Patent ID: 6727346
Filing Date: 2004-04-27
Classification: A61K,A61P,C07K

Abstract:
A process for obtaining an insulin or an insulin derivative thereof having correctly bonded cystine bridges in the presence of cysteine or cysteine hydrochloride and a chaotropic auxiliary, which comprises the following steps:(a) mixing an aqueous suspension of a precursor of an insulin or an insulin derivative with an amount of cysteine or cysteine hydrochloride which results in approximately 1 to 15 SH residues of the cysteine or cysteine hydrochloride per cysteine residue of the precursor; (b) adding the cysteine- or cysteine hydrochloride-containing suspension of the precursor into an approximately 4 to 9 molar solution of the chaotropic auxiliary at a pH of approximately 8 to 11.5 and a temperature of approximately 15Â° C. to 55Â° C., keeping the mixture obtained at this temperature for approximately 10 to 60 minutes; (c) diluting the cysteine or the cysteine hydrochloride concentration to approximately 1 to 5 mM which results in a chaotropic auxiliary concentration of approximately 0.2 to 1.0 M in the mixture at a pH of approximately 8 to 11.5 and a temperature of approximately 5Â° C. to 30Â° C.; (d) cleaving with trypsin or a trypsin-like enzyme the precursor of the insulin or the insulin derivative obtained by this process; and (e) purifying the insulin or the insulin derivative having correctly bonded cystine bridges, wherein the precursor of the insulin or the insulin derivative of step (a) is obtained by expressing a fusion protein in a microorganism.