AU624894B2

AU624894B2 - Process for the preparation of an insulin precursor

Info

Publication number: AU624894B2
Application number: AU48621/90A
Authority: AU
Inventors: Michael Dorschug
Original assignee: Hoechst AG
Current assignee: Hoechst AG
Priority date: 1989-01-21
Filing date: 1990-01-19
Publication date: 1992-06-25
Anticipated expiration: 2010-01-19
Also published as: IL93115A0; EP0381958A3; NO178861C; ES2078249T3; HUT54389A; FI900296A7; JPH02233699A; EP0381958B1; PT92907B; NZ232178A; DE3901719A1; CA2008245A1; FI900296A0; ZA90396B; PT92907A; NO900278D0; IL93115A; IE900217L; HU900197D0; HU208704B

Abstract

An insulin precursor of the formula I <IMAGE> in which R1 = H, an amino-acid or peptide residue which can be eliminated chemically or enzymatically, R2 = OH or an amino-acid or peptide residue, X = a radical joining the insulin A and B chain, Y = residue of a genetically encodable amino acid, Z = residue of a genetically encodable amino acid, A1-A20 and B1-B29 = insulin peptide sequences which are non-mutated or mutated by exchange of one or more amino acids, is prepared by reacting a precursor in which the disulphide 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. The particularly preferred organic redox system is the pair of compounds ascorbic acid + dehydroascorbic acid. The insulin precursor of the formula I can be converted into insulin either enzymatically or chemically by known techniques.

Description

1394 Form COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged; Complete Specificatioi Lodged: Accepted: Published: Pr1ority o eae Ar 0 Name of Applicant HOECHST AKTPIENGESELLSCHAFT o 'A(Idress of ApplicantAO Bruningstrasse, D-6230 Frankfurt/Main 80, Federal Republic of Germany Actal nvnto :MICHAEL

DORSCHUG

4 ~dross for ServicA WATERMARK PATENT TRADEMARK ATTORNEYS.

290 rwod RadHawthorn, Victoria, Australia Complete Specification for the inverition entitled: PROCESS FOR 'THE PREPARATION OF AN INSULIN PRECURSOR The following statement Is a full description of this Invention, Including the best method of performing it known to 1.

I I

I

HOECHST AKTIENGESELLSCHAFT HOE 89/F 021 Dr. ME/rh Description Process 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 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 process 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- .ao 96, 1988). In addition to the naturally occurring C- S: 15 peptides, a large number of bonding possibilities between the A chain and B chain are described in the literature i o° (Yanaihara et al., Diabetes 27, 149-160 (1978), Busse et a° al., Biochemistry 15, 1649-1657 (1971) and Geiger et al., Biochem. Biophys. Res. Com. 55, 60-66 (1973)).

In the contaxt of genetic engineering, it is now possible to prepare insulin from microorganisms modified by ,0 genetic engineering. If E. coli is used as the microorganism, the product is often expressed as fosion protein, that is to say the product is coupled with a protein endogenous to the bacteria, for example with pgalactosidase. This fusion protein precipitates out in 0 the cell and is in this way protected from proteolytic degradation. After breakdown of the cell, the fusion protein content is split off chemically or enzymatically and the 6 cysteines of the insulin precursor are converted into their S-sulfonates by means of oxidative sulfitolysis. Natural preproinsulin .mist be produced from this so-called preproinsulin S-sulfonate in a subsequent step, the 3 correct disulfide bridges being formed.

r I 2 This step is carried out, for example, by the process described in EP-B-0,037,255 by reaction of the starting S-sulfonate with a mercaptan in an amount which results in 1 to 5 SH radicals per SS0 3 radical, in an aqueous medium at a pH of 7 to 11.5 and a S-sulfonate concentration 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 bridges from A6 to All, from A7 to B7 and from A20 to B19) it is necessary to maintain the stated narrowly limited SH/SS0 3 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.

0 0 4 Surprisingly, it has now been found that high folding r yields which are virtually independent of the SH/SS03 ratio within wide limits are obtained if the procedure is carried out at a higher that is to say above 5:1

SH/SS

3 ratio in the presence of an organic redox system o0 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.

I

The higher SH/SS03" (or S-protective groups) ratio means that as long as only a certain minimum amount of mercaptan is 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 SSO3 (or S-protective) groups in the corresponding insulin starting substance is in this way also superfluous, which represents a con- 3 i siderable advantage of the process.

Carrying out the process virtually independently of the level of the mercaptan excess is made possible by the presence of an organic redox system or of compounds which form such an organic redox system under the reaction Sconditions.

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 precursors containing S-protective groups by means of mercaptans.

i' In detail, the invention thus relates to a process for S the preparat on of an insulin precursor of the formula I i~h- C -I 4 00 .0 4 5 00 0 0 00 0000 0 t0o 0 0 Q0 o 0 1 0 4 4'r Gly-NH X Cys--S- I I(A-20) (A-21) Cys Z R 2 I (A-11) I S

S

S S (B-1)

R

1 (B-19) in which R, H or an amino acid or peptide radical which can be split off chemically or enzymatically,

R

2 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 Al-A20 and B1-B29 peptide sequences of insulin which are non-mutated or mutated by replacement of one or more amino acid radicals, preferably non-mutated peptide sequences of human, porcine or bovine insulin, in particular human or porcine insulin, 4It 44 by reaction of a precursor having protected Cys-S groups with a mercaptan in aqueous medium; the process comprises reacting a precursor having protected Cys-S groups, of i the formula II Gly-NH X Cys-S-R 3

S-R

3 (A-21) Cys Z R 2 (A-11) I(II)

S-R

3

S-R

3

S-R

3 (B-i) (B-19) in which Ri, Rz, X, Y, Z, A1-A20 and Bl-B29 have the same Smeaning as in formula I and 5 R3 a Cys-S protective group, 0' preferably the -SO 3 or the tert.-butyl group, with a mercaptan in an amount corresponding to a (mer- "oo" captan) SH/(insulin precursor) Cys-S-R 3 ratio of more than 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.

0001 4.

If R 2 in formula I and II H, the substances are proinsulin or products derived from proinsulin; if R, an amino acid or peptide radical which can be split off chemically or enzymatically, the substances are preproinsulin 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 spli off chemically or 6enzymatically are peptide radicals having at least 2 amino acids.

1 All the amino acids possible for R, are preferably from the group of naturally occurring amino acids, that is to say mainly Gly, Ala, Ser, Thr, Val, Leu, Ile, Asn, Gin, Cys, Met, Tyr, Phe, Pro, Hyp, Arg, Lys, Hyl, Orn, Cit and His.

R

2 is OH or similarly to R i likewise an amino acid 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 R, 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 2" 5 and Ser, in particular Thr.

Z can like Y likewise denote the radical of a genetically encodable amino acid, but in this case Asn, Gin, Asp, Glu, Gly, Ser, Thr, Ala and Met, in particular Asn, are preferred.

Al A20 and Bi B29 can in principle be the peptide sequences, which are non-mutated or mutated by i i i ii i i i :i i i _I _I 7 replacement of one or more amino acids, of all possible insulinE; the mutants can be produced by known processes of genetic engineering (site directed mutagenesis).

However, the non-mutated peptide sequences of human, 5 porcine or bovine insulin, in particular of human or porcine insulin (the Al A20 and B1 B29 sequences of human and porcine insulin are identical) are preferred.

The radical R 3 which occurs only in formula II denotes virtually any desired Cys-S-protective group, but preferably the -S0 3 or the tert,-butyl group, the -S,0 3 group being of somewhat greater importance here.

I 44 t 15 a a a a, a.

a t a a a1a a)0 The starting substance of the formula II can on principle be employed within wide concentration range advantageously between about 10 pg and 10 mg/ml of solution lower concentrations as is known leading to higher renaturing yields, since at low protein concentrations the tendency towards aggregation is reduced. Preferred concentrations are between about 0.1 mg and 0.5 mg/ml.

9 t Suitable mercaptans for the reaction according to the invention are in principle all the possible organic compounds having SH groups; mercaptoethanol, thioglycolic acid, glutathione and cysteine, in particular mercaptoethanol and cysteine, are preferred. The mercaptans can be employed individually or as a mixture.

The amount of mercaptan is (to be) chosen so that the ratio of its SH groups to the Cys-SR3 groups of the starting material of the formula II is, as far as possible, greater than 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 abc~t 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 1i -8 chosen SH/Cys-SR 3 ratio.

I -Profrrod -poesibI, 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 0 C C 0 OH OH (II 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' S(III') 0 o S0 0 0 C C Sor 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, C-C 4 -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, 0, it being possible for the ring in turn to be substituted by groups which are inert vider 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 0 H -C C -C H AxK' u I -r I- L ~1 9 reductic acid OH OH I I i i HC CH

H

2 C C 2 Yc/ o C 0

H

2 OH OH I I HC CH I I H2 0 methylreductic acid ascorbic acid (vitamin C)

HOCH.

H CH 3 OH OH I i OH HC CH S- H CH C0=0 0 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'.

10 Suitable aromatic o- and p-dihydroxy compounds are in principle all the possible aromatic compounds having two OH groups in the o- or p-position, it merely also being necessary that the o- or p-quinone formation from the oand p-dihydroxy compounds cannot be prv'rented 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 anthrahydroquinone; the corresponding quinones are formed therefrom on oxidation.

In the reaction according to the invention, the particular organic redox systems, consisting, for example, I I C- _111 10 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 (preferably in approximately the equimolar ratio). However, it is also possible for the particular individual components of these pairs of compounds that is to say, for example, only ascorbic acid or only dehydroascorbic acid or only hydroquinone and the like to be added, because in each case the othe imnponent (dehydroascorbic acid or ascorbic acid or p-quinone and the like) belonging to the redox pair of co:~.pounds is formed in the reaction medium.

Preferred organic redox systems are the combinations consisting of the pairs of compounde 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 compounds.

Ascorbic acid and/or dehydroascorbLc 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

!H

11 of the buffer having a certain influence on the folding yield. It is advantageous to keep the ionic strength low, a range of about 1 mM (mM millimolar) to 1 M (M molar), in perticular one of about 5 mM to 50 mM, being preferred. Examples of buffer substances which can be used are brate buffer, carbonate buffer or glycine buffer, the latter being preferred.

A range between about 0 and 45°C can be stated as a general range for the reaction temperature; a range from 'out 4 to 8"C is preferred.

Covering the renaturing solution with a layer of certain t..i gases, such as, for example, oxygen, nitrogen or helium, go has no noticeable influence on the renaturing yield.

9 II The duration of the reaction is in general between about 2 and 24 hours, preferably between about 6 and 16 hours.

9'o 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 °ox. also described, for example, in the abovementioned EP-B- 20 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.

I The following examples are now intended to explain the invention further, and also to illustrate the advantages over the prior art.

Preproinsulin-SSOs" obtained by genetic engineering was used as the starting material for the renaturing experiments.

E. coli was used as the expression system, In E. coli, the gene for proinsulin is coupled with a part of the pi--irr=-~-n9 C- *rs~ uaa*r-- -12 galactosidase gene and is synthesized as fusion protein, which precipitates in the cell and is deposited in the j 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 subjected to oxidative sulfitolysis 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 prpproinsulin-S-S0 3 (the prefix "pre" means that the proinsulin is extended on its N-terminus by 5 amino acids) prepared in this way is then concentrated by means of ion S. J exchangers in a manner which is known per se, precipita- 6 15 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 oot a 1. Dependence of the folding yield on the mercaptan/ ascorbic acid ratio Preproinsulin-S-S0 3 O is dissolved in a concentration of 0.33 mg/ml in 20 mM glycine buffer, pH 10.5, which corresponds to a preproinsulin-S-S0 3

O

concentration of 0.2 mg/ml. 20 ml are employed per batch, to which 480 pl of a 0.1 M cysteine solution molar excess per S-S0 3 group) and between 0 and 480 pl of a 0.1 M ascorbic acid solution are added. The renaturing temperature is 8OC and the I ot« duration of the reaction is 16 hours.

4 3C Ascorbic acid (0.1 M) Folding yield 0 25% comparison 24 .1 28% 48 U1 53% according to the 96 i1 78% invention 480 1p 81% This exampla shows the influence of the redox compound 13 on the folding yield. Whereas predominantly only formation of incorrectly folded proteins occurs in the i batch without ascorbic acid, the folding yield increases in the presence of the redox compound.

2. Dependence of the folding yield on the mercaptan/ascorbic acid excess 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-S0 3 group being between 2.5 and 100.

Amount of cysteine Excess per Folding yield c an.i ascorbic acid added S-S0 3 group 1p 2.5 38% comparison 120 1, 5 .'.240 pl 10 83% S0480 l 20 85% according to 1200 pl 50 91% the inven- 2400 pl 100 72%J tion This example shows that as soon as a certain minimum 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 tii" and temperature correspond to experiment 1. In the experiment, 480 pl of 0.1 M cysteine solution and 480 pl of 0.1 M ascorbic acid solution are added 20-fold molar excess per S-SO3 group) 14 pH 11.0 10.5 10.0 Folding yield 79% 81% 66% 53% 46% 33% 24% 4. Dependence of the folding yield on the type of mercaptan 0 *0Q0 04 15 0000 0 0 o a& S00 00 0 0 00 20 The amount weighed out, volume, buffer concentration, reaction time and temperature and pH correspond to experiment 1. In the experiment, in each case 480 pl of the corresponding 0.1 M mercaptan solution and 480 pl of 0.1 M arcorbic acid solution are added 20-fold molar excess per S-SO,- group) Mercaptan Cysteine Mercaptoethanol Glutathione Thioglycolic acid 3-Mercapto-1,2-propanediol Folding yield 81% 86% 74% 76 Dependence of the folding yield on the buffer substance and on the ionic strength of the buffer The amount weighed out, volume, reaction time and temperatui:e and pH correspond to experiment 1. In the experiment, in each case 4,o0 pl of the corresponding 0.1 M mercaptan solution and 480 pu of 0.1 M ascorbic acid solution are added 20-fold molar excess per S-S03" group).

Buffer mM glycine 100 mM glycine mM borate Folding yield 89% 82% 82% F 7

V~

-I 15 100 100 mM borate rmM carbonate mM carbonate 51% 88% 72% 1

III.

6. Dependence of the folding yield on the reaction time The amount weighed out, volume, buffer concentration, pH and reaction temperature correspond to experiment 1. In each case 480 pl of a 0.1 M mercaptoethanol solution and 480 pl of a 0.1 M ascorbic acid solution are added 20-fold molar excess per S-SO 3 group) Reaction time (hours) 0.25 Folding yield 46% 61% 77% 83% 79% ooo *20 ttIt I 7. Dependence of the folding yield on the preproinsulin- S-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 preproinsulin-S-sulfonate employed for a 20-fold molar excess per S-S0 3 group to exist.

1 t Preproinsulin-S-SO 3 concentration 0.1 ml/ml 0.2 ml/ml 0.3 ml/ml 0.4 ml/ml ml/ml ml/ml ml/ml Folding yield 86% 84% 78% 19% 1- I 15

T

16 8. Dependence of the folding yield on the ascorbic 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 and Excess per S-S0 3 group 50 Folding yield 56% comparison 61% 74%] according to 75% the invention I I 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 mercaptoethanol/S-S03 group and a 20-fold molar excess of the corresponding redox partner are added.

Redox partner Ascorbic acid Dehydroascorbic acid Pyrocatechol Hydroquinone Benzo(-p-)quinone Folding yield 77% 66% 23%

L

L .i

Claims

1. A process for the preparation of an insulin precursor of the formula I Gly-NH X Cys-S-S (A-21) Z R2 (A-11) (I) S S (B- 1 R 1 -HN-Phe--Cy (B-19) in which R 1 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, X a radical which bonds the insulin A and B chain, Y the radical of a genetically encodable amino acid, preferably Thr, Ala or Ser, Z the radical of a genetically encodable amino acid, and A1-A20 and B1-429 peptide sequences of insulin which are non-mutated or mutated by replacement of one or more amino acid radicals, 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 /m u-\ lSM^^0 \1T_ I 18 Gly-NH Cys-S-R 3 S-R 3 I (A-20) (A-21) Cys Z R (A-11) (II) S-R 3 S-R 3 S-R3 S-R3 (B-1) R 1 (B-19) S" in which R 1 R 2 X, Y, Z, A1-A20 and B1-B29 have the same meaning as in formula I and Rs a Cys-S protective group, o preferably the -SO 3 or the tert.-butyl group, with a mercaptan in an amount corresponding to a (mercaptan) SH/(insulin precursor) Cys-S-R 3 ratio of more than in the presence of an organic redox system being a pair of compounds ascorbic acid dehydroascorbic acid, *6 pyrocatechol o-quinone or S. i 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 compounds, or organic compounds which forms such an organic redox system under the reaction conditions. C1 I

2. The process as claimed in claim 1, wherein mercaptoethanol and/or cystelne are used as the mercaptan.

3, The process as claimed in either of claims 1 and 2, carried out at a (mercaptan) SH/Cys-SRa (in formula II) ratio of more than 5 to 100. k?4 qrl JL-IL I I-L^LI lriil- rpcli----- b 19

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. OH OH 0 I I II C- C- C II (III) 0 OH OH II II II C= C- 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' 0 0 0 S II II II (IrI') S- C C or an o- or p-quinone. *9

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. i

6. The process as claimed in any one of claims 1 to 5, carried out in the presence of ascorbic acid and/or dehydroascorbic acid.

7. The process as claimed in any one of claims 1 to 6, wherein the mercaptan and the compound(s) which form(s) the organic redox system are employed in a ratio of 1 ii gram equivalent of mercaptan to 1/10,000 to 10,000 moles of the compound(s) which form(s) the organic redox system.

8. The process as claimed In any one of claims 1 to 7, wherein the reaction is carried out at a pH of between about 7 and 12.

9. A process as claimed in any one of claims 1 to 8 in which R 2 is OH 1-t ;T l i QI, X is an amino acid or peptide radical ,Y is Thr and/or *Z is Asn, Gin, Asp, Glu, Gly, Ser, Thr, Ala or Met.

A process as claimed in claim 9 in which Z is Asn.

11. A process as claimed in any one of claims 1 to 9 in which the peptide sequences of insulin are non mutated peptide sequences of human, porcine, or bovine insulin.

12. A process as claimed in claim 6 in which the peptide sequences are from human or porcine insulin.

S13. A process as claimed in claim 3 in which the ratio is about 10 to

14. A process as claimed in claim 12 in which the ratio is about 10 to

15. A process as claimed in claim 7 in which the ratio is 1/10 to i

16. A process as claimed in claim 8 in which the pH is between about 9.5 and 11. DATED this 12th day of February 1992 t C t t HOECHST AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRAUA DOC 008 4862190.WPC DBM/IAS/BS