B30-h-In was 83%.
Example 35 The process described in Example 34 was repeted with the proviso that the reaction was carried out at 50C
for 4 hours. Analysis by HPLC showed that the conversion into (Thr-OMe) -h-In was 23%.
Example 36 20 mg of porcine insulin was dissolved in 0.1 ml of 3 M acetic acid. 0.3 ml of 0.33 M Thr-O(CH2)2-SO2-CH3, CH3COOH (threonine 2-(methylsulfonyl)ethylester, hydroace-tate) in N,N-dimethylacetamide was added. The mixture was cooled to 12C and 2 mg of trypsin in 0.025 ml of 0.05 M
calcium acetate was added. HPLC showed after 24 hours at 12 C a 77% conversion into (Thr-O(~H2)2-SO2-CH3) ~ -h-In- me product elutes approximately at the position of (Thr-OMe) h-In.
Example 37 20 mg of porcine insulin was dissolved in 0.1 ml of 10 M acetic acid. 0.2 ml of 2 M Thr-OEt (Et is ethyl) in N,N-dimethylacetamide was added and the mixture was cooled to 12 C. 2 mg of trypsin in 0.025 ml of 0.05 M calcium acetate was added. HPLC showed after 24 hours at 12C a .
~1~%8~
75% conversion into (Thr-O~t)B30-h-In. The product eluted in a position slightly after that o (Thr-OMe)B30-h-In.
ExampLe 38 20 mg of porcine insulin was dissolved in 0.1 ml of 6 M acetic acid. 0.3 ml of 0.67 M Thr(Bu )-OBu (Bu is ter~iary ~utyl) in N,N-dimethylacetamide was added. The mixture was cooled to 12C and 2 mg of trypsin in 0.025 ml of 0.05 M calcium acetate was added. After 24 hours at 12C the conversion into (Thr(But)-OBu~B30-h-In was 77~l The product was eluted from the HPLC column by applying a gradient in acetonitrile from 27% to 40%.
Example 39 20 mg of porcine insulin was dissolved in 0.1 ml of 4 M acetic acid. 0.2 ml of 1.5 M Thr-OMe dissolved in tetrahydrofuran was added and the mixture was cooled to 12C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M
calcium acetate was added. After 4 hours at 12C the analysis by HPLC showed a conversion o~ 75% into (Thr-OMe)B30-h-In~
Example 40 20 mg of porcine insulin was dissolved in 0.1 ml of 4 M acetic acid. 0.8 ml o~ 2 M Thr-OMe dissolved in 1,2-ethanediol was added and the mixture was cooled to 12C.
2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate wa's added. After 4 hours a~ 12C the analysis by HPLC showed a conversion of 48% into (Thr-OMe)B30-h-In~
~z~
Example 41 20 mg of porcine insulin was dissolved in 0.1 ml of 4 M acetic acid. 0.2 ml of 2 M Thr-OMe dissolved in ethanol was added and the mixture was cooled to 12C~ 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added and the reaction was carried out for 4 hours at 12 C. Analysis by HPLC showed a 46% conversion into (Thr-OMe)B30-h-In - Example 42 20 mg of porcine insulin was dissolved in 0.1 ml of 4 M acetic acid. 0.2 ml of 2 M Thr-OMe in acetone was added and the mixture ~as cooled to 12C. 2 mg of trypsin dis-solved in 0.025 ml of 0.05 M calcium acetate was added.
After 4 hours at 12C the analysis by HPLC showed a con-version of 48% into (Thr-OMe) -h-In.
~ . . .
..
I
By this process human insulin of an accep-table pharmaceutical purity can be obtained and be further ~purified, if desired.
Furthermore, the present invention relates to novel threonineB30 esters of human insulin wherein the ester moiety is different from tert-butyl and methyl.
Abbreviations- used are in accordance with the rules approved (1974) by the IUPAC-IUB Commission on Biochemical Nomenclature, vide Collected Tentative Rules & Recommendations of the Commission on Bio-chemical Nomenclature IUPAC-IUB, 2nd ed., Maryland 1975.
~L~62~
Analytical tests:
The conversion of porcine in.sulin and porcine proinsulin into human insulin esters can be demonstrated by DISC PAGE electrophoresis in 7.5% polyacrylamide gel in a buffer consisting of 0.375 M Tris, 0.06 M HCl, and 8 M
urea. The pH of the buffer is 8.7. ESters of the formula III migrate with a speed of 75~ of that of porcine insulin. Porcine proinsulin migrating with 55% of the speed of porcine insulin is by the process according to the present invention converted into the same product.
Identification of the conversion product as compounds of the formula III is due to the following criteria:
(a) The electrophoretic migration of the human insulin esters of formula III in relation to porcine insulin corresponds to the loss of one negative charge.
(b) The amino acid composition of the stained protein bonds in the gel representing compounds of the formula III is identical with that of human insulin, i.e. 3 mol threonine and l mol alanine per mol insulin, and the composition of porcine insulin is 2 mol threonine and 2 mol alanine per mol insulin. The technique for analyzing amino acid compositions of protein bonds in bolyacrylamide gels has been described in Eur.J.Biochem. 25, 147.
(c) The proof that the incorporated threonine is placed as C-terminal amino acid in the B chain, is proved by oxidative sulfitolysis of the S-S bridges o~
insulin in 6 M guanidinium hydrochloride followed by separation of ~ and B chains by ion exchange chromatography on "SP Sephadex". DigeStion of the B
chain S-sulfonate with carboxypeptidase A liberates .~
!~
only the C-terminal amino acid. The technique has been described by Markussen in Proceedings of the Symposium on Proinsulin, Insulin and C-peptide, Tokushima, 12 - 14 July, 1978, (Editor: saba, Kaneko & Y~niahara) Int.Congress Series No. 468, Excerpta Medica, Amsterdam-Oxford. The analysis is performed after the ester group has been split from compounds of the formula III.
Those three analyses prove unambiguously that the conversion into human insulin has taken place.
The conversion of poxcine insulin and porcine proinsulin into human insulin esters can be followed quantitatively by E~PLC (high pressure liquid chromato-graphy) on reverse phase. ~ 4 x 300 mm~/u Bondapak C18 column" (Waters Ass.) was used and the elution was performed with a buffer comprising 0.2 M ammonium sulphate (adjusted to a pH value of 3.5 with sulphuric acid) and containing 26 - 50% acetonitrile. The optimal acetonitrile concentration depends on which ester of the formula III one desires to separate from porcine insulin. In case R4 is methyl, and R5 is hydrogen, separation is achieved in 26~ (v/v) of acetonitrile. Porcine insulin and (Thr-0~4e)B30-h-In (Me is methyl) elute after 4.5 and 5.9 column volumes, respectivily, as well separated symmetrical peaks.
Before the application on the HPLC column the proteins in the reaction mixture were precipitated by addition of 10 volumes of acetone. The precipitate was isolated by centrifugation, dried in vacuo, and dissolved in 0.02 M sulph~ric acid.
The process for preparing human insulin esters and human insulin is illustrated by the following examples which, however, are not to be construed as limiting. The examples illustrate some preferred em-bodiments of the process according to the invention.
.
.... . ........ . . . . . . ....... ... .... . .. _ .... _ .. _ _ _ _ __ _ _ _ . . _.. .... . ... ... .
Example 1 200 mg crude porcine insulin, crystallized once, was dissolved in 1.8 ml 3.33 M acetic acid. 2 ml of a 2 M solution of Thr-OMe (Me is methyl) in N,N-di~.ethyl-acetamide, and 20 mg of trypsin dissolved in 0.2 ~nl of water were added. After storage for 18 hours at 37C
the proteins were precipitated by the addition OI 40 ml of acetone, and the precipitate was isolated by centri--fugation. The supernatent was discarded. Analysis of the precipitate by ~IPLC using 26~ acetonitrile (vide Analytical tests) showed a 60% conversion of porcine insulin into(Thr-~e)B30-h-In . The precipitate was dis-solved in 8 ml freshly deionized 8 M urea, the pH value was adjusted to 8.0 with 1 M ammonia, and the solution was applied to a 2.5 x 25 cm column packed with "PAE A-25 Sephadex",-equilibrated with a 0.1 M ~nium chloride buf~er, which contained 60% (v/v) ethanol, the pH value of which was adjusted to 8.0 with ammonia. Elution ~7as carried out with the same buffer, and fractions of 15 ml were collected.
(Thr-CMe) -h-In was found in the fractions Nos. 26 - 46, and unreacted porcine insulin in the fractions Nos.
90 - 120. The fractions Nos. 26 - 46 were pooled, the ethanol evaporated _ vacuo, and the (Thr-OMe)B30--h-In was crystallized in a citrate buffer as described by Schlichtkrull et al., Handbuch der inneren Medizin, 7/2A, 96, Berlin, Heidelberg, New York 1975. The yield was 95 mg of crystals having the same rhombic shape as porcine insulin crystallized in the same manner. The amino acid composition was found to be identical with that of human insulin. Further analytical tests descri-bed in the above section: "Analytical Tests", prooved that the resulting product was ~Thr-OMe)B30-h-In.
~xample 2 100 mg porcine insulin fulfilling the puri-ty requirements stated in British Patent No. 1,285,023 was dissolved in 0.9 ml 3.33 M acetic acid and, there-.
. . ~ .
. ~ ,___,,~ _, ___,.. ... . _ . .. . . . . .. ... _ .. ~ ......... ... _ _ .. _.. _. _ .. __ ... __.. _ .. _ . ___.__ ___._.___._.. _____.____ .. ____.___._.. --- -- -- - ~a~ ~ ~ -~2~
after, L ml of a 2 M solution of threonine methyl ester in N,N-dimethylfon~ude and 12 mg TFCK (tosylphenylaL~L~hlorcmeth ketone) treated trypsin dissolved in 0.1 ml water were added. After an incubation for 24 hours at 37C the reaction was stopped by the addition of 4 ml 1 M
phosphoric acid. The(Thr-oMe)B3o-h~In obtained was separated from non-reacted porcine insulin by lon ex-change chromatography on a 2.5 x 25 cm column of ~SP-Sephadex~ with an eluent comprising 0.09 ~ sodium chloride and 0.02 M sodium dihydrogen phosphate(pH value of the buffer: 5.5) in 60~ ethanol. Frac~ions containing (Thr-o~e)B30-h-In were collected, the ethanol was removed Ln vacuo, and the product was crystallized as described in Example 1. The yield was 50 mg of ~Thr-OMe3B30-h-In.
Example 3 100 mg porcine proinsuiin was dissolved in 0.9 ml of 3.33 M acetic acid and converted into 5Thr-OMe)B30-h--In and purified as described for porcine insulin in Example 1. The conversion of proinsulin into (Tpr-OMe) --h-In was found to be 73% by HPLC analysis of the acetone precipitate. The yield of crystalline (Thr-O~e)B30--h-In was 54 mg.
Example 4 100 mg porcine insulin was dissolved in 0.9 ml of 2.77 M:acetic acid in water and reacted analogously ~ the process described in Example 2. After completion o~ the reaction the proteins were precipitated by the addition of 10 volumes of acetone. Analysis by DISC PAGE showed a conversion into (Thr-OMe)B30-h-In of 70%.
Example 5 1~0 mg porclne insulin was dissolved in 0.9 ml of ~, 1~6~
3.33 ~. acetic acid and 1 ml 2 M Thr-OMe in N-methylpyrro-lidone was added. The reaction was performed in a mar.ner ~loyous ~o that described in Example 4 and the conversion into (Thr-OMe)B30-h-In was 20%.
Example 6 100 mg porcine insulin was dissolved in 0.9 ml of 2.77M acetic acid in~water and 1 ml 2 M Thr-OMe in HMPA (hexamethylphosphortriamide) was added. The reaction was performed in a m~n~er analogous to'that described in Example 4. The convers~on into (Thr-OMe)B30-h-In was 80~.
Example 7 100 mg porcine insulin was dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-dimethyl-acetamide was added. The reaction was performed in a manner ana,logous to that described in Ex,ample 4. The conversion into (Thr~OMe)B30-h-In was 80%
Example 8 100 mg porcine insulin was dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-dimethylacetamide was added. Thereafter, 200 U trypsin activity (measured against the substrate BAEE) immobi-lized on 1 g of glass beads was added and after incubation at 37C during 24 hours the trypsin bound to the glass was filtred off. After completion of the reaction the proteins were precipitated by the addition of 10 volumes of acetone. Analysis by DISC PAGE showed a conversion into (Thr-OMe) 3,-h-In of 40~.
Example 9 100 mg porcine insulin was dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-dimethyl-; ~ . ., ~ -,, acetamide was added. Thereafter, 300 U trypsin (activity measured with the substrate BAEE) immobilized on 200 mg CNBr activated "Sephadex G-150" was added.
~fter incubation at 37C during 24 hours the trypsin bound to "S~phadex" was filtred off. After completion of the reaction the proteins were precipitated by the addition of 10 volumes of acetone. Analysis by DISC PAGE
showed a conversion into (Thr-OMe) 30-h-In of 70~.
Example 10 The process described in Example 7 was repeted, provided that the ester used was 2 M Thr-OBut (But is tert-butyl) in N,N-dimethylacetamide. The conversion into (Thr oBu!t)B30 h In was 80%
Example 11 The process described in Example 8 was repeted, provided that the ester used ~as 2 M Thr-OBut in N,N-di-methylacetamide. The convers~on into (Thr-OBut)B30-h-In was 30%.
Example 12 The process described in Example 9 was repeted, provided that the ester used was 2 M Thr-OBut in N,N-di-methylacetamide. The convers~on into (Thr-OBut) 3 -h-In was 70~.
Example 13 100 mg porcine proinsulin was dissolved in 0.9 ml :of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-dimethyl-acetamide was added. The reaction was perfonm~d in a manner analogous to that described in Example 4. The convers'ion into (Thr OMe)B30 h In was 80~
s ~.~
6~3 Example 14 100 mg porcine proinsulin was dissolved in 0.9ml of 3.33M ace~c acid and 1 ml 2 M Thr-OMe in N,N-dimethyl-acetamide was added. Themixture was treated with immobi-lized trypsin analogically to the ~rocess described in Example B. The convexs\~on into ~Thr-OMe) -h-In was 40~.
Example 15 100 mg porcine proinsulin was dissolved in 0.9ml O~ 3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-di-methylacetamide was added. The mixture was treated analogically to the process described in Example 9 with immobilized trypsin. The convers~on into (Thr-OMe)R30-h-In was 70%.
Example 16 100 mg porcine proinsulin was dissol~ed in 0.9 ml of 3.33 M acetic acid and 1 ml 2 M Thr-OBut in N,N-di-methylacetamide was added. The reaction was performed in a manner anahogous to that described in Example 4.
The conversion into (Thr-OBut)B30-h-In was 80%
.
Example 17 100 mg porcine proinsulin was dissolved in 0.9 ml Of 3.33 M acetic acid and 1 ml 2 M Thr-OBu in N,N-di-methylacetamide was added. The mixture was treated with trypsin immobilized to glass beads analogically to process described in Example 8. The conver~ion into (Thr-OBut)B3 -h-In was 40%.
Example 18 100 mg poxcine proinsulin was dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml 2 M Thr-OBut in N,N-di-methylacetamide was added. The mixture was treated ~Z8~
wit,. trypsin immob~lized to CNBr activated "Sephadex G-150" analogically with the process described in Example 9. The conversion into ~Thr-OBu )B30-h-In was 70%.
Example 19 100 mg porcine insulin was dissolved in 0.5 mlof 6 M acetic acid and 1 ml 1 M Thr-OTmb (Tmb is 2,4,6-tri~
methylbenzyl) in N,N-dimethylac~tamide was added.
Furthermore, 0.5 ml N,N-dimethylacetamide and 5 mg TPCK
treated trypsin in 0.1 ml water were added. The reaction mixture was stored at 32C for 44 hours. After completion of the reaction the proteins were precipitated by the addition of 10 volumes of acetone. Analysis by DISC
PAGE showed a conversion into (Thr-OTmb)B30-h-In of 50%.
Example 20 100 mg porcine insulin was dissolved in 0.9 ml of 3 M acetic acid and 1 ml 2 M Thr-OMe in dioxane was added.
The reaction was performed in a manner analogous to that descri~ed in Example 4 and ~he conversion i~to (Thr-OMe)B30_ -h-In was 10%.
Example 21 100 mg porcine insulin was dissolved in 0.9 ml of 3 M acetic acid and 1 ml 2 M Thr-OMe in acetonitrile was added. The reaction was performed in a manner analogous to that described in Example 4 and the conversion into (Th~-OMe)B30--h-In was 10~.
'~' ''I
24 ~ i~
Example 22 250 mg of crystallin~(Thr-oMe)B3o-h-In was dis-persed in 25 ml of water and dissolved by the addition of 1 N sodium hydroxide solution to a pH value of 10Ø The pH value was kept constant at 10.0 for 24 hours at 25 C.
The human insulin formed was crystallized by the addition of 2 g of sodium chloride, 350 mg of sodium acetate tri-hydrate and 2.5 mg of zinc acetate dihydrate followed by the addition of 1 N hydrochloric acid to obtain a pH value of 5.52. After storage for 24 hours at 4 C the rhombohedral crystals were isolated by centrifugation, washed with 3 ml of water, isolated by centrifugation, and dried in vacuo. Yield: 220 mg of human insulin.
Example 23 10Q mg (Thr-OTmb~B30-h-In was dissolved in 1 ml of ice cold trifluoroacetic acid and the solution was stored for 2 hours at 0 C. The human insulin formed was precipitated by the addition of 10 ml of tetrahydro-furan and 0.97 ml o~ 1.03 M hydrochloric acid in tetra-hydrofuran. The precipitate formed was isolated by centri-fugation, washed with 10 ml of tetrahydrofuran, isolated by centrifugation, and dried in vacuo. The precipitate was dissolved in 10 ml of water and the pH value of the solution was adjusted to 2.5 with 1 N sodium ~ydroxide solution. The human insulin was precipitated by the addition of 1.5 g of sodium chloride and isolated by centrifugation. The precipitate was dissolved in 10 ml of water, and the human insulin was precipitated by the addition of 0.8 g of sodium chloride, 3.7 mg of zinc acetate dih~drate and a.l4 g of sodium acetate trihydrate followed by the addition of 1 N
sodium hydroxide solution to obtain a p~ value of 5.52.
After storage for 24 hours at 4 C, the precipitate was isolated by centrifugation, washed with 0.9 ml of water, isolated by centrifugation and dried in vacuo. Yield:
90 mg of human insulin.
2~
Example 24 100 mg of ~orcine insulin was dissolved in 0.5 ml of 10 M acetic acid and 1.3 ml of 1.54 M Thr-OMe in N,N-dimethylacetamide was added. The mixture was cooled to 12C. 10 mg of trypsin dissolved in 0.2 ml of 0.05 M
calcium acetate was added. After 48 hours at 12C the proteins were precipitated by addition of 20 ml of acetone. The conversion of porcine insulin into (Thr-OMe) -h-In was 97% by HPLC.
Example 25 20 mg of porcine insulin was dissolved in a mix-ture of 0.08 ml of 10 M acetic acid and 0.14 ml of water.
0.2 ml of 2 M Thr-OMe in N,N-dimethylacetamide was added and the mixture was cooled to -10C. 2 mg of trypsin dis-solved in 0.025 ml of 0.05 M calcium acetate was added.
After 72 hours at -10C the proteins were precipitated by addi~ion of 5 ml of~acetone. The conversion of porcine insulin into (Thr-OMe) -h-In was 64% by HPLC.
Example 26 20 mg of porcine insulin was dispensed in 0.1 ml of water. Addition of 0.6 ml of 2 M Thr-O~e in N,N-dimethylacetamide caused the insulin to go into solution.
The mixtu~e was cooled to 7C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added. After 24 hours at 7C the proteins were precipitated by addition of 5 ml of acetone. The conversion of porcine insulin into (Thr-OMe)B30-h-In was 62~ by HPLC.
.1 ~
25a ~162~
Example 27 20 mg of porcine insulin was dissolved in 0.135 ml of 4.45M propionic acid. 0.24 ml of 1.67 M Thr-OMe in N,N-dimethylacetamide was added. 2 mg of trypsin in 0.025 ml of ~.., ~L6;2 ~
o.05 M calcium acetate was added and the mixture was kept at 37C for 24 hours. The proteins were precipitated by addition of 10 vol~nes of 2-propanol. The conversion of porcine insulin into (Thr-OMe)B30-h-In was 75~ by HPLC.
Example 28 20 mg of porcine insulin was dispersed in 0.1 ml of water. 0~4 ml 2 M T~Me in N,N-dimethylace~de was added follcwed by 0.04 ml of 10 N hydro~hLoric acid caused the insulLn to go in~4 solution. 2mg ~f trypsin dissolved in 0.025 ml of 0.05 M
calcium acetate was added and the mixture was ~ept at 37C
for 4 hours. Analysis by HPLC showed a 46% conversion of porcine insulin into (Thr-OMe)B30-h-In.
Example 29 20 mg of porcine insulin was dissolved in 0.175 ml of 0.57 M acetic acid. 0.2 ml of 2 M Thr-OMe in N,N-dimethylacetamide was added followed by the addition of 0.025 ml of 0.05 M calcium acetate. 10 mg of a crude preparation of AchromObacter lyticus protease was added and the mixture was kept for 22 hours at 37C. The proteins were precipi-tated by the addition of 10 volumes of acetone. The conver-sion of porcine insulin into (Thr-OMe)B30-h-In waS 12% ~EP~C.
.
Example 30 20 mg of porcine insulin was dispersed in 0.1 ml of 0.5 M acetic acid. Addition of 0.2 ml of 0.1 M Thr-OMe in N,N-dlmethylacetamide dissolved the insulin. The mix-ture was cooled to 12C and 2 mg of trypsin in 0.025 ml of 0.05 M caLcium acetate was added. After 24 hours a~ 12C
the analysis by HPLC showed a 42% conversion of porcine insulin into (Thr-OMe)B30-h-In ~.
. ~;
~z~
Example 31 2 mg of rabbit insulin was dissolved in 0.135 ml of 4.45 M acetic acid. 0.24 ml of 1.67 M Thr-OMe in N,N-dimethylacetamide was added followed by the addition of 1.25 mg trypsin in 0.025 ml of 0.05 M calcium acetate.
The mixture was kept at 37C for 4 hours. Analysis by HPLC showed an 88% conversion of rabbit insulin into (Th~OMe)B30-h-In. Rabbit insulin elutes before porcine insulin from the HPLC column, the ratio between the elu-tion volumes of rabbit insulin to (Thr-OMe)B30-h-In being 0.72.
Example 32 2 mg of porcine diarginine insulin (Arg 31-ArgB32-insulin) was dissolved in 0.135 ml of 4.45 M acetic acid.
0.24 ml of 1.67 M Thr-OMe in N,N-dimethylacetamide was added followed by the addition of 1.25 mg of trypsin in 0.025 ml of 0.05 M calcium acetate. The mixture was kept at 37C for 4 hours. Analysis by HPLC showed a 91% conver-sion of diarginine insulin into (Thr-OMe) -h-In. Diarginine insulin elutes before procine insulin from the HPLC column, the ratio between the elution volumes of diarginine insulin to (Thr-OMe)B30-h-In being 0.50.
Example 33 2 mg of porcine intermediates (i.e. a mixture of des-dipeptide (Lys62-Arg63)proinsulin and desdipeptide(Arg31-Arg32)proinsulin) was reacted analogously to the reaction described in Example 32. Analysis by HPLC showed 88% (Thr-OMe)B30-h-In .
Z8~i~
Example 34 20 mg of porcine insulin was dissolved in 0.1 ml of 2 M acetic acid. 0.2 ml of 2 M Thr-OMe in N,N-dimethyla-cetamide was added and the mixture was cooled to -18C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added and the mixture was kept for 120 hours at -18C.
Analysis by HPLC showed that the conversion into (Thr-OMe)B30-h-In was 83%.
Example 35 The process described in Example 34 was repeted with the proviso that the reaction was carried out at 50C
for 4 hours. Analysis by HPLC showed that the conversion into (Thr-OMe) -h-In was 23%.
Example 36 20 mg of porcine insulin was dissolved in 0.1 ml of 3 M acetic acid. 0.3 ml of 0.33 M Thr-O(CH2)2-SO2-CH3, CH3COOH (threonine 2-(methylsulfonyl)ethylester, hydroace-tate) in N,N-dimethylacetamide was added. The mixture was cooled to 12C and 2 mg of trypsin in 0.025 ml of 0.05 M
calcium acetate was added. HPLC showed after 24 hours at 12 C a 77% conversion into (Thr-O(~H2)2-SO2-CH3) ~ -h-In- me product elutes approximately at the position of (Thr-OMe) h-In.
Example 37 20 mg of porcine insulin was dissolved in 0.1 ml of 10 M acetic acid. 0.2 ml of 2 M Thr-OEt (Et is ethyl) in N,N-dimethylacetamide was added and the mixture was cooled to 12 C. 2 mg of trypsin in 0.025 ml of 0.05 M calcium acetate was added. HPLC showed after 24 hours at 12C a .
~1~%8~
75% conversion into (Thr-O~t)B30-h-In. The product eluted in a position slightly after that o (Thr-OMe)B30-h-In.
ExampLe 38 20 mg of porcine insulin was dissolved in 0.1 ml of 6 M acetic acid. 0.3 ml of 0.67 M Thr(Bu )-OBu (Bu is ter~iary ~utyl) in N,N-dimethylacetamide was added. The mixture was cooled to 12C and 2 mg of trypsin in 0.025 ml of 0.05 M calcium acetate was added. After 24 hours at 12C the conversion into (Thr(But)-OBu~B30-h-In was 77~l The product was eluted from the HPLC column by applying a gradient in acetonitrile from 27% to 40%.
Example 39 20 mg of porcine insulin was dissolved in 0.1 ml of 4 M acetic acid. 0.2 ml of 1.5 M Thr-OMe dissolved in tetrahydrofuran was added and the mixture was cooled to 12C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M
calcium acetate was added. After 4 hours at 12C the analysis by HPLC showed a conversion o~ 75% into (Thr-OMe)B30-h-In~
Example 40 20 mg of porcine insulin was dissolved in 0.1 ml of 4 M acetic acid. 0.8 ml o~ 2 M Thr-OMe dissolved in 1,2-ethanediol was added and the mixture was cooled to 12C.
2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate wa's added. After 4 hours a~ 12C the analysis by HPLC showed a conversion of 48% into (Thr-OMe)B30-h-In~
~z~
Example 41 20 mg of porcine insulin was dissolved in 0.1 ml of 4 M acetic acid. 0.2 ml of 2 M Thr-OMe dissolved in ethanol was added and the mixture was cooled to 12C~ 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added and the reaction was carried out for 4 hours at 12 C. Analysis by HPLC showed a 46% conversion into (Thr-OMe)B30-h-In - Example 42 20 mg of porcine insulin was dissolved in 0.1 ml of 4 M acetic acid. 0.2 ml of 2 M Thr-OMe in acetone was added and the mixture ~as cooled to 12C. 2 mg of trypsin dis-solved in 0.025 ml of 0.05 M calcium acetate was added.
After 4 hours at 12C the analysis by HPLC showed a con-version of 48% into (Thr-OMe) -h-In.
~ . . .
..
I
Claims (39)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for preparing threonineB30 esters of human insulin or a salt or complex thereof, characterized by transpeptidizing at position B30 an insulin compound or a salt or complex thereof convertible thereinto with an L-threonine ester or a salt thereof in a mixture of water, a water miscible organic solvent, and trypsin, the content of water in the reaction mixture being less than 50 per cent (volume/volume), and the reaction temperature being below 50°C, and in the optional presence of an-acid.
2. A process according to claim 1, wherein the concentration of L-threonine ester in the reaction mixture exceeds 0.1 molar, that the reaction temperature is above the freezing point of the reaction mixture, and that the reaction mixture contains from 0 (zero) to 10 equivalents of an acid per equivalent of the L-threonine ester.
3. A process according to claim 1 or claim 2, wherein the insulin compound is a compound selected from the group consisting of porcine, dog, finwhale, sperm-whale, and rabbit insulin, porcine diarginine insulin, porcine split proinsulin, porcine desdipeptide proinsulin, porcine, dog, monkey, and human proinsulin, and mixtures thereof.
4. A process according to claim 1, wherein the insulin compound is of porcine origin.
5. A process according to claim 4, wherein relatively crude insulin is used as the insulin compound.
6. A process according to claim 2, wherein the reaction temperature is from 0-37°C.
7. A process according to claim 6, wherein the reaction temperature is below ambient.
8. A process according to claim 1, wherein the content of water in the reaction mixture is between 40 and 10 per cent (volume/volume).
9. A process according to claim 2, wherein the molar ratio of the L-threonine ester to the insulin compound is above 5:1.
10. A process according to claim 2, wherein the molar ratio of the L-threonine ester to the insulin compound is from about 5:1 to about 1000:1.
11. A process according to claim 4, claim 6 or claim 8, wherein the molar ratio of the L-threonine ester to the insulin compound is from 20:1 to 250:1.
12. A process according to claim 1, wherein the organic solvent is a polar solvent.
13. A process according to claim 12, wherein the solvent is methanol, ethanol, 2-propanol, 1,2-ethanediol, acetone, dioxane, tetrahydrofuran, formamide, N,N-dimethyl-formamide, N,N-dimethylacetamide, N-methylpyrrolidone, hexamethylphosphortriamide, or acetonitrile.
14. A process according to claim 2, wherein the acid is an organic acid.
15. A process according to claim 4, claim 6 or claim 12, wherein the acid is formic acid, acetic acid, propionic acid, and butyric acid.
16. A process according to claim 14, wherein the amount of acid in the reaction mixture is between 0.5 and 5 equivalent(s) per equivalent of the L-threonine ester.
17. A process according to claim 16, wherein the weight ratio between trypsin and the insulin compound in the reaction mixture is above 1:200.
18. A process according to claim 17, wherein said weight ratio is above 1:50 and below 1:1.
19. A process according to claim 1, wherein the transpeptidation is carried out in the presence of calcium ions.
20. A process according to claim 1, wherein the transpeptidation is carried out in a buffer system.
21. A process according to claim 1, wherein the L-threonine ester is added as an acetate, propionate, butyrate, or hydrohalide.
22. A process according to claim 21, wherein the L-threonine ester is added as a hydrochloride.
23. A process according to claim 2, wherein the reaction temperature and the content of water and acid in the reaction mixture are chosen so that the yield of threonineB30 ester of human insulin is higher than 60 per cent.
24. A process according to claim 2, wherein the reaction temperature and the content of water and acid in the reaction mixture are chosen so that the yield of threonineB30 ester of human insulin is higher than 80 per cent.
25. A process according to claim 2, wherein the reaction temperature and the content of water and acid in the reaction mixture are chosen so that the yield of threonineB30 ester of human insulin is higher than 90 per cent.
26. A process according to claim 2, wherein the insulin compound is a compound having the formula I
(I) wherein represents the human des(ThrB30)-insulin moiety wherein GlyA1 is connected to the substituent designated R1 and LysB29 is connected to the substituent designated R2, R2 represents an amino acid or a peptide chain containing not more than 36 amino acids, and R1 represents hydrogen or a group of the general formula R3-X-, wherein X
represents arginine or lysine, and R3 represents a peptide chain containing not more than 35 amino acids, or R2 together with R3 represents a peptide chain containing not more than 35 amino acids, with the proviso that the number of amino acids present in R1 plus R2 is less than 37.
(I) wherein represents the human des(ThrB30)-insulin moiety wherein GlyA1 is connected to the substituent designated R1 and LysB29 is connected to the substituent designated R2, R2 represents an amino acid or a peptide chain containing not more than 36 amino acids, and R1 represents hydrogen or a group of the general formula R3-X-, wherein X
represents arginine or lysine, and R3 represents a peptide chain containing not more than 35 amino acids, or R2 together with R3 represents a peptide chain containing not more than 35 amino acids, with the proviso that the number of amino acids present in R1 plus R2 is less than 37.
27. A process according to claim 26, wherein R is hydrogen, and R2 is -Ala, -Ser, , or , R3 is , and R2 is , or R3 together with R2 is , wherein the terminal alanyl is connected to LysB29, , wherein the terminal alanyl is connected to , wherein the terminal threonyl is connected to LysB29, or , wherein the terminal threonyl is connected to LysB29.
28. A process according to claim 27, wherein R1 is hydrogen, and R2 represents alanine.
29. A process according to claim 1, wherein the L-threonine ester has the general formula II
Thr(R5)-OR (II) wherein R4 represents a carboxyl protecting group, and R5 represents hydrogen or a hydroxyl protecting group.
Thr(R5)-OR (II) wherein R4 represents a carboxyl protecting group, and R5 represents hydrogen or a hydroxyl protecting group.
30. A process according to claim 29, wherein R4 is lower alkyl, substituted benzyl or diphenylmethyl or a group of the general formula -CH2CH2SO2R6 , wherein R6 represents lower alkyl.
31. A process according to claim 30, wherein R4 is methyl, ethyl, tert-butyl, p-methoxy-benzyl, 2,4,6- trimethylbenzyl, or a group of the formula -CH2CH2SO2R6, wherein R6 is methyl, ethyl, propyl, or n-butyl.
32. A process according to claim 1, wherein the insulin compound is porcine insulin or porcine proinsulin, that the L-threonine ester is Thr-OMe, Thr-OBut or Thr-OTmb, that the water miscible organic solvent is N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, hexamethylphosphortriamide, dioxane or acetonitrile, that the reaction temperature is in the range from about 32 to 37°C, that the content of water in the reaction mixture is in the range from about 20 to 43 per cent, that the weight ratio between trypsin and the insulin compound is in the range from about 1:8 to 1:27, that the molar ratio between the L-threonine ester and the insulin compound is in the range from about 60:1 to 180:1, and that there is added between about 1.25 and 3.0 equivalents of acetic acid per equivalent of the L-threonine ester.
33. A process according to claim 32, wherein the insulin compound is porcine insulin, that the L-threonine ester is Thr-OMe or Thr-OBut, that the water miscible organic solvent is N,N-dimethylformamide or N,N-dimethylacetamide, that the reaction temperature is about 37°C, that the content of water in the reaction mixture is between 41 and 43%, that the weight ratio between trypsin and the insulin compound is about 1:8, that the molar ratio between the L-threonine ester and the insulin compound is about 120:1, and that there is added between 1.2 and 1.5 equivalents of acetic acid per equivalent of the L-threonine ester.
34. A process according to claim 1, claim 2 or claim 3, wherein the content of water in the reaction mixture is in the range from 43 to 20 per cent (volume/volume).
35. A process for preparing human insulin according to claim 1, wherein as a subsequent step, the resulting threonineB30 ester of human insulin is deblocked.
36. A process according to claim 35, wherein the deblocking is performed in an aqueous medium in the presence of a base.
37. A process according to claim 35 or claim 36, wherein the deblocking is performed at a pH value of from 8 to 12.
38. A process according to claim 35, wherein the deblocking is performed by acidolysis.
39. A process according to claim 35, wherein the deblocking is performed by trifluoroacetic acid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK57480A DK147109C (en) | 1980-02-11 | 1980-02-11 | METHOD FOR PREPARING HUMAN INSULIN OR HUMAN INSULINESTERS OR A SALT OR COMPLEX THEREOF |
| DK80.574 | 1980-02-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1162868A true CA1162868A (en) | 1984-02-28 |
Family
ID=8095095
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000370483A Expired CA1162868A (en) | 1980-02-11 | 1981-02-10 | Process for preparing insulin esters |
Country Status (19)
| Country | Link |
|---|---|
| JP (1) | JPS56135452A (en) |
| AR (1) | AR231277A1 (en) |
| AU (1) | AU541528B2 (en) |
| BE (1) | BE887480A (en) |
| CA (1) | CA1162868A (en) |
| CH (1) | CH655949A5 (en) |
| DE (1) | DE3104949C2 (en) |
| DK (3) | DK147109C (en) |
| ES (1) | ES8205858A1 (en) |
| FI (1) | FI77876C (en) |
| GB (1) | GB2069502B (en) |
| GR (1) | GR73844B (en) |
| IT (1) | IT1195038B (en) |
| NL (1) | NL178797C (en) |
| NO (1) | NO156247C (en) |
| NZ (1) | NZ196224A (en) |
| PT (1) | PT72485B (en) |
| SE (2) | SE427025B (en) |
| ZA (1) | ZA81898B (en) |
Families Citing this family (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK147437A (en) | 1980-02-11 | 1900-01-01 | Process for preparing human insulin or threonine B30 esters of human insulin, or a salt or complex thereof | |
| DE3101382A1 (en) * | 1981-01-17 | 1982-09-02 | Hoechst Ag, 6000 Frankfurt | "METHOD FOR PRODUCING HUMANISULIN OR ITS DERIVATIVES FROM PIG INSULIN OR ITS DERIVATIVES" |
| CA1174623A (en) * | 1981-09-15 | 1984-09-18 | Finn H. Andresen | Process for preparing human insulin or b-30 esters thereof |
| DK149824C (en) * | 1982-01-22 | 1987-03-16 | Carlsberg Biotechnology Ltd | PROCEDURE FOR ENZYMATIC REPLACEMENT OF B-30 AMINO ACIDS IN INSULINES |
| WO1983002772A1 (en) * | 1982-02-08 | 1983-08-18 | Robin Ewart Offord | An improved method for preparing human insulin from non-human insulin |
| DE3209184A1 (en) * | 1982-03-13 | 1983-09-15 | Hoechst Ag, 6230 Frankfurt | METHOD FOR CONVERTING PRAEPROINSULIN ANALOGS TO INSULINES |
| NL8201650A (en) * | 1982-04-21 | 1983-11-16 | Akzo Nv | SEMISYNTHETIC PREPARATION OF HUMANE INSULIN. |
| DK182483A (en) * | 1982-04-23 | 1983-10-24 | Wako Pure Chem Ind Ltd | METHOD OF SEMI SYNTHESIS OF HUMAN INSULIN AND ALKALIC PROTEASE FOR USE THEREOF |
| DK55183D0 (en) * | 1983-02-09 | 1983-02-09 | Nordisk Insulinlab | METHOD OF MANUFACTURING HUMAN INSULIN |
| DE3334407A1 (en) * | 1983-09-23 | 1985-04-04 | Hoechst Ag, 6230 Frankfurt | INSULINE DERIVATIVES MODIFIED IN POSITION B 30, METHOD FOR THE PRODUCTION AND USE THEREOF AND PHARMACEUTICAL AGENTS FOR THE TREATMENT OF THE DIABETES MELLITUS |
| US5157021A (en) * | 1985-03-15 | 1992-10-20 | Novo Nordisk A/S | Insulin derivatives and pharmaceutical preparations containing these derivatives |
| DK119785D0 (en) * | 1985-03-15 | 1985-03-15 | Nordisk Gentofte | INSULIN PREPARATION |
| SE449472B (en) * | 1985-10-15 | 1987-05-04 | Mth Gruppen Ab | EQUIPMENT AT FLOOR STORES |
| AU612141B2 (en) * | 1987-02-25 | 1991-07-04 | Novo Nordisk A/S | Novel insulin derivatives |
| DK134189D0 (en) * | 1989-03-20 | 1989-03-20 | Nordisk Gentofte | INSULIN COMPOUNDS |
| FI96503C (en) * | 1994-08-26 | 1996-07-10 | Hymatic Ltd Oy | Method and apparatus for controlling a mast |
| EP1711220A1 (en) | 2004-01-16 | 2006-10-18 | Biodel, Inc. | Sublingual drug delivery device |
| US20080090753A1 (en) | 2004-03-12 | 2008-04-17 | Biodel, Inc. | Rapid Acting Injectable Insulin Compositions |
| US7713929B2 (en) | 2006-04-12 | 2010-05-11 | Biodel Inc. | Rapid acting and long acting insulin combination formulations |
| US8084420B2 (en) | 2005-09-29 | 2011-12-27 | Biodel Inc. | Rapid acting and long acting insulin combination formulations |
| WO2007121256A2 (en) | 2006-04-12 | 2007-10-25 | Biodel, Inc. | Rapid acting and long acting insulin combination formulations |
| US9060927B2 (en) | 2009-03-03 | 2015-06-23 | Biodel Inc. | Insulin formulations for rapid uptake |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NO153061C (en) * | 1979-04-13 | 1986-01-08 | Shionogi & Co | PROCEDURE FOR THE PREPARATION OF A B30 THREON INSULIN |
| DK147437A (en) | 1980-02-11 | 1900-01-01 | Process for preparing human insulin or threonine B30 esters of human insulin, or a salt or complex thereof | |
| DE3101382A1 (en) * | 1981-01-17 | 1982-09-02 | Hoechst Ag, 6000 Frankfurt | "METHOD FOR PRODUCING HUMANISULIN OR ITS DERIVATIVES FROM PIG INSULIN OR ITS DERIVATIVES" |
-
0
- DK DK147437D patent/DK147437A/en unknown
-
1980
- 1980-02-11 DK DK57480A patent/DK147109C/en not_active IP Right Cessation
-
1981
- 1981-02-09 DK DK54081A patent/DK147437C/en not_active IP Right Cessation
- 1981-02-09 ES ES499238A patent/ES8205858A1/en not_active Expired
- 1981-02-10 JP JP1758081A patent/JPS56135452A/en active Granted
- 1981-02-10 SE SE8100926A patent/SE427025B/en not_active IP Right Cessation
- 1981-02-10 NZ NZ196224A patent/NZ196224A/en unknown
- 1981-02-10 NO NO810443A patent/NO156247C/en unknown
- 1981-02-10 NL NLAANVRAGE8100624,A patent/NL178797C/en not_active IP Right Cessation
- 1981-02-10 FI FI810385A patent/FI77876C/en not_active IP Right Cessation
- 1981-02-10 PT PT72485A patent/PT72485B/en not_active IP Right Cessation
- 1981-02-10 CA CA000370483A patent/CA1162868A/en not_active Expired
- 1981-02-10 SE SE8100928A patent/SE451143B/en not_active IP Right Cessation
- 1981-02-10 AR AR284258A patent/AR231277A1/en active
- 1981-02-10 IT IT19620/81A patent/IT1195038B/en active
- 1981-02-10 GB GB8104114A patent/GB2069502B/en not_active Expired
- 1981-02-11 DE DE3104949A patent/DE3104949C2/en not_active Expired
- 1981-02-11 GR GR64097A patent/GR73844B/el unknown
- 1981-02-11 ZA ZA00810898A patent/ZA81898B/en unknown
- 1981-02-11 BE BE6/47396A patent/BE887480A/en not_active IP Right Cessation
- 1981-02-11 CH CH910/81A patent/CH655949A5/en not_active IP Right Cessation
- 1981-02-11 AU AU67180/81A patent/AU541528B2/en not_active Ceased
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