Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Insulin; related peptides
Patent
1986-09-02
1989-01-31
Schain, Howard E.
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Insulin; related peptides
530304, 530305, C07K 740
Patent
active
048016845
DESCRIPTION:
BRIEF SUMMARY
A process for the preparation of an insulin precursor of the formula I (see patent claim) is known, in which R is hydrogen, an amino acid residue which can be eliminated chemically or enzymatically, or a peptide residue which can be eliminated chemically or enzymatically and has at least two amino acid residues, Y represents --Lys.sup.B29 --Z.sup.B30 --, in which Z denotes Ala, Thr or Ser, the bridge extending from A-1 to A-21 being an insulin A-chain, the bridge extending from B-1 to B-30 representing an insulin B-chain, and X being a bridge which is bonded to the insulin A-chain at the amino group of A-1 and is bonded to the insulin B-chain at the carboxyl group of B-30, it being possible to cleave this bridge enzymatically or chemically without destroying the A-chain and the B-chain.
In this process, a S-sulfonate of the formula II (see patent claim 1), in which R, X and Y have the abovementioned meanings, is reacted, in an aqueous medium at a pH of 7 to 11.5 and at a S-sulfonate concentration of up to 10 mg per ml of the aqueous medium, with a quantity of a mercaptan sufficient to result in 1-5 SH groups per SSO.sub.3.sup.- unit (cf. European Pat. No. 37 255, corresponding to Japanese Published Specification No. 81-150051 and U.S. Pat. No. 4,430,266). The folding yield in this process depends on several parameters, such as pH, ratio of SSO.sub.3.sup.- to --SH, the concentration, the nature of the mercaptan, the temperature and the reaction time. Under conditions approximating to those in practice, the process can be optimized to a folding yield of about 60%.
In other words, this entails the preparation of an insulin precursor by folding, with the formation of the natural spatial structure with three disulfide bridges, from which free insulin can then be obtained by subsequent transformation with proteases. During the conversion with the proteases, the falsely linked insulin precursors resulting as byproducts are broken down into fragments which cannot now be processed to give the desired insulin precursors with the aid of the same process. The term insulin precursors is defined in this context as both proinsulins and preproinsulins, the prefix "pre" being intended to relate to one or more additional amino acids on the N-terminal end of the proinsulin, and the proinsulin moiety itself preferably having the sequence of human or monkey proinsulin. Of course, in principle, other proinsulins are also possible, for example porcine, bovine or ovine, which can be isolated from pancreas, or those having synthetic sequences containing the human insulin sequence and being preparable by genetic engineering processes or, in the case of porcine insulin, being processable semi-synthetically to give human insulin.
It is known that not all the disulfide bridges in insulin are equally reactive, on the contrary there being, on mild reduction of the disulfide, initial opening of the disulfide bridge between cysteines A7 and B7 before the second disulfide bridge between cysteines B19 and A20 is opened. In our own investigations, the preferred reduction of the A7-B7 disulfide bridge was also observed on reaction of insulin precursor S-sulfonate with a small excess of mercaptan.
The further processing of the folded insulin precursor is carried out, according to the literature, with chromatographic processes, for example by removal of salts by gel chromatography, for example on .sup.(R) Sephadex G 25, followed by gel chromatography on Sephadex G50 superfine, there being separation in the second stage of "aggregated forms" from natural insulin precursors.
It has now been found, surprisingly, that the false recombinants which result from the folding of insulin precursors from the corresponding S-sulfonates and whose contribution usually amounts to 30 to 50% of the quantity used can be precipitated directly from the folding medium at pH 4 to 6, while, in contrast, the natural form of the insulin precursor remains almost entirely in solution. This applies not only when about 2 equivalents of --SH are used per S-sulfonate group fo
REFERENCES:
patent: 4421685 (1983-12-01), Chance et al.
patent: 4430266 (1984-02-01), Frank
patent: 4654324 (1987-03-01), Chance et al.
patent: 4701440 (1987-10-01), Grau
Chem.-Abstracts, 181618h, (1982), Frank et al.
Chem.-Abstracts, 110370f (1982), Dahno et al.
Chem.-Abstracts, 220286z, Losse et al. (1981).
Chem.-Abstracts, 220291x, Naithani et al. (1981).
Tetrahedron Letters, No. 12 (1973), Robinson et al., pp. 1-4.
Chem.-Abstracts, 181617g, (1981), Chance et al.
Hoechst Aktiengesellschaft
Schain Howard E.
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