Process for obtaining insulin precursors from reaction mixtures resulting from the folding of insulin precursors from the corresponding S-sulfonates
First Claim
1. A process for the preparation of correctly-recombined insulin precursors of the formula (I) ##STR4## in which R is hydrogen or the presequence Pm --Qn --, in which P is a sequence of naturally occurring amino acids with m being from 0 to 50, Q are basic natural amino acids, and n is an integer from 1 to 4, Y represents --LysB29 --ZB30 --, in which Z denotes Ala, Thr or Ser, and the bridge extending from A-1 to A-21 is an insulin A-chain, the bridge extending from B-1 to B-30 represents an insulin B-chain, and X is 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 ε
- -amino group of B-29--in which case it is bonded to the free bond of ZB30 OH--or at the carboxyl group of B-30, comprising the steps offolding, in a reaction mixture, S-sulfonates of the formula (II) ##STR5## in which R, X and Y have the abovementioned meanings, under conditions sufficient to correctly recombine the insulin precursors of formula (I),precipitating false recombinants co-formed in the folding step by adjustment of the reaction mixture to pH 4 to 6,removing the precipitate,converting the precipitate by sulfitolysis into S-sulfonates of the formula (II), andsubjecting the sulfitolyzed S-sulfonates to folding under conditions sufficient to form the insulin precursors of formula (I).
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Abstract
A process for the preparation of insulin precursors of the formula (I) ##STR1## in which R is hydrogen or the presequence Pm --Qn --, in which P is a sequence of naturally occurring amino acids with m being from 0 to 50, Q are basic natural amino acids, and n is an integer from 1 to 4, Y represents --LysB29 --ZB30 --, in which Z denotes Ala, Thr or Ser, and the bridge extending from A-1 to A-21 is an insulin A-chain, the bridge extending from B-1 to B-30 represents an insulin B-chain, and X is 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 ε-amino group of B-29-- in which case it is bonded to the free bond of ZB30 OH-- or at the carboxyl group of B-30, from reaction mixtures which result from folding of insulin precursors from S-sulfonates of the formula (II) ##STR2## in which R, X and Y have the abovementioned meanings, which comprises the false recombinants contained in the reaction mixture being precipitated by adjustment of the reaction mixture to pH 4 to 6, the precipitate being removed in customary manner and converted by sulfitolysis into the S-sulfonate of the formula (II), and the latter then being subjected to renewed folding.
10 Citations
12 Claims
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1. A process for the preparation of correctly-recombined insulin precursors of the formula (I) ##STR4## in which R is hydrogen or the presequence Pm --Qn --, in which P is a sequence of naturally occurring amino acids with m being from 0 to 50, Q are basic natural amino acids, and n is an integer from 1 to 4, Y represents --LysB29 --ZB30 --, in which Z denotes Ala, Thr or Ser, and the bridge extending from A-1 to A-21 is an insulin A-chain, the bridge extending from B-1 to B-30 represents an insulin B-chain, and X is 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 ε
- -amino group of B-29--in which case it is bonded to the free bond of ZB30 OH--or at the carboxyl group of B-30, comprising the steps of
folding, in a reaction mixture, S-sulfonates of the formula (II) ##STR5## in which R, X and Y have the abovementioned meanings, under conditions sufficient to correctly recombine the insulin precursors of formula (I), precipitating false recombinants co-formed in the folding step by adjustment of the reaction mixture to pH 4 to 6, removing the precipitate, converting the precipitate by sulfitolysis into S-sulfonates of the formula (II), and subjecting the sulfitolyzed S-sulfonates to folding under conditions sufficient to form the insulin precursors of formula (I). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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4. The process as claimed in claim 3, wherein the polymer is added in an amount of 1 to 400, preferably 2 to 200 mg/l.
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5. The process as claimed in claim 1, wherein the folding step is carried out in the presence of a physiologically acceptable surface-active substance effective to prevent the binding of correctly-recombined insulin precursors of formula (I) to co-formed false recombinants.
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6. The process as claimed in claim 5, wherein an excess of mercaptan is used in the folding, and the recombined insulin precursors are reoxidized with oxygen.
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7. The process as claimed in claim 2, wherein the folding step is carried out in the presence of a physiologically acceptable surface-active substance effective to prevent the binding of corectly-recombined insulin precursors of formula (I) to co-formed false recombinants.
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8. The process as claimed in claim 3 wherein the folding step is carried out in the presence of a physiologically acceptable surface-active substance effective to prevent the binding of correctly-recombined insulin precursors of formula (I) to co-formed false recombinants.
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9. The process as claimed in claim 4 wherein the folding step is carried out in the presence of a physiologically acceptable surface-active substance effective to prevent the binding of correctly-recombined insulin precursors of formula (I) to co-formed false recombinants.
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10. The process as claimed in any one of claims 1, 2, 3, 4, 5 or 6 wherein the folding is carried out using a ratio of --SH groups to --SSO3- groups of 1 to 5, in an aqueous medium, at a pH of 7 to 11.5 and a S-sulfonate insulin precursor concentration of up to 10 mg per ml, and at a temperature of 0°
- to 37°
C.
- to 37°
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11. The process as claimed in claim 7, further comprising at least one of the following process modifications:
- the reaction is carried out (1) with a S-sulfonate insulin precursor concentration of 0.2 to 1 mg per ml of aqueous medium, (2) at a pH of 9.5 to 11, (3) at a reaction temperature of 2°
to 10°
C., (4) with a reaction time of 5 to 20 hours, (5) using a ratio of --SH groups to --SSO3- groups of 2 to 3, and (6) with exclusion of oxygen to a large extent.
- the reaction is carried out (1) with a S-sulfonate insulin precursor concentration of 0.2 to 1 mg per ml of aqueous medium, (2) at a pH of 9.5 to 11, (3) at a reaction temperature of 2°
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12. The process as claimed in any one of claims 1, 2, 3, 4, 5 or 6 wherein the mercaptan is 2-mercaptoethanol, thioglycolic acid or its methyl ester, 3-mercapto-1,2-propane-diol or 3-mercaptopropionic acid or its esters.
- -amino group of B-29--in which case it is bonded to the free bond of ZB30 OH--or at the carboxyl group of B-30, comprising the steps of
Specification