CA2008245A1 - Process for the preparation of an insulin precursor - Google Patents
Process for the preparation of an insulin precursorInfo
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- CA2008245A1 CA2008245A1 CA002008245A CA2008245A CA2008245A1 CA 2008245 A1 CA2008245 A1 CA 2008245A1 CA 002008245 A CA002008245 A CA 002008245A CA 2008245 A CA2008245 A CA 2008245A CA 2008245 A1 CA2008245 A1 CA 2008245A1
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- insulin
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- mercaptan
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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
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- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
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- Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
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- Ultra Sonic Daignosis Equipment (AREA)
Abstract
Abstract of the Disclosure: HOE 89/F 021 Process for the preparation of an insulin precursor Preparation of an insulin precursor of the formula I
(A-1) (A-6) (A-20) (A-21) (A-11) (A-7) (I) (B-1) (B-7) (B-19) (B-30) in which R1 = H or an amino acid or peptide radical which can be split off chemically or enzymatically, R2=OH or an amino acid or peptide radical, X = a radical which bonds the insulin A and B
chain, Y = the radical of a genetically encodable amino acid, Z = the radical of a genetically encodable amino acid and A1-A20 and B1-B29 = peptide sequences of insulin which are non-mutated or mutated by replacement of one or more amino acids, by reaction of a precursor in which the disulfide bridges between positions A6 and A11, A7 and B7 and A20 and B19 have not yet been formed, with excess mercaptan in the presence of an organic redox system or at least one organic compound which forms such an organic redox system under the reaction conditions. A particularly preferred organic redox system is the pair of compounds ascorbic acid + dehydroascorbic acid.
(A-1) (A-6) (A-20) (A-21) (A-11) (A-7) (I) (B-1) (B-7) (B-19) (B-30) in which R1 = H or an amino acid or peptide radical which can be split off chemically or enzymatically, R2=OH or an amino acid or peptide radical, X = a radical which bonds the insulin A and B
chain, Y = the radical of a genetically encodable amino acid, Z = the radical of a genetically encodable amino acid and A1-A20 and B1-B29 = peptide sequences of insulin which are non-mutated or mutated by replacement of one or more amino acids, by reaction of a precursor in which the disulfide bridges between positions A6 and A11, A7 and B7 and A20 and B19 have not yet been formed, with excess mercaptan in the presence of an organic redox system or at least one organic compound which forms such an organic redox system under the reaction conditions. A particularly preferred organic redox system is the pair of compounds ascorbic acid + dehydroascorbic acid.
Description
2~ 2~5 HOECHST AKTIENGESELLSCHAFT HOE 89/F 021 Dr. ME/rh Description Pro~ess for the preparation of an insulin precursor Insulin is a molecule which consists of 2 polypeptide chains linked to one another via disulfide bridges. The A chain consists of 21 amino acids and the B chain of 30 amino acids. These two chains are linked to one another in the precursor molecule, the proinsulin, by a peptide, the C-peptide. The C-peptide in human proinsulin consists of 35 amino acids. In the context of the maturation proeess of the hormone, the C-peptide is split off by specific proteases and the proinsulin is in this way converted into insulin (Davidson et al., Nature 333, 93-96, 1988). In addition to the naturally occurring C-peptides, a large number of bonding possibilities betweenthe A chain and B chain are described in the literature (Yanaihara et al., Diabetes 27, 149-160 (1978), Busse et al., Biochemistry 15, 1649-1657 (1971) and Geiger et al., Biochem. Biophys. Res. Com~ 55, 60-66 (1973)).
In the context of genetic engineering, it is now possible to prepare insulin from microorganisms modified by genetic engineering. If E. coli is used as the micro-organism, the product is often expre~sed as fusion protein, that is to say the product is coupled with a protein endogenous to the bacteria, for example with ~-galactosidase. This fusion protein precipitates out in the cell and i8 in this way protected from proteolytic degradation. After breakdown of the cell, the fu&ian protein content is split off chemically or enzymatically and the 6 cysteines of the insulin precursor are con-verted into their S-sulfonates (-S-S03- ) by means of oxidative sulfitolysis. Natural preproinsulin must be produced from this so-called preproinsulin S-sulfonate in a subsequent step, the 3 correct disulfide bridges being formed.
2~ 5 This step is carried out, for example, hy the process described in EP-B-0,037,255 by reaction of the starting 5-sulfonate with a mercaptan in an amount which results in 1 to 5 SH radicals per SS03- radical, in an aqueous medium at a pH of 7 to 11.5 and a S-sulfonate concentra-tion of up to 10 mg per ml of aqueous medium, preferably in the absence of an oxidizing agent.
However, to obtain high folding yields - that is to say high yields of preproinsulin with the "correct" peptide sequence linkages (-S-S- bridges from A6 to A11, from A7 to ~7 and from ~20 to Bl9) - it is necessary to maintain the stated - narrowly limited - SH/SS03- ratio, which requires a not inconsiderable care in carrying out the process and in particular a - not entirely simple -quantitative determination of the SS03- groups in the starting S-sulfonate.
Surprisingly, it has now been found that high folding yields - which are virtually independent of the SH/SS03-ratio within wide limits - are obtained if the procedure ~0 is carried out at a higher - that is to say above 5:1 -SH/SS03- ratio in the presence of an organic redox system or compounds which form such an organic redox system under the reaction conditions; instead of the SS03-groups, it is also possible for other S-protective groups to be present in the corresponding starting substance; in addition, the peptide sequences of the insulin A and B
chain can also be modified by replacement of one or more amino acids.
The higher SH/SS03- (or S-protective groups) ratio means that - as long as only a certain minimum amount of mercaptan i8 exceeded - the procedure can be carried out virtually independently of the level of mercaptan excess without substantial impairment in the yield; exact quantitative determination of the Ss03- (or S-protective~
groups in the corresponding insulin starting substance is in this way also superfluous, which represents a con-82~S
-- 3 --siderable advantage of the process.
Carrying out the process virtually independently of the level of the mercaptan excess is made possib~e by the presence of an organic redox system or of compounds which form such an organic redox system under the reaction conditions.
Although it is known that reduced proinsulin - that is to say proinsulin, the S-S bridges of which, for example, have been split reductively with a mercaptan to give SH
groups - can be reoxidized to the original proinsulin and that this reoxidation is accelerated by the presence of dehydroascorbic acid - cf. D.F. Steiner and J.L. Clark, Proc. Natl. Acad.Sci. USA 60, 622-629 (1968) - even if a redox system of dehydroascorbic acid and ascorbic acid should be formed from dehydroascorbic acid under the reaction conditions described therein, it is a matter there of oxidation of reduced proinsulin with unprotected S groups, whereas in the present case according to the invention only insulin precursors having protected S
groups are suitable starting substances. In addition, the literature reference by D.F. Steiner and J.L. Clark loc.
cit. mentioned contains no indication or suggestion at all in the direction of the use and the action of an organic redox system in recombination of insulin precur-sors containing S-protective groups by means of mercap-tans.
In detail, the invention thus relates to a process for the preparation of an insulin precursor of the formula I
200~5 (A-1) Gl~-NH X
I
(A-6) Cys-S-S
1 l (A-20) (A-21) (A-7) Cys---Cys~ ---- Cys - Z - R2 ¦ (A~ (I) S S
S S
(B-1) 1 1 R1-HN-Phe---Cys--------------- Cys------------Y
(B-7) (B-19) (B-30) in which R1 = H or an amino acid or peptide radical which can be split off chemically or enzymatically, S R2 = OH or an amino acid or peptide radical, preferably OH, X = a radical which bonds the insulin A and chain, preferably an amino acid or peptide radical, Y = the radical of a genetically encodable amino acid, preferably Thr, Ala or Ser, in particular Thr, Z = the radical of a genetically encodable amino acid, preferably Asn, Gln, Asp, Glu, Gly, Ser, Thr, Ala or Met, in particular Asn, and Al-A20 and Bl-B29 = peptide sequences of insulin which are non-mutated or mutated by re-placement of one or more amino acid radi-cals, preferably non-mutated peptide ~equences of human, porcine or bovine insulin, in particular human or porcine insulin, by reaction of a precursor having protected Cys-S groups with a mercaptan in aqueous medium; the process comprises reactinq a precursor having protected Cys-S groups, of s - s -the formula II
(A-1) Gly-NH X
I
(A-6) Cys-S-R3 S-R3 I l (A-20) (A-21) (A-7) Cys-------Cys--------- Cys - Z - R2 ¦ (A~ (II) (B-1) ¦ l R1-HN-Phe---Cys------------------- Cys--------------Y
(~~7) (B-19) (B-30) in which Rl, Rz, X, Y, Z, Al-A20 and Bl-B29 have the same meaning as in formula I and R3 = a Cyæ-S protective group, preferably the -S03- or the tert.-butyl group, with a mercaptan in an amount corresponding to a (mer-captan) SH/(insulin precursor) Cys-S-R3 ratio of more than 5 in the presence of an organic redox system or at least one organic compound which forms such an organic redox system under the reaction conditions.
If R2 in formula I and II = H, the substances are proin-sulin or products derived from proinsulin; if R1 = an amino acid or peptide radical which can be split off chemically or enzymatically, the substances are prepro-insulin and products derived therefrom.
Amino acid radicals which can be split off chemically are those which can be split off, for example, by means of BrCN or N-bromosuccinimide; these are, for example, methionine (Met) or tryptophan (Trp).
Amino acid radicals which can be split off enzymatically are those which can be split off, for example, by means of trypsin (such as, for example, Arg or Lys).
Peptide radicals which can be split off chemically or - 6 ~ 2~5 enzymatically are peptide radicals having at least 2 amino acids.
All the amino acids possible for Rl are preferably from the group of naturally occurring amino acids, that is to say mainly Gly, Ala, Ser, Thr, Val, ~eu, Ile, Asn, Gln, Cys, Met, Tyr, Phe, Pro, Hyp, Arg, Lys, Hyl, Orn, Cit and His.
R2 is OH or - similarly to Rl - likewise an amino scid or peptide radical, the meaning of OH being preferred. The amino acids (including those which form the peptide radical - consisting of at least 2 amino acid radicals) preferably originate - as for Rl - from the group of naturally occurring amino acids.
X is a radical which bonds the insulin A and B chains, preferably an amino acid or peptide radical.
If X is an amino acid radical, the radical of Arg or Lys is preferred; if X is a peptide radical, the radical of a naturally occurring C-peptide - in particular of human, pork or bovine insulin C-peptide - is preferred.
Genetically encodable amino acids - for Y - are (in each case in the L form): Gly, Ala, Ser, Thr, Val, Leu, Ile, Asp, Asn, Glu, Gln, Cys, Met, Arg, Lys, His, Tyr, Phe, Trp and Pro.
Preferred genetically encodable amino acids are Thr, Ala and Ser, in particular Thr.
Z can - like Y - likewise denote the radical of a geneti-cally encodable amino acid, but in this case A~n, Gln, Asp, Glu, Gly, Ser, Thr, Ala and Met, in particular Asn, are preferred.
Al - A20 and B1 - B29 can in principle be the peptide sequences, which are non-mutated or mutated by - 7 - Z ~ S
replacement of one or more amino acids, of all possible insulins; the mutants can be produced by known processes of genetic engineering (site directed mutagenesi~).
Howe~er, the non-mutated peptide sequences of human, S porcine or bovine insulin, in particular of human or porcine insulin (the A1 - A20 and Bl - B29 sequences of human and porcine insulin are identical) are preferred.
The radical R3 which occurs only in formula II denotes virtually any desired Cys-S-protective group, but prefer-ably the -S03- or the tert.-butyl group, the -S03- group being of somewhat greater importance here.
The starting substance of the formula II can on principle be employed within a wide concentration range - advan-tageously between about 10 ~g and 10 mg/ml of solution -lower concentrations as is known leading to higher renaturing yields, 8 ince at low protein concentrations the tendency towards aggregation is reduced. Preferred concentrations are between about 0.1 mg and 0.5 mg/ml.
Suitable mercaptans for the reaction according to the20 invention are in principle all the possible organic compounds having SH groups; mercaptoethanol, thioglycolic acid, glutathione and cysteine, in particular mercap-toethanol and cysteine, are preferred. The mercaptans can be employed individually or as a mixture.
The amount of mercaptan is (to be) chosen 80 that the ratio of its SH groups to the Cys-SR3 groups of the starting material of the formula II is, as far as pos-8 ible, greater than 5.
The upper limit of this ratio is set virtually only by economic considerations; an upper limit of about 100 is advantageous. A ratio of about 10 - 50, in particular about 10 - 30, is preferred. The mercaptan concentration in the reaction batch then depends on the amount of starting material of the formula II employed and the zo~ s chosen SH/Cys-SR3 ratio.
Preferred possible organic redox systems are pairs of compounds, one component of which is an organic compound having the structural element of the formula III
OH OH O
-- C = C -- C --O OH OH (III) - ~ - C = ~ -or an aromatic o- or p-dihydroxy compound and the other component of which i6 an organic compound having the structural element of the formula III in oxidized form =
structural element of the formula III' O O ~
C ~ C (III') or an o- or p-quinone.
The free valencies of the structural element of the formula III and III' can be satisfied by hydrogen or organic groups, such as, for example, Cl-C4-alkyl groups.
However, the structural element can also be part of a ring having preferably 4, 5 or 6 C ring atoms and if appropriate also one or two heteroatoms, such as, for example, O, it being possible for the ring in turn to be substituted by groups which are inert under the reaction conditions, such as, for example, alkyl or hydroxyalkyl groups.
Examples of compounds having the structural element of the formula III are:
reductone OH OH O
~1 H - C = C - C - H
- g - ;~ 2~r.
reductic acid OH OH
HC CH
H2C~ ~ ~o methylreductic acidOH OH
HC CH
\C ~ O
H CH~
ascorbic acid OH OH
(vitamin C)OH HC - CH
~ O~
S The formulae are written here in each case in only one of the tautomeric forms.
All the compounds are reducing. In the oxidized form, the structural element of the formula III becomes that of the formula III'.
Suitable aromatic o- and p-dihydroxy compounds are in principle all the possible aromatic compounds having two OH groups in the o- or p-po~ition, it merely al~o being necessary that the o- or p-quinone formation from the o-and p-dihydroxy compounds cannot be prevented by any particular substituents or the like. Examples of aromatic o- and p-dihydroxy compounds are 1,2-dihydroxybenzene = pyrocatechol, 1,4-dihydroxybenzene = hydroquinone, methyl-hydroquinone, naphtho-1,4-hydroquinone and anthra-hydroquinone; the corresponding quinones are formedtherefrom on oxidation.
In the reaction according to the invention, the par-ticular organic redox systems, consisting, for example, - 10 - ~ ~('8 X ~S
of the pairs of compounds ascorbic acid + dehydroascorbic acid, pyrocatechol + o-quinone, hydroquinone + p-quinone, naphthohydroquinone + naphthoquinone and the like, can thus be employed in virtually any desired ratio (prefer-ably in approximately the equimolar ratio). However, itis also possible for the particular individual components of these pairs of compounds - that is to ~ay, for ex-ample, only ascorbic acid or only dehydroascorbic acid or only hydroquinone and the like - to be added, because in each case the other component (dehydroascorbic acid or ascorbic acid or p-quinone and the like) belonging to the redox pair of compounds i6 formed in the reaction medium.
Preferred organic redox systems are the combinations con-~isting of the pairs of compounds ascorbic acid + dehydroascorbic acid, pyrocatechol + o-quinone and hydroquinone + p-quinone, and preferred individual compounds which form such a redox system under the reaction conditions are the individual components of these pairs of compound~.
Ascorbic acid and/or dehydroascorbic acid are especially preferred.
The amount employed of the compound(s) which form(s) the organic redox system can be varied within wide limits.
The number of moles of the compound(s) which form(s) the organic redox system can be chosen between about 1/10,000 and 10,000, preferably between about 1/10 and 10, based on one gram equivalent of mercaptan (= molecular weight of the mercaptan employed in g/number of SH groups in the mercaptan molecule).
The reaction according to the invention is advantageously carried out in the alkaline pH range, preferably between about 7 and 12, in particular between about 9.5 and 11.
To maintain the desired pH, the addition of a buffer substance is advantageous, the nature and ionic strength X~ 32~5 of the buffer having a certain influence on the folding yield. It is advantageous to keep the ionic strength low, a range of about ~ mM (mM = millimolar) to 1 M (M =
molar~, in particular one of about 5 mM to 50 mM, being preferred. Examples of buffer substances which can be used are borate buffer, carbonate buffer or glycine buffer, the latter being preferred.
A range between about 0 and 45C can be fitated as a general range for the reaction temperature; a range from about 4 to 8C is preferred.
Covering the renaturing solution with a layer of certain gases, such as, for example, oxygen, nitrogen or helium, has no noticeable influence on the renaturing yield.
The duration of the reaction is in general between about 2 and 24 hours, preferably between about 6 and 16 hours.
When the reaction has ended (which can be ascertained, for example, by high performance liquid chromatography), the mixture is worked up in a known manner, such as is also described, for example, in the abovementioned EP-B-0,037,255.
The "correctly" folded product of the formula I can then be converted into the corresponding insulin enzymatically or chemically by known techniques.
The following examples are now intended to explain the invention further, and also to illustrate the advantages over the prior art.
Preproinsulin-S-S03~ obtained by genetic engineering was used as the starting material for the xenaturing experi-ments.
E. coli was used as the expression system. In E. coli, the gene for proinsulin is coupled with a part of the ~-2~ 2~S
galactosidase gene and i6 synthesized as fusion protein, which precipitates in the cell and is deposited in the polar caps ~by the process of DE-A-3,805,150). After breakdown of the cell, the fusion protein content is split off by means of cyanogen halide (in accordance with the process of DE-A-3,440,988) and is then sub~ected to oxidative sulfitolysis (R.C. Marshall and A.S. Ingles in A. Darbre (publishers) ~'Practical Protein Chemistry - A
Handbook" (1986), pages 49 - 53), in order to convert the 6 cysteines into their S-sulfonate form. The prepro-insulin-S-SO3~ (the prefix ~pre~ means that the proinsulin is extended on it~ N-terminus by 5 amino acids) prepared in this way is then concentrated by means of ion exchangers in a manner which i5 known per ~e, precipita-ted and freeze-dried. According to determination by high performance liquid chromatography, the freeze-dried starting material obtained in this way has a content of 60%.
1. Dependence of the folding yield on the mercaptan/
ascorbic acid ratio Preproinsulin-S-SO3~ (60%) is dissolved in a con-centration of 0.33 mg/ml in 20 mM glycine buffer, pH
10.5, which corresponds to a preproinsulin-S-SO3~
concentration of 0.2 mg/ml. 20 ml are employed per batch, to which 480 ~1 of a 0.1 M cysteine solution (~
20-fold molar exce6s per S-S03- group) and between 0 and 480 ~1 of a 0.1 M ascorbic acid solution are added. The renaturing temperature is 8C and the duration of the reaction is 16 hours.
30Ascorbic acid (0.1 M) Folding yield 0 25~ ] comparison 24 ~1 28% ~
48 ~1 53% according to the 96 ~1 78% invention 35480 ~1 81%
This example shows the influence of the redox compound z~
_ 13 -on the folding yield. Whereas predominantly only formation of incorrectly folded proteins occurs in the batch without ascorbic acid, the folding yield in-crease~ in the presence of the redox compound.
2. Dependence of the folding yield on the mercaptan/ascorbic acid exces~
The amount weighed out, volume, buffer, reaction time and temperature and pH correspond to experiment 1. In each case equimolar amounts of cysteine and ascorbic acid are added, the molar excess per S-S03- group being between 2.5 and 100.
Amount of cysteine Excess per Folding yield and ascorbic acid added S-S03- group 60 ~1 2.5 38%~ comparison 15 120 ~1 5 70% J
240 ~1 10 83~
480 ~1 20 85~ according to 1200 ~1 50 81~ the inven-2400 ~1 100 72~ tion This example shows that - as soon as a certain minLmum amount of mercaptan is exceeded - the folding yield is largely independent of the mercaptan excess within a wide range.
In the context of genetic engineering, it is now possible to prepare insulin from microorganisms modified by genetic engineering. If E. coli is used as the micro-organism, the product is often expre~sed as fusion protein, that is to say the product is coupled with a protein endogenous to the bacteria, for example with ~-galactosidase. This fusion protein precipitates out in the cell and i8 in this way protected from proteolytic degradation. After breakdown of the cell, the fu&ian protein content is split off chemically or enzymatically and the 6 cysteines of the insulin precursor are con-verted into their S-sulfonates (-S-S03- ) by means of oxidative sulfitolysis. Natural preproinsulin must be produced from this so-called preproinsulin S-sulfonate in a subsequent step, the 3 correct disulfide bridges being formed.
2~ 5 This step is carried out, for example, hy the process described in EP-B-0,037,255 by reaction of the starting 5-sulfonate with a mercaptan in an amount which results in 1 to 5 SH radicals per SS03- radical, in an aqueous medium at a pH of 7 to 11.5 and a S-sulfonate concentra-tion of up to 10 mg per ml of aqueous medium, preferably in the absence of an oxidizing agent.
However, to obtain high folding yields - that is to say high yields of preproinsulin with the "correct" peptide sequence linkages (-S-S- bridges from A6 to A11, from A7 to ~7 and from ~20 to Bl9) - it is necessary to maintain the stated - narrowly limited - SH/SS03- ratio, which requires a not inconsiderable care in carrying out the process and in particular a - not entirely simple -quantitative determination of the SS03- groups in the starting S-sulfonate.
Surprisingly, it has now been found that high folding yields - which are virtually independent of the SH/SS03-ratio within wide limits - are obtained if the procedure ~0 is carried out at a higher - that is to say above 5:1 -SH/SS03- ratio in the presence of an organic redox system or compounds which form such an organic redox system under the reaction conditions; instead of the SS03-groups, it is also possible for other S-protective groups to be present in the corresponding starting substance; in addition, the peptide sequences of the insulin A and B
chain can also be modified by replacement of one or more amino acids.
The higher SH/SS03- (or S-protective groups) ratio means that - as long as only a certain minimum amount of mercaptan i8 exceeded - the procedure can be carried out virtually independently of the level of mercaptan excess without substantial impairment in the yield; exact quantitative determination of the Ss03- (or S-protective~
groups in the corresponding insulin starting substance is in this way also superfluous, which represents a con-82~S
-- 3 --siderable advantage of the process.
Carrying out the process virtually independently of the level of the mercaptan excess is made possib~e by the presence of an organic redox system or of compounds which form such an organic redox system under the reaction conditions.
Although it is known that reduced proinsulin - that is to say proinsulin, the S-S bridges of which, for example, have been split reductively with a mercaptan to give SH
groups - can be reoxidized to the original proinsulin and that this reoxidation is accelerated by the presence of dehydroascorbic acid - cf. D.F. Steiner and J.L. Clark, Proc. Natl. Acad.Sci. USA 60, 622-629 (1968) - even if a redox system of dehydroascorbic acid and ascorbic acid should be formed from dehydroascorbic acid under the reaction conditions described therein, it is a matter there of oxidation of reduced proinsulin with unprotected S groups, whereas in the present case according to the invention only insulin precursors having protected S
groups are suitable starting substances. In addition, the literature reference by D.F. Steiner and J.L. Clark loc.
cit. mentioned contains no indication or suggestion at all in the direction of the use and the action of an organic redox system in recombination of insulin precur-sors containing S-protective groups by means of mercap-tans.
In detail, the invention thus relates to a process for the preparation of an insulin precursor of the formula I
200~5 (A-1) Gl~-NH X
I
(A-6) Cys-S-S
1 l (A-20) (A-21) (A-7) Cys---Cys~ ---- Cys - Z - R2 ¦ (A~ (I) S S
S S
(B-1) 1 1 R1-HN-Phe---Cys--------------- Cys------------Y
(B-7) (B-19) (B-30) in which R1 = H or an amino acid or peptide radical which can be split off chemically or enzymatically, S R2 = OH or an amino acid or peptide radical, preferably OH, X = a radical which bonds the insulin A and chain, preferably an amino acid or peptide radical, Y = the radical of a genetically encodable amino acid, preferably Thr, Ala or Ser, in particular Thr, Z = the radical of a genetically encodable amino acid, preferably Asn, Gln, Asp, Glu, Gly, Ser, Thr, Ala or Met, in particular Asn, and Al-A20 and Bl-B29 = peptide sequences of insulin which are non-mutated or mutated by re-placement of one or more amino acid radi-cals, preferably non-mutated peptide ~equences of human, porcine or bovine insulin, in particular human or porcine insulin, by reaction of a precursor having protected Cys-S groups with a mercaptan in aqueous medium; the process comprises reactinq a precursor having protected Cys-S groups, of s - s -the formula II
(A-1) Gly-NH X
I
(A-6) Cys-S-R3 S-R3 I l (A-20) (A-21) (A-7) Cys-------Cys--------- Cys - Z - R2 ¦ (A~ (II) (B-1) ¦ l R1-HN-Phe---Cys------------------- Cys--------------Y
(~~7) (B-19) (B-30) in which Rl, Rz, X, Y, Z, Al-A20 and Bl-B29 have the same meaning as in formula I and R3 = a Cyæ-S protective group, preferably the -S03- or the tert.-butyl group, with a mercaptan in an amount corresponding to a (mer-captan) SH/(insulin precursor) Cys-S-R3 ratio of more than 5 in the presence of an organic redox system or at least one organic compound which forms such an organic redox system under the reaction conditions.
If R2 in formula I and II = H, the substances are proin-sulin or products derived from proinsulin; if R1 = an amino acid or peptide radical which can be split off chemically or enzymatically, the substances are prepro-insulin and products derived therefrom.
Amino acid radicals which can be split off chemically are those which can be split off, for example, by means of BrCN or N-bromosuccinimide; these are, for example, methionine (Met) or tryptophan (Trp).
Amino acid radicals which can be split off enzymatically are those which can be split off, for example, by means of trypsin (such as, for example, Arg or Lys).
Peptide radicals which can be split off chemically or - 6 ~ 2~5 enzymatically are peptide radicals having at least 2 amino acids.
All the amino acids possible for Rl are preferably from the group of naturally occurring amino acids, that is to say mainly Gly, Ala, Ser, Thr, Val, ~eu, Ile, Asn, Gln, Cys, Met, Tyr, Phe, Pro, Hyp, Arg, Lys, Hyl, Orn, Cit and His.
R2 is OH or - similarly to Rl - likewise an amino scid or peptide radical, the meaning of OH being preferred. The amino acids (including those which form the peptide radical - consisting of at least 2 amino acid radicals) preferably originate - as for Rl - from the group of naturally occurring amino acids.
X is a radical which bonds the insulin A and B chains, preferably an amino acid or peptide radical.
If X is an amino acid radical, the radical of Arg or Lys is preferred; if X is a peptide radical, the radical of a naturally occurring C-peptide - in particular of human, pork or bovine insulin C-peptide - is preferred.
Genetically encodable amino acids - for Y - are (in each case in the L form): Gly, Ala, Ser, Thr, Val, Leu, Ile, Asp, Asn, Glu, Gln, Cys, Met, Arg, Lys, His, Tyr, Phe, Trp and Pro.
Preferred genetically encodable amino acids are Thr, Ala and Ser, in particular Thr.
Z can - like Y - likewise denote the radical of a geneti-cally encodable amino acid, but in this case A~n, Gln, Asp, Glu, Gly, Ser, Thr, Ala and Met, in particular Asn, are preferred.
Al - A20 and B1 - B29 can in principle be the peptide sequences, which are non-mutated or mutated by - 7 - Z ~ S
replacement of one or more amino acids, of all possible insulins; the mutants can be produced by known processes of genetic engineering (site directed mutagenesi~).
Howe~er, the non-mutated peptide sequences of human, S porcine or bovine insulin, in particular of human or porcine insulin (the A1 - A20 and Bl - B29 sequences of human and porcine insulin are identical) are preferred.
The radical R3 which occurs only in formula II denotes virtually any desired Cys-S-protective group, but prefer-ably the -S03- or the tert.-butyl group, the -S03- group being of somewhat greater importance here.
The starting substance of the formula II can on principle be employed within a wide concentration range - advan-tageously between about 10 ~g and 10 mg/ml of solution -lower concentrations as is known leading to higher renaturing yields, 8 ince at low protein concentrations the tendency towards aggregation is reduced. Preferred concentrations are between about 0.1 mg and 0.5 mg/ml.
Suitable mercaptans for the reaction according to the20 invention are in principle all the possible organic compounds having SH groups; mercaptoethanol, thioglycolic acid, glutathione and cysteine, in particular mercap-toethanol and cysteine, are preferred. The mercaptans can be employed individually or as a mixture.
The amount of mercaptan is (to be) chosen 80 that the ratio of its SH groups to the Cys-SR3 groups of the starting material of the formula II is, as far as pos-8 ible, greater than 5.
The upper limit of this ratio is set virtually only by economic considerations; an upper limit of about 100 is advantageous. A ratio of about 10 - 50, in particular about 10 - 30, is preferred. The mercaptan concentration in the reaction batch then depends on the amount of starting material of the formula II employed and the zo~ s chosen SH/Cys-SR3 ratio.
Preferred possible organic redox systems are pairs of compounds, one component of which is an organic compound having the structural element of the formula III
OH OH O
-- C = C -- C --O OH OH (III) - ~ - C = ~ -or an aromatic o- or p-dihydroxy compound and the other component of which i6 an organic compound having the structural element of the formula III in oxidized form =
structural element of the formula III' O O ~
C ~ C (III') or an o- or p-quinone.
The free valencies of the structural element of the formula III and III' can be satisfied by hydrogen or organic groups, such as, for example, Cl-C4-alkyl groups.
However, the structural element can also be part of a ring having preferably 4, 5 or 6 C ring atoms and if appropriate also one or two heteroatoms, such as, for example, O, it being possible for the ring in turn to be substituted by groups which are inert under the reaction conditions, such as, for example, alkyl or hydroxyalkyl groups.
Examples of compounds having the structural element of the formula III are:
reductone OH OH O
~1 H - C = C - C - H
- g - ;~ 2~r.
reductic acid OH OH
HC CH
H2C~ ~ ~o methylreductic acidOH OH
HC CH
\C ~ O
H CH~
ascorbic acid OH OH
(vitamin C)OH HC - CH
~ O~
S The formulae are written here in each case in only one of the tautomeric forms.
All the compounds are reducing. In the oxidized form, the structural element of the formula III becomes that of the formula III'.
Suitable aromatic o- and p-dihydroxy compounds are in principle all the possible aromatic compounds having two OH groups in the o- or p-po~ition, it merely al~o being necessary that the o- or p-quinone formation from the o-and p-dihydroxy compounds cannot be prevented by any particular substituents or the like. Examples of aromatic o- and p-dihydroxy compounds are 1,2-dihydroxybenzene = pyrocatechol, 1,4-dihydroxybenzene = hydroquinone, methyl-hydroquinone, naphtho-1,4-hydroquinone and anthra-hydroquinone; the corresponding quinones are formedtherefrom on oxidation.
In the reaction according to the invention, the par-ticular organic redox systems, consisting, for example, - 10 - ~ ~('8 X ~S
of the pairs of compounds ascorbic acid + dehydroascorbic acid, pyrocatechol + o-quinone, hydroquinone + p-quinone, naphthohydroquinone + naphthoquinone and the like, can thus be employed in virtually any desired ratio (prefer-ably in approximately the equimolar ratio). However, itis also possible for the particular individual components of these pairs of compounds - that is to ~ay, for ex-ample, only ascorbic acid or only dehydroascorbic acid or only hydroquinone and the like - to be added, because in each case the other component (dehydroascorbic acid or ascorbic acid or p-quinone and the like) belonging to the redox pair of compounds i6 formed in the reaction medium.
Preferred organic redox systems are the combinations con-~isting of the pairs of compounds ascorbic acid + dehydroascorbic acid, pyrocatechol + o-quinone and hydroquinone + p-quinone, and preferred individual compounds which form such a redox system under the reaction conditions are the individual components of these pairs of compound~.
Ascorbic acid and/or dehydroascorbic acid are especially preferred.
The amount employed of the compound(s) which form(s) the organic redox system can be varied within wide limits.
The number of moles of the compound(s) which form(s) the organic redox system can be chosen between about 1/10,000 and 10,000, preferably between about 1/10 and 10, based on one gram equivalent of mercaptan (= molecular weight of the mercaptan employed in g/number of SH groups in the mercaptan molecule).
The reaction according to the invention is advantageously carried out in the alkaline pH range, preferably between about 7 and 12, in particular between about 9.5 and 11.
To maintain the desired pH, the addition of a buffer substance is advantageous, the nature and ionic strength X~ 32~5 of the buffer having a certain influence on the folding yield. It is advantageous to keep the ionic strength low, a range of about ~ mM (mM = millimolar) to 1 M (M =
molar~, in particular one of about 5 mM to 50 mM, being preferred. Examples of buffer substances which can be used are borate buffer, carbonate buffer or glycine buffer, the latter being preferred.
A range between about 0 and 45C can be fitated as a general range for the reaction temperature; a range from about 4 to 8C is preferred.
Covering the renaturing solution with a layer of certain gases, such as, for example, oxygen, nitrogen or helium, has no noticeable influence on the renaturing yield.
The duration of the reaction is in general between about 2 and 24 hours, preferably between about 6 and 16 hours.
When the reaction has ended (which can be ascertained, for example, by high performance liquid chromatography), the mixture is worked up in a known manner, such as is also described, for example, in the abovementioned EP-B-0,037,255.
The "correctly" folded product of the formula I can then be converted into the corresponding insulin enzymatically or chemically by known techniques.
The following examples are now intended to explain the invention further, and also to illustrate the advantages over the prior art.
Preproinsulin-S-S03~ obtained by genetic engineering was used as the starting material for the xenaturing experi-ments.
E. coli was used as the expression system. In E. coli, the gene for proinsulin is coupled with a part of the ~-2~ 2~S
galactosidase gene and i6 synthesized as fusion protein, which precipitates in the cell and is deposited in the polar caps ~by the process of DE-A-3,805,150). After breakdown of the cell, the fusion protein content is split off by means of cyanogen halide (in accordance with the process of DE-A-3,440,988) and is then sub~ected to oxidative sulfitolysis (R.C. Marshall and A.S. Ingles in A. Darbre (publishers) ~'Practical Protein Chemistry - A
Handbook" (1986), pages 49 - 53), in order to convert the 6 cysteines into their S-sulfonate form. The prepro-insulin-S-SO3~ (the prefix ~pre~ means that the proinsulin is extended on it~ N-terminus by 5 amino acids) prepared in this way is then concentrated by means of ion exchangers in a manner which i5 known per ~e, precipita-ted and freeze-dried. According to determination by high performance liquid chromatography, the freeze-dried starting material obtained in this way has a content of 60%.
1. Dependence of the folding yield on the mercaptan/
ascorbic acid ratio Preproinsulin-S-SO3~ (60%) is dissolved in a con-centration of 0.33 mg/ml in 20 mM glycine buffer, pH
10.5, which corresponds to a preproinsulin-S-SO3~
concentration of 0.2 mg/ml. 20 ml are employed per batch, to which 480 ~1 of a 0.1 M cysteine solution (~
20-fold molar exce6s per S-S03- group) and between 0 and 480 ~1 of a 0.1 M ascorbic acid solution are added. The renaturing temperature is 8C and the duration of the reaction is 16 hours.
30Ascorbic acid (0.1 M) Folding yield 0 25~ ] comparison 24 ~1 28% ~
48 ~1 53% according to the 96 ~1 78% invention 35480 ~1 81%
This example shows the influence of the redox compound z~
_ 13 -on the folding yield. Whereas predominantly only formation of incorrectly folded proteins occurs in the batch without ascorbic acid, the folding yield in-crease~ in the presence of the redox compound.
2. Dependence of the folding yield on the mercaptan/ascorbic acid exces~
The amount weighed out, volume, buffer, reaction time and temperature and pH correspond to experiment 1. In each case equimolar amounts of cysteine and ascorbic acid are added, the molar excess per S-S03- group being between 2.5 and 100.
Amount of cysteine Excess per Folding yield and ascorbic acid added S-S03- group 60 ~1 2.5 38%~ comparison 15 120 ~1 5 70% J
240 ~1 10 83~
480 ~1 20 85~ according to 1200 ~1 50 81~ the inven-2400 ~1 100 72~ tion This example shows that - as soon as a certain minLmum amount of mercaptan is exceeded - the folding yield is largely independent of the mercaptan excess within a wide range.
3. Dependence of the folding yield on the pH
The amount weighed out, volume, buffer concentration and reaction time and temperature correspond to experiment 1. In the experiment, 480 ~1 of 0.1 N
cysteine solution and 480 ~1 of 0.1 M ascorbic acid solution are added ~~ 20-fold molar excess per S-S03-group) 32~5 pH Folding yield 1~.0 79~
10.5 81%
10.0 66%
9.5 53%
9.0 46%
8.5 33%
8.0 24%
The amount weighed out, volume, buffer concentration and reaction time and temperature correspond to experiment 1. In the experiment, 480 ~1 of 0.1 N
cysteine solution and 480 ~1 of 0.1 M ascorbic acid solution are added ~~ 20-fold molar excess per S-S03-group) 32~5 pH Folding yield 1~.0 79~
10.5 81%
10.0 66%
9.5 53%
9.0 46%
8.5 33%
8.0 24%
4. Dependence of the folding yield on the 10type of mercaptan The amount weighed out, volume, buffer concentration, reaction time and temperature and pH correspond to experiment 1. In the experiment, in each case 480 ~1 of the corresponding 0.1 M mercaptan solution and 480 ~1 of 0.1 M ascorbic acid solution are added (z 20-fold molar excess per S-S03- group) Mercaptan Folding yield Cysteine 81%
Mercaptoethanol 86%
Glutathione 7s%
Thioglycolic acid 74%
3-Mercapto-1,2-propanediol 76%
Mercaptoethanol 86%
Glutathione 7s%
Thioglycolic acid 74%
3-Mercapto-1,2-propanediol 76%
5. Dependence of the folding yield on the buffer fiubstance and on the ionic ~trength of the buffer The amount weighed out, volume, reaction time and temperature and pH correspond to experiment 1. In the experiment, in each case 480 ~1 of the corresponding 0.1 M mercaptan solution and 480 ~1 of 0.1 M ascorbic acid solution are added (~ 20-fold molar excess per S-S03- group).
Buffer Folding yield 10 mM glycine 89~
100 mM glycine 82%
10 mM borate 82%
~ 2 100 mM borate 51%
10 mM carbonate 88~
100 mM carbonate 72%
Buffer Folding yield 10 mM glycine 89~
100 mM glycine 82%
10 mM borate 82%
~ 2 100 mM borate 51%
10 mM carbonate 88~
100 mM carbonate 72%
6. Dependence of the folding yield on the reaction tL~e S The amount weighed out, volume, buffer concentration, pH and reaction temperature correspond to experiment 1. In each case 480 ~1 of a 0.1 M mercaptoethanol solution and 480 ~1 of a 0.1 M ascorbic acid solution are added (- 20-fold molar excess per S-S03- group) 10Reaction time (hours) Folding yield 0.25 25%
0.5 40%
1 46%
2 61%
4 70%
6 77%
8 83%
24 79%
0.5 40%
1 46%
2 61%
4 70%
6 77%
8 83%
24 79%
7. Dependence of the folding yield on the preproinsulin-20S-sulfonate concentration The volume, buffer composition, pH, reaction time and temperature correspond to experiment 1. In each case enough mercaptoethanol stock solution and ascorbic acid stock solution are added to the amount of prepro-insulin-S-~ulfonate employed for a ~ 20-fold molar excess per S-S03- group to exist.
Preproinsulin-S-S03~ concentration Folding yield 0.1 ml/ml 86%
0.2 ml/ml 84%
0.3 ml/ml 78 0.4 ml/ml 65~
0.5 ml/ml 55%
1.0 ml/ml 19~
2.5 ml/ml 5%
~2~)G~4~, _ 16 -8. Dependence of the folding yield on the ascorbi~ acid/
mercaptoethanol excess using modified starting material A precursor molecule in which the A and B chain of the insulin are linked only by an arginine is employed.
The production of this molecule by genetic engineering is carried out as described at the start of the descriptions of the experiments. The freeze-dried material has a content of 60% and is dissolved in a concentration of 0.5 mg/ml in 20 mM glycine buffer, pH
10.5, which corresponds to a precursor concentration of 0.3 mg/ml. The reaction time and temperature correspond to experiment 1 and the molar excess of the ascorbic acid/mercaptan mixture varies between 2.5-and 50-fold.
Excess per S-S03 group Folding yield 2.5 56%~ comparison 61%1 74~ according to 75% the invention 55~
Preproinsulin-S-S03~ concentration Folding yield 0.1 ml/ml 86%
0.2 ml/ml 84%
0.3 ml/ml 78 0.4 ml/ml 65~
0.5 ml/ml 55%
1.0 ml/ml 19~
2.5 ml/ml 5%
~2~)G~4~, _ 16 -8. Dependence of the folding yield on the ascorbi~ acid/
mercaptoethanol excess using modified starting material A precursor molecule in which the A and B chain of the insulin are linked only by an arginine is employed.
The production of this molecule by genetic engineering is carried out as described at the start of the descriptions of the experiments. The freeze-dried material has a content of 60% and is dissolved in a concentration of 0.5 mg/ml in 20 mM glycine buffer, pH
10.5, which corresponds to a precursor concentration of 0.3 mg/ml. The reaction time and temperature correspond to experiment 1 and the molar excess of the ascorbic acid/mercaptan mixture varies between 2.5-and 50-fold.
Excess per S-S03 group Folding yield 2.5 56%~ comparison 61%1 74~ according to 75% the invention 55~
9. Dependence of the folding yield on the redox system The amount weighed out, volume, buffer composition, pH
and reaction time and temperature correspond to experiment 1. A ~ 20-fold molar excess of mercapto-ethanol/S-SO3~ group and a 20-fold molar excess of the corresponding redox partner are added~
Redox partner Folding yield Ascorbic acid 77%
Dehydroascorbic acid 70%
Pyrocatechol 66%
Hydroquinone 40%
Benzo(-p-)quinone 23%
and reaction time and temperature correspond to experiment 1. A ~ 20-fold molar excess of mercapto-ethanol/S-SO3~ group and a 20-fold molar excess of the corresponding redox partner are added~
Redox partner Folding yield Ascorbic acid 77%
Dehydroascorbic acid 70%
Pyrocatechol 66%
Hydroquinone 40%
Benzo(-p-)quinone 23%
Claims (10)
1. A process for the preparation of an insulin precursor of the formula I
(A-1) (A-6) (A-20) (A-21) (A-7) (A-11) (I) (B-1) (B-7) (B-19) (B-30) in which R1 = H or an amino acid or peptide radical which can be split off chemically or enzy matically, R2 = OH or an amino acid or peptide radical, preferably OH, X = a radical which bonds the insulin A and B chain, preferably an amino acid or peptide radical, Y = the radical of a genetically encodable amino acid, preferably Thr, Ala or Ser, in particular Thr, Z = the radical of a genetically encodable amino acid, preferably Asn, Gln, Asp, Glu, Gly, Ser, Thr, Ala or Met, in particular Asn, and A1-A20 and B1-B29 = peptide seqyences of insulin which are non-mutated or mutated by replacement of one or more amino acid radi-cals, preferably non-mutated peptide sequences of human, porcine or bovine insulin, in particular human or porcine insulin, by reaction of a precursor having protected Cys-S
groups with a mercaptan in aqueous medium; which comprises reacting a precursor having protected Cys-S
groups, of the formula II
(A-1) (A-6) (A-20) (A-21) (A-7) (II) (A-11) (B-1) (B-7) (B-19) (B-30) in which R1, R2, X, Y, Z, A1-A20 and B1-B29 have the same meaning as in formula I and R3 = a Cys-S protective group, preferably the -SO3- or the tert.-butyl group, with a mercsptan in an amount corresponding to a (mer-captan) SH/(insulin precursor) Cys-S-R3 ratio of more than 5 in the presence of an organic redox system or at least one organic compound which forms such an organic redox system under the reaction conditions.
(A-1) (A-6) (A-20) (A-21) (A-7) (A-11) (I) (B-1) (B-7) (B-19) (B-30) in which R1 = H or an amino acid or peptide radical which can be split off chemically or enzy matically, R2 = OH or an amino acid or peptide radical, preferably OH, X = a radical which bonds the insulin A and B chain, preferably an amino acid or peptide radical, Y = the radical of a genetically encodable amino acid, preferably Thr, Ala or Ser, in particular Thr, Z = the radical of a genetically encodable amino acid, preferably Asn, Gln, Asp, Glu, Gly, Ser, Thr, Ala or Met, in particular Asn, and A1-A20 and B1-B29 = peptide seqyences of insulin which are non-mutated or mutated by replacement of one or more amino acid radi-cals, preferably non-mutated peptide sequences of human, porcine or bovine insulin, in particular human or porcine insulin, by reaction of a precursor having protected Cys-S
groups with a mercaptan in aqueous medium; which comprises reacting a precursor having protected Cys-S
groups, of the formula II
(A-1) (A-6) (A-20) (A-21) (A-7) (II) (A-11) (B-1) (B-7) (B-19) (B-30) in which R1, R2, X, Y, Z, A1-A20 and B1-B29 have the same meaning as in formula I and R3 = a Cys-S protective group, preferably the -SO3- or the tert.-butyl group, with a mercsptan in an amount corresponding to a (mer-captan) SH/(insulin precursor) Cys-S-R3 ratio of more than 5 in the presence of an organic redox system or at least one organic compound which forms such an organic redox system under the reaction conditions.
2. The process as claimed in claim 1, wherein mercap-toethanol and/or cysteine are used as the mercaptan.
3. The process as claimed in either of claims 1 and 2, carried out at a (mercaptan) SH/Cys-SR3 (in formula II) ratio of more than 5 to 100, preferably about 10 to 50 and in particular about 10 to 30.
4. The process as claimed in any one of claims 1 to 3, wherein the organic redox system used is a pair of compounds, one component of which is an organic compound having the structural element of the formula III
(III) or an aromatic o- or p-dihydroxy compound and the other component of which is an organic compound having the structural element of the formula III in oxidized form = structural element of the formula III' (III') or an o- or p-quinone.
(III) or an aromatic o- or p-dihydroxy compound and the other component of which is an organic compound having the structural element of the formula III in oxidized form = structural element of the formula III' (III') or an o- or p-quinone.
5. The process as claimed in any one of claims 1 to 3, wherein the organic compound used which forms an organic redox system under the reaction conditions is one or more of the individual components mentioned in claim 4.
6. The process as claimed in any one of claims 1 to 5, wherein the organic redox system used is the pair of compounds ascorbic acid + dehydroascorbic acid, pyrocatechol + o-quinone or hydroquinone + p-quinone and the organic compound employed which can form such a redox system under the reaction conditions is in each case only one component of this pair of com-pounds.
7. The process as claimed in any one of claims 1 to 5, carried out in the presence of ascorbic acid and/or dehydroascorbic acid.
8. The process as claimed in any one of claims 1 to 7, wherein the mercaptan and the compound(s) which form(6) the organic redox system are employed in a ratio of 1 gram equivalent of mercaptan to 1/10,000 to 10,000, preferably 1/10 to 10, moles of the com-pound(s) which form(s) the organic redox system.
9. The process as claimed in any one of claims 1 to 8, wherein the reaction is carried out at a pH of between about 7 and 12, preferably between about 9.5 and 11.
10. The process as claimed in claim 1, and substantially as described herein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3901719A DE3901719A1 (en) | 1989-01-21 | 1989-01-21 | METHOD FOR PRODUCING AN INSULIN PREVENTE |
| DEP3901719.2 | 1989-01-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2008245A1 true CA2008245A1 (en) | 1990-07-21 |
Family
ID=6372512
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002008245A Abandoned CA2008245A1 (en) | 1989-01-21 | 1990-01-22 | Process for the preparation of an insulin precursor |
Country Status (17)
| Country | Link |
|---|---|
| EP (1) | EP0381958B1 (en) |
| JP (1) | JPH02233699A (en) |
| AT (1) | ATE126808T1 (en) |
| AU (1) | AU624894B2 (en) |
| CA (1) | CA2008245A1 (en) |
| DE (2) | DE3901719A1 (en) |
| DK (1) | DK0381958T3 (en) |
| ES (1) | ES2078249T3 (en) |
| FI (1) | FI900296A0 (en) |
| GR (1) | GR3017591T3 (en) |
| HU (1) | HU208704B (en) |
| IE (1) | IE900217L (en) |
| IL (1) | IL93115A (en) |
| NO (1) | NO178861C (en) |
| NZ (1) | NZ232178A (en) |
| PT (1) | PT92907B (en) |
| ZA (1) | ZA90396B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6590072B2 (en) | 1999-12-22 | 2003-07-08 | Nna/S | Method for extractive refolding of scrambled single-chain polypeptides |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3901718A1 (en) * | 1989-01-21 | 1990-07-26 | Hoechst Ag | METHOD FOR RENATURING INCORRECT RECOMBINANT OF INSULIN PREFERRED |
| AU664021B2 (en) * | 1990-09-05 | 1995-11-02 | Natinco Nv | Solubilization of proteins in active forms |
| EP0600372B1 (en) * | 1992-12-02 | 1997-02-05 | Hoechst Aktiengesellschaft | Process for the preparation of proinsulin with correctly linked cystin bridges |
| JP4624495B2 (en) * | 1994-12-29 | 2011-02-02 | フェリング・インターナショナル・センター・エス．・エー． | Production of human insulin |
| KR100253916B1 (en) * | 1997-12-29 | 2000-05-01 | 김충환 | A process for preparing human proinsulin |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RO81951B (en) * | 1980-03-27 | 1984-02-28 | Eli Lilly And Company | Process for the preparation of an insulin precursory |
| DE3501641A1 (en) * | 1985-01-19 | 1986-07-24 | Hoechst Ag, 6230 Frankfurt | METHOD FOR OBTAINING INSULIN PRECURSORS FROM REACTION MIXTURES WHICH ARE INCLUDED IN THE FOLDING OF INSULIN PRECURSORS FROM THE CORRESPONDING S-SULPHONATES |
| DE3901718A1 (en) * | 1989-01-21 | 1990-07-26 | Hoechst Ag | METHOD FOR RENATURING INCORRECT RECOMBINANT OF INSULIN PREFERRED |
-
1989
- 1989-01-21 DE DE3901719A patent/DE3901719A1/en not_active Withdrawn
-
1990
- 1990-01-17 ES ES90100889T patent/ES2078249T3/en not_active Expired - Lifetime
- 1990-01-17 DE DE59009540T patent/DE59009540D1/en not_active Expired - Lifetime
- 1990-01-17 AT AT90100889T patent/ATE126808T1/en not_active IP Right Cessation
- 1990-01-17 EP EP90100889A patent/EP0381958B1/en not_active Expired - Lifetime
- 1990-01-17 DK DK90100889.6T patent/DK0381958T3/en active
- 1990-01-18 FI FI900296A patent/FI900296A0/en not_active Application Discontinuation
- 1990-01-19 IE IE900217A patent/IE900217L/en unknown
- 1990-01-19 NO NO900278A patent/NO178861C/en unknown
- 1990-01-19 JP JP2008568A patent/JPH02233699A/en active Pending
- 1990-01-19 IL IL9311590A patent/IL93115A/en not_active IP Right Cessation
- 1990-01-19 HU HU90197A patent/HU208704B/en not_active IP Right Cessation
- 1990-01-19 PT PT92907A patent/PT92907B/en not_active IP Right Cessation
- 1990-01-19 NZ NZ232178A patent/NZ232178A/en unknown
- 1990-01-19 ZA ZA90396A patent/ZA90396B/en unknown
- 1990-01-19 AU AU48621/90A patent/AU624894B2/en not_active Ceased
- 1990-01-22 CA CA002008245A patent/CA2008245A1/en not_active Abandoned
-
1995
- 1995-09-29 GR GR950402704T patent/GR3017591T3/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6590072B2 (en) | 1999-12-22 | 2003-07-08 | Nna/S | Method for extractive refolding of scrambled single-chain polypeptides |
Also Published As
| Publication number | Publication date |
|---|---|
| DE3901719A1 (en) | 1990-07-26 |
| JPH02233699A (en) | 1990-09-17 |
| NO178861C (en) | 1996-06-19 |
| PT92907B (en) | 1995-12-29 |
| NO900278L (en) | 1990-07-23 |
| PT92907A (en) | 1990-07-31 |
| IE900217L (en) | 1990-07-21 |
| NO900278D0 (en) | 1990-01-19 |
| FI900296A0 (en) | 1990-01-18 |
| IL93115A0 (en) | 1990-11-05 |
| EP0381958B1 (en) | 1995-08-23 |
| EP0381958A3 (en) | 1991-07-24 |
| ZA90396B (en) | 1990-09-26 |
| NO178861B (en) | 1996-03-11 |
| HU208704B (en) | 1993-12-28 |
| GR3017591T3 (en) | 1995-12-31 |
| AU624894B2 (en) | 1992-06-25 |
| DE59009540D1 (en) | 1995-09-28 |
| DK0381958T3 (en) | 1995-12-18 |
| AU4862190A (en) | 1990-07-26 |
| HUT54389A (en) | 1991-02-28 |
| EP0381958A2 (en) | 1990-08-16 |
| IL93115A (en) | 1995-07-31 |
| ATE126808T1 (en) | 1995-09-15 |
| ES2078249T3 (en) | 1995-12-16 |
| HU900197D0 (en) | 1990-03-28 |
| NZ232178A (en) | 1991-10-25 |
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