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|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|
|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,
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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