NO178861B - Process for preparing an insulin precursor - Google Patents

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

The present invention relates to a method for producing an insulin precursor.

Insulin is a molecule consisting of 2 polypeptide chains linked by disulfide bridges. The A chain consists of 21 amino acids and the B chain of 30 amino acids. Both of these chains are in the precursor molecule, proinsulin, connected to each other through a peptide, the C-peptide. The C-peptide in human proinsulin consists of 35 amino acids. The C-peptide is cleaved by specific proteases during the maturation process of the hormone and the proinsulin is then converted to insulin (Davidson et al., Nature 333, 93-96, 1988). Apart from the naturally occurring C-peptides, many possibilities are described in the literature regarding the connection between the A-chain and the B-chain (Yanaihara et al., Diabetes 27, 149-160

(1978), Busse et al., Biochemistry 15, 1649-1657 (1971), Geiger et al., Biochem, Biophys. Res. Com. 55, 60-66 (1973)).

Within the framework of genetic technology, it is now possible to produce insulin from genetically modified microorganisms. If the microorganism E. coli is used, the product is expressed in large quantities as a fusion protein, i.e. the product is linked with a protein from the bacterium, e.g. with p<->galactosidase. This fusion protein precipitates into the cell and is thus protected from proteolytic degradation. After processing the cells, the fusion protein portion is cleaved off chemically or enzymatically and the 6 cysteines of the insulin precursor are transferred to S-sulfonates (-S-SO3") by means of oxidative sulfitolysis". From this so-called preproinsulin S-sulfonate, native preproinsulin must be formed of the 3 correct disulphide bridges be provided in a following step.

This step takes place e.g. according to the method described in EP-B-00 37 255 by reacting the starting S-sulfonate with a mercaptan in an amount which gives 1 to 5 SH residues per SS03~-residue in aqueous medium at a pH value of 7-11.5 at an S-sulfonate concentration of up to 10 mg per ml aqueous medium

preferably in the absence of an oxidizing agent.

For the achievement of higher folding yield, i.e. higher yield of preproinsulin with the "correct" peptide sequence linkage (-S-S bridges from A6 to All, from A7 to B7 and from A20 to B19), the obtained, narrowly limited, The SH/SS03~ ratio, which necessitates not insignificant care when carrying out the method and a not entirely easy quantitative determination of the SS03~ groups in the starting S-sulfonate.

It was now surprisingly found that high folding yields are achieved practically independently of the SH/SSC"3_ ratio when working at a higher, i.e. above 5:1 - SH/SS03~ ratio in the presence of an organic redox- system or from compounds which, under the reaction conditions, form such an organic Redox system. Instead of the SSO*^__ groups, other S-protecting groups may be present in the corresponding starting product. The peptide sequences of the insulin A and B chain may be changed by substitution of one or more amino acids.

The higher SH/SS03~-(resp. S protection group ) ratio means that, if one only exceeds a certain minimum mercaptan amount, one can work practically regardless of the height of the mercaptan excess without significant impact on the yield, and this also does an accurate quantitative determination of the SS03~-(or S-protecting) group determination in the corresponding insulin starting product is redundant, and this is a significant method advantage.

It is possible to work practically independently of the height of the mercaptan excess in the presence of an organic redox system, resp. of compounds which under the reaction conditions form such an organic Redox system.

It is even known that reduced proinsulin, i.e. proinsulin where the S-S bridges e.g. has been cleaved with a mercaptan reducing agent to SH groups, can be reoxidized to the original proinsulin and that this reoxidation is accelerated in the presence of dehydroascorbic acid, cf. D.F. Steiner and J.L. Clark, Proe. Nati. Mod. Pollock. USA 60, 622-624 (1968); even when under the reaction conditions described there from dehydroascorbic acid a redox system is formed from dehydroascorbic acid and ascorbic acid, it is about the oxidation of reduced proinsulin with unprotected S groups, while in the present case according to the invention it is about only insulin precursors which starting products with protected S groups. The aforementioned literature reference to D.F. Steiner and J.L. Clark does not refer anywhere in the direction of the addition and effect of an organic redox system by recombination of insulin precursors containing S-protecting groups by means of mercaptans.

The present invention therefore relates to a method for producing an insulin precursor of formula I

where R1 = E,

a chemically or enzymatically cleavable amino acid or peptide residue,

Rg = OH or an amino acid or peptide residue,

preferably OH

X = a residue connecting the insulin A and B chain,

preferably an amino acid or peptide residue,

Y = the residue of a genetically coded amino acid,

preferably Thr, Ala, Ser, especially Thr,

Z = the residue of a genetically coded amino acid,

preferably Asn, Gin, Asp, Glu, Gly, Ser, Thr, Ala or Met, especially Asn,

A1-A20 and B1-B29 = unmutated or mutated peptide sequences to insulin by replacement of one or more amino acids, preferably the unmutated peptide sequences of human, porcine or bovine insulin, especially human or porcine insulin, by reaction of a precursor with protected Cys- S groups and a mercaptan in aqueous medium,

characterized by reacting a precursor with protected Cys-S groups of formula II

wherein Rlt R2, X, Y, Z, A1-A20 and B1-B29 have the same meaning as in formula I and

R3 = a Cys-S protecting group, preferably -SO3" group, with a mercaptan in an amount corresponding to a (mercaptan)-SH/Cys-S-R3 (in formula II) ratio of 10 to 30, a pH -value between 9.5 and 11, in the presence of an organic redox system or at least one organic compound, which under the reaction conditions forms such an organic redox system, selected from the group of ascorbic acid, dehydroascorbic acid, brenzcatechin, o-quinone, hydroquinone and p -quinone, in which the mercaptan and the compound(s) forming the organic redox system are added in a ratio of 1 gamma equivalent mercaptan to 1/10 to 10 mol of the compound(s) forming the organic redox system.

When R-^ = H in formulas I and II, it concerns proinsulin, resp. proinsulin-derived products. When R^ = chemically or enzymatically cleavable amino acid or peptide residue, it concerns preproinsulin and products derived therefrom.

Chemically cleavable amino acid residues are such as e.g. with the help of BrCN or N-bromosuccinimide is split off, these are e.g. methionine (Met) or tryptophan (Trp).

Enzymatically cleavable amino acid residues are such as e.g. can be cleaved using trypsin (such as Arg or Lys).

Chemically or enzymatically cleavable peptide residues are peptide residues with at least 2 amino acids.

All amino acids relating to R^ are preferably from the group of natural amino acids, 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 - like R 2 - likewise an amino acid or peptide residue, where the meaning OH is preferred. The amino acids (which consist of at least 2 amino acid residues forming peptide residues) derive - as in R^ - preferably from the group of the natural amino acids.

X is an insulin A and B chain binding residue, preferably an amino acid or peptide residue.

When X is an amino acid residue, a residue of Arg or Lys is preferred, when X is a peptide residue, the residue of a naturally occurring C-peptide, especially human, porcine or bovine insulin C-peptides is preferred.

Genetically codeable amino acids are for Y (in L form): Gly, Ala, Ser, Thr, Val, Leu, Ile, Asp, Asn, Glu, Gin, Cys, Met, Arg, Lys, His, Tyr, Phe, Trp , Pro.

Preferred genetically encoded amino acids are Thr, Ala and Ser, especially Thr.

Z can - like Y - mean the remainder of a genetically codeable amino acid, but here Asn, Gin, Asp, Glu, Gly, Ser, Thr, Ala and Met, especially Asn, are preferred.

Al - A20 and Bl - B29 can in principle be the unmutated or through replacement of one or more amino acids mutated peptide sequences all possible insulins, the mutants can be confirmed according to known genetic engineering methods (site-directed mutagenesis). The unmutated peptide sequences of human, porcine or bovine insulin, especially human or porcine insulin (the A1 - A20 and B1 - B29 sequences of human and porcine insulin are identical).

The residue R 3 , which only occurs in formula II, means practically any Cys-S protecting group, on the other hand, preferably -SO 3 - or tert-butyl group, where the -SO 3 - group here has a somewhat greater meaning.

As starting product, formula II can in principle be added in a wide concentration range, suitably between approximately 10 pg and 10 mg/ml solution, where as is known, lower concentrations lead to higher renaturation yield, as lower protein concentrations reduce the tendency to aggregation. Preferred concentrations are between about 0.1 mg and 0.5 mg/ml.

As mercaptans for the reaction according to the invention, in principle all possible organic compounds with SH groups come into consideration, mercaptoethanol, thioglycolic acid, dithioerythritol, dithioerythritol, glutathione and cysteine, especially mercaptoethanol and cysteine are preferred. The mercaptans can be added individually or in a mixture.

The amount of mercaptan is measured in such a way that the ratio between the SH groups and the Cys-SRs groups of the starting materials with formula II is above 5 if possible.

Above, this ratio is almost only limited by economic considerations and an upper limit of about 100 is appropriate. A ratio of about 10-50, especially about 10-30, is preferred. The mercaptan concentration in the reaction addition then adjusts according to the amount of the added starting material with formula II and the selected SH/Cys-SR3~ ratio.

Organic redox systems are preferably compound pairs whose one component is an organic compound with a structural element of formula III

or an aromatic o- or p-dihydroxy compound and whose second component is an organic compound with structural

intended with formula III in oxidized form = structural element with formula III'

or an o- or p-quinone.

The free valences of the structural elements with formula III, respectively III', can be desaturated with hydrogen or organic groups such as e.g. C 1 -C 4 alkyl groups. On the other hand, the structural element can also be part of a ring with preferably 4.5 or 6 C-ring atoms, as well as possibly one or two more heteroatoms such as e.g. 0, where the ring can further be substituted with groups inert under the reaction conditions such as e.g. alkyl or hydroxyalkyl groups.

Examples of compounds with the structural element of formula III are:

The formulas are only described here in the one tautomeric form.

All connections are reduced. In the oxidized form, structural element with formula III becomes formula III'.

Aromatic o- and p-dihydroxy compounds in principle include all possible aromatic compounds with two OH groups in the o- or p-position, where it is only necessary that the o-respectively p-quinone formation from the o- and p-dihydroxy compounds must not be prevented by any special substitutes etc. Examples of aromatic o- and p-dihydroxy compounds are

1,2-dihydroxybenzene = fuel catechin

1,4-dihydroxybenzene = hydroquinone,

methyl-hydroquinone, naphtho-hydroquinone-1,4 and

anthra-hydroquinone; whereby the oxidation occurs in the corresponding quinones.

In the turnover according to the invention, the organic Redox systems, consisting e.g. from the compound pairs ascorbic acid + dehydroascorbic acid, benzcatechin + o-quinone, hydroquinone + p-quinone, naphthohydroquinone + naphthoquinone, etc. in practical conditions (preferably in approximately equimolar conditions) be added. However, it is also possible to only add the individual components of these connection pairs, also e.g. only ascorbic acid or only dehydroascorbic acid or only hydroquinone etc. - due to the formation in the reaction medium of the other components belonging to the Redox compound pair (dehydroascorbic acid or ascorbic acid or p-quinone).

Preferred organic redox systems are those based on the compound pairs

ascorbic acid + dehydroascorbic acid,

brenzcatechin + o-quinone and

hydroquinone + p-quinone

existing combinations

and preferred single compounds which, under the reaction conditions, form such a Redox system, are the individual components of these compound pairs.

Particularly preferred is ascorbic acid and/or dehydroascorbic acid.

The added amounts of the organic redox system-forming compound(s) can be varied within wide limits. With regard to a gram-equivalent mercaptan (= the molecular weight of added mercaptan in g/number of the SH group in the mercaptan molecule), the molar number of the organic Redox system-forming compound(s) can be chosen between approximately 1/10,000 and 10,000, preferably between about 1/10 and 10.

The reaction according to the invention is preferably carried out in an alkaline pH range, preferably between about 7 and 12, especially between about 9.5 and 11. To maintain the desired pH value, it is necessary to add a buffer compound, the type and ionic strength of the buffer has some influence on the folding yield. It is advantageous to keep the ionic strength low, and a range of about 1 mM (mM = millimolar) to 1 M (M = molar), especially of about 5 mM to 50 mM is preferred. As buffer connections, e.g. borate buffer, carbonate buffer or glycine buffer can be used, the latter being preferred.

As a general range for the reaction temperature, one between approximately 0 and 45°C can be used and a range of approximately 4 to 80C is preferred.

Covering the renaturation solution with specific gases such as e.g. oxygen, nitrogen or helium have no noticeable influence on the renaturation yield.

The turnover time is generally between about 2 and 24 hours, preferably between about 6 and 16 hours.

After completion of turnover (which can, for example, be determined by HPLC), this is worked up in a known manner, such as e.g. described in EP-B-00 37 255.

The "correctly" folded product of formula I can then be enzymatically or chemically transferred according to known techniques to the corresponding insulin.

The following examples further explain the invention and clarify the advantages according to the state of the art.

As starting material for the renaturation experiments, preproinsulin-S-SO3~ was used, and this was isolated according to genetic engineering techniques.

E.coli was used as an expression system. In E. coli, the gene for proinsulin is linked with part of the ε-galactosidase gene and is synthesized as a fusion protein which falls out of the cell and which is deposited on "polar caps") according to the method in DE-Å-38 05 150). After suspension of the cells, the fusion protein portion is cleaved by means of halogen cyan according to the method in DE-A34 40 988) and finally subjected to oxidative sulphitolysis (R.C. Marshall and A.S. Ingles in A.

Darbre (Eds. ) "Practical Protein Chemistry - A Handbook"

(1986), pp.49-53), to transfer the 6 cysteines to their S-sulfonate form. The preproinsulin-S-SO3~ produced in this way (the prefix "pre" means that the proinsulin is extended by 5 amino acids at the N-terminal end) is then enriched by means of ion exchange in a manner known per se and precipitated and freeze-dried. The obtained freeze-dried starting material has, according to HPLC determination, a content of 60%.

1. The dependence of the folding yield on

the mercaptan/ascorbic acid ratio.

Preproinsulin-S-SOø (605é) is dissolved in a concentration 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. Per mixture, 20 ml is added, as well as 480 µl of a 0.1 M cysteine solution (~20 times molar excess per S-SO3~ group) and between 0 and 480 µl of a 0.1 M ascorbic acid solution. The renaturation temperature is 8°C, and the reaction lasts for 16 hours.

This example shows the influence of the Redox compound on the folding yield. Due to the fact that in the mixture without ascorbic acid it predominantly leads to the formation of proteins that are not folded correctly, the folding yield rises in the presence of the Redox compound.

2. Dependence of the folding yield on

mercaptan/ascorbic acid excess

The measured amounts, volumes, buffer, reaction time and pH value correspond to those in experiment 1. Equimolar amounts of cysteine and ascorbic acid are added, the molar excess per S-SC>3_ group lies between 2.5 and 100.

This example shows that when exceeding a certain mercaptan minimum amount, the folding yield in a large area is largely independent of the mercaptan excess.

3. Dependence of the folding yield on the pH value.

The weighed quantities, volumes, buffer concentration, reaction time and temperature correspond to experiment 1. In the experiment, 480 µl 0.1 M cysteine solution and 480 µl 0.1 M ascorbic acid solution are added (~20 times molar excess per S-SO3_-group)

4. Dependence of the folding yield on the type of mercaptan. The weighed amount, volumes, buffer concentration, reaction time and temperature and pH value correspond to experiment 1. In the experiment, 480 µl of the corresponding 0.1 M mercaptan solution and 480 µl of 0.1 M ascorbic acid solution (~20 times molar) are added each time excess per S-S03_ group) 5. Dependence of folding yield on the buffer compound and the ionic strength of the buffer.

The amount weighed, the volumes, the reaction time and the temperature and the pH value correspond to experiment 1. In the experiment, 480 µl of the corresponding 0.1 M mercaptan solution and 480 µl of the 0.1 M ascorbic acid solution (~20 times molar excess) are added each time per S-S03~ group).

6. Dependence of folding yield on

the reaction time.

The amount weighed, the volumes, the buffer concentration, the pH value and the reaction temperature are the same as in experiment 1. 480 µl of a 0.1 M mercaptoethanol solution and 480 µl of a 0.1 M ascorbic acid solution (~20 times the molar excess per S-S03~ group).

7. Dependence of the folding yield on

preproinsulin S-sulfonate concentration

Volumes, buffer composition, pH value, reaction time and temperature correspond to experiment 1. To the added amount of preproin-sul in-S-sulfonate, so much of a mercaptoethanol stock solution and ascorbic acid stock solution is added that a ~20-fold molar excess occurs per S-S03~ group.

8. Dependence of folding yield on ascorbic acid/mer- the excess captoethanol in modified starting material A precursor molecule is added where the A and B chains of insulin are linked with arginine. The genetic engineering isolation of this molecule follows as described at the beginning of the experimental description. The freeze-dried material has a content of 60% and is dissolved at 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 times. 9. Dependence on the folding yield for the Redox system Weighed amount, volumes, buffer composition, pH value, reaction time and temperature correspond to experiment 1. A ~20-fold molar excess of mercaptoethanol/S-SO3_ group and a 20-fold molar excess are added excess of the corresponding Redox partners.

The folding yield of preproinsulin-S-SO3 is dissolved in 20 mM glycine buffer, pH 10.8, 250 ml buffer, 0.2 mg prepro-insulin-S-SO3/ml buffer at about 25°C. Different amounts of excess mercaptan and ascorbic acid according to the following table are used in the reaction solution. The reaction solution is stirred in an open glass.

The following table shows the results

The folding yield was determined in the following high-pressure liquid chromatograph system:

column waters C-^; 5 p.m.; flux 1.5 ml/min; detection at 210 nm, gradient from 28% to 40% buffer B over 41 minutes.

The results show compared to Steiner et al. (Biochemistry, Vol 60, 1968, pp 622 to 629 ) a surprisingly higher folding yield in a shorter time.

It was surprising that this higher yield could be obtained within a pH range that is very different from the best reaction conditions described by Steiner et al.

Claims (3)

1. Process for the preparation of an insulin precursor of formula I where Ri = H, a chemically or enzymatically cleavable amino acid or peptide residue, Rg = OH or an amino acid or peptide residue, preferably OH X = a residue connecting the insulin A and B chain, preferably an amino acid or peptide residue, Y = the residue of a genetically codeable amino acid, preferably Thr, Ala, Ser, especially Thr, Z = the residue of a genetically codeable amino acid, preferably Asn, Gin, Asp, Glu, Gly, Ser, Thr, Ala or Met, especially Asn, A1-A20 and B1-B29 = untested or when replacing one or several amino acid mutated peptide sequences of insulin, preferably the unmutated peptide sequences of human, porcine or bovine insulin, especially human or porcine insulin, by reacting a precursor with protected Cys-S groups and a mercaptan in an aqueous medium, characterized by reacting a precursor with protected Cys-S groups of formula II wherein R-^ , R 2 , X, Y, Z, A1-A20 and B1-B29 have the same meaning as in formula I and R3 = a Cys-S protecting group, preferably -SC>3_ group, with a mercaptan in an amount corresponding to a (mercaptan)-SH/Cys-S-R3 (in formula II) ratio of 10 to 30, a pH value between 9.5 and 11, in the presence of an organic redox system or at least one organic compound, which under the reaction conditions forms such an organic redox system, selected from the group of ascorbic acid, dehydroascorbic acid, benzcatechin, o-quinone, hydroquinone and p-quinone, in which the mercaptan and the compound(s) forming the organic redox system are added in a ratio of 1 gamma equivalent mercaptan to 1/10 to 10 mol of the compound(s) forming the organic redox system. 2. Method according to claim 1, characterized in that mercaptoethanol and/or cysteine are used as mercaptan. 3. Method according to one or more of claims 1 to 2, characterized by working in the presence of ascorbic acid and/or dehydroascorbic acid.