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Details for Patent: 5,095,092

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Details for Patent: 5,095,092

Title: Process for the isolation and purification of hirudin
Abstract:The invention relates to a process for the isolation and purification of hirudin from complex and salt-containing solutions by hydrophobic chromatography, using as stationary phase porous adsorber resins and as mobile phase organic solvents which are miscible with water.
Inventor(s): Badziong; Werner (Bad Soden am Taunus, DE), Crause; Peter (Offenbach, DE), Habermann; Paul (Eppstein/Taunus, DE), Tripier; Dominique (Eppstein/Taunus, DE)
Assignee: Hoechst Aktiengesellschaft (Frankfurt am Main, DE)
Filing Date:Sep 27, 1990
Application Number:07/590,581
Claims:1. A process for isolation and purification of a hirudin from complex and salt-containing solutions by hydrophobic chromatography, which comprises carrying out the hydrophobic chromatography on porous adsorber resins selected from the group consisting of a copolymer of styrene and divinylbenzene and a copolymer of acrylate ester and divinylbenzene, using as eluent a 10 to 40% strength solution of one or more organic solvents which are miscible with water selected from the group consisting of methanol, ethanol, n-propanol, isopropanol and acetone.

2. The process as claimed in claim 1, which results in a hirudin of the formula 1: ##STR2## In which m=0-50,

n=0-100 and

R denotes phenolic hydrogen or a phenolic ester group,

X represents identical or different radicals of naturally occurring alpha-amino acids,

Z represents identical or different radicals of naturally occurring alpha-amino acids and

A represents Ile or the absence of an amino acid,

B represents Ile or Thr or the absence of an amino acid,

C denotes Thr, Val, Ile, Leu, or Phe,

D denotes Glu or the absence of an amino acid,

E denotes Glu or Pro,

F denotes Thr or Ile,

G denotes Lys or Lys-Asp and

I denotes Ala or Leu and

J denotes Gln or Lys

in which the 6 Cys radicals are linked in pairs via disulfide bridges, and physiologically acceptable salts thereof.

3. The process as claimed in claim 2, which results in a hirudin of the formula I ##STR3## wherein m is 0 or 1,

n is 0,

A represents Val, Ile, Leu or Ala,

D represents the absence of an amino acid,

E represents the absence of an amino acid,

G represents Thr or Val,

L represents Thr,

M represents Thr,

N represents Gln or Lys,

Q represents Gly,

R represents Glu,

T represents Lys,

X represents Glu or Pro,

Z represents Leu, and

R.sub.1 represents a phenolic hydrogen,

wherein Cys 7 and Cys 15, Cys 17 and Cys 29, and Cys d23 and Cys 40 are linked in pairs via disulfide bridges,

or a physiologically acceptable salt thereof.

4. The process as claimed in claim 2, which results in the hirudin ##STR4## or a physiologically acceptable salt thereof.

5. The process as claimed in claim 1, wherein a copolymer of styrene and divinylbenzene is used as porous adsorber resin.

6. The process as claimed in claim 1, wherein isopropanol is used as an organic solvent.
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