Method of purifying preproinsulin

1. A method for the chromatographic purification of preproinsulin of the formula 1,

wherein

X a) is a genetically encodable amino acid residue or

b) is a peptide having from 2 to 35 amino acid residues, which starts and ends with in each case a basic amino acid residue, in particular Arg, and which, if it consists of more than 3 amino acid residues, starts and ends with in each case two basic amino acid residues, in particular Arg and/or Lys,

R 1 a) is hydrogen,

b) is a genetically encodable amino acid residue or

c) is a peptide having from 2 to 15 amino acid residues,

R 2 is a genetically encodable amino acid residue, and

the residues A1-A20 correspond to the amino acid sequence of the A chain of human insulin or of an insulin analog and the residues B1-B30 correspond to the amino acid sequence of the B chain of human insulin or of an insulin analog;

wherein said method for chromatographic purification of preproinsulin comprises:

removing higher molecular weight substances from an aqueous solution of said preproinsulin by means of a first chromatography on an anion exchanger in flow-through mode and a subsequent second chromatography on a cation exchanger in adsorption mode and

wherein said preproinsulin of formula (l) is consisting of the following amino acid sequence:

(SEQ ID NO: 3)

Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg-Phe-Val-

Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-

Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-

Thr-Pro-Lys-Thr-Arg-Arg-Glu-Ala-Glu-Asp-Pro-Gln-

Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-

Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-

Gln-Lys-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-

Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Gly

and wherein

X is a peptide chain having 35 amino acid residues with the sequence of simian C peptide,

R1 is a peptide chain having 10 amino acid residues with the sequence Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg (SEQ ID NO: 5),

R2 is the amino acid residue Gly,

A1-A20 is a peptide chain with the sequence (only A1 to A20) of the A chain of human insulin,

B1-B30 is a peptide chain having the sequence of the B chain of human insulin.

2. The method of claim 1 , which comprises separating foreign substances from said aqueous solution of preproinsulin which induce insulin denaturation.

3. The method of claim 1 wherein said second chromatography is carried out at a pH of from 3.0 to 5.5.

4. The method of claim 1 wherein said second chromatography is carried out under a pressure of from 1 to 30 bar.