Process for enzymatic replacement of the B-30 amino acid in insulins

• US 4,645,740 A
• Filed: 03/23/1982
• Issued: 02/24/1987
• Est. Priority Date: 07/24/1980
• Status: Expired due to Fees

• Current Assignee: Carlsberg Biotechnology Limited A/S
• Original Assignee: Carlsberg Biotechnology Limited A/S
• Inventors: Widmer, Fred, Johansen, Jack T., Breddam, Klaus
• Primary Examiner(s): Kepplinger, Esther M.
• Application Number:
  US06/364,856
• Time in Patent Office: 1,799 Days
• Field of Search: 435/68, 435/69, 435/70, 435/174, 435/212, 435/219, 435/224, 435/71
• US Class Current: 435/68.1
• CPC Class Codes: C07K 14/62 Insulins

Abstract

The B-30 amino acid in insulins is replaced enzymatically by

reacting as substrate component the selected insulin Ins-X, wherein X represents the B-30 amino acid

with an amine component selected from the group consisting of

(a) amino acids of the formula

H--B--OH

wherein B is an amino acid residue,

(b) optionally N-substituted amino acid amides of the formula

H--B--NR.sup.1 R.sup.2

wherein B is an amino acid residue and R¹ and R² are independently selected from the group consisting of hydrogen, amino, hydroxy, alkyl, cycloalkyl, aryl, heteroaryl and aralkyl or R¹ and R² together with the nitrogen atom form a heterocyclic group which may contain a further hetero atom, and

(c) amino acid esters of the formula

H--B--OR.sup.3, H--B--SR.sup.3 or H--B--SeR.sup.3

wherein B is am amino acid residue and R³ represents alkyl, cycloalkyl, aryl, heteroaryl or aralkyl

in the presence of an L-specific serine or thiol carboxypeptidase enzyme, preferably carboxypeptidase-Y, in an aqueous solution or dispersion having a pH from about 7 to 10.5, thereby to form an insulin derivative

Ins--B--OH, Ins--B--NR¹ R², Ins--B--B--NR¹ R²,

Ins--B--OR³, Ins--B--SR³ or Ins--B--SeR³

and subsequently cleaving a group --NR¹ R², --B--NR¹ R², --OR³, --SR³ or SeR³, if desired, preferably by using a carboxypeptidase enzyme. The cleaving may also be performed on derivatives obtained by other methods.

By using porcine insulin as substrate component and threonine as the amino acid forming part of the amine component human insulin is obtained.

Claims

1. A process for enzymatic replacement of the B-30 amino acid in insulins, characterized by reacting as substrate component the selected insulin Ins-X, wherein X represents the B-30 amino acid,

with an amine component selected from the group consisting of

(a) optionally N-substituted amino acid amides of the formula

View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)

2. The process according to claim 1, characterized by using as the carboxypeptidase enzyme a serine or thiol carboxypeptidase from yeast or of animal, vegetable or microbial origin.

3. The process according to claim 2, characterized by using carboxypeptidase Y from yeast as the carboxypeptidase enzyme.

4. The process according to claim 3, characterized by using a carboxypeptidase Y which has been purified by affinity chromatography on an affinity resin comprising a polymeric resin matrix with a plurality of coupled benzylsuccinyl groups.

5. The process according to claim 2, characterized by using a carboxypeptidase enzyme selected from the group consisting of penicillocarboxypeptidase S-1 and S-2 from Penicillium janthinellum, carboxypeptidases from Aspergillus saitoi or Aspergillus oryzae, carboxypeptidases C from orange leaves or orange peels, carboxypeptidase C_N from Citrus natsudaidai Hayata, phaseolain from french bean leaves, carboxypeptidases from germinating barley, germinating cotton plants, tomatoes, watermelons and Bromelain(pineapple)powder.

6. The process according to claim 1, characterized by using an immobilized carboxypeptidase enzyme.

7. The process according to claim 1, characterized by maintaining the pH in the solution or dispersion at 7.5 to 10.5.

8. The process according to claim 1, characterized by using an organic solvent selected from the group consisting of alkanols, dimethyl sulfoxide, dimethyl formamide, dioxane, tetrahydrofurane, dimethoxy ethane, ethylene glycol and polyethylene glycols.

9. The process according to claim 1, characterized by using an aqueous solution or dispersion containing urea or guanidine hydrochloride in a concentration of up to 3 molar.

10. The process according to any of claims 1, or 2, characterized in that the substrate component is porcine insulin and the amino acid forming part of the amine component is threonine, the amine component being threonine amide.

11. The process of claim 1 further comprising cleaving from said insulin derivative a group --NR¹ R², --B--NR¹ R², --OR³, --SR³, or SeR³.

12. The process according to claim 11 characterized in that the group --NR¹ R², --B--NR¹ R², --OR³, --SR³ or --SeR³ is removed by treating said insulin derivative with an L-specific serine or thiol carboxypeptidase enzyme from yeast or of animal, vegetable or microbial origin in an aqueous solution or dispersion having a pH from about 7 to about 10.5.