Note: Descriptions are shown in the official language in which they were submitted.
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BEHRINGWERICE ARTIENGESELLSCHAFT HOE 89/B 012 - Ma 745
Dr. Ha/Sd
A process for removing toxins from protein solutions
The invention relates to a process for removing of toxins
from solutions of proteins, in particular from lipo-
cortins PP4, PP4-X (PAP II), PA.D III, p68 and lipocortins
I and II.
Placental tissue protein PP4, proteins PP4-X, PAP III and
p68, as well as lipocortins I and II, display homology in
the amino acid sequences and belong to a family of
proteins called lipocortins.
These proteins have antiinflammatory and anticoagulant
effects. They have been detected in many organs and can
be isolated from the latter.
Preparation from tissues, for example from human placen-
ta, or of proteins prepared by gene manipulation, for
example expressed in E. coli, such as rPP4 or rPP4-X, is
associated with isolation, together with these proteins,
of substances which are toxic for humans, such as bac-
terial lipopolysaccharides. Despite high purity (greater
than 95~ based on the protein content), the isolated
proteins showed heavy contamination with toxic substances
in toxicity tests such as the Limulus test or after
administration of therapeutic doses (1 mg of protein/kg
of body weight) to rabbits.
It was not possible to remove these evidently protein-
associated contaminants either by chromatographic
processes or filtration techniques such as sterile
filtration or by the use of non-ionic detergents or of
chelating reagents, alone or in combination.
Hence the object of the invention was to develop proces-
ses for removing toxins deriving from organs, tissues and
cell cultures in proteins of the lipocortin family which
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have been isolated as well as prepared by gene manipula-
tion, which do not impair the biological activity of the
proteins and thus permit potential use as therapeutics
for coagulation and/or inflammatory disorders.
It has been found, surprisingly, that toxic substances
can be removed from these proteins by ion exchange
chromatography in the presence of chelating reagents in
combination with ionic detergents, without an adverse
effect on the biological activity.
Thus the invention relates to a process for removing
toxins from a protein solution, which comprises subjec-
ting a protein in an aqueous buffer solution in the
presence of a chelating agent and of an ionic detergent
to an ion exchange chromatography.
This process is especially applicable to lipocortins,
which can be of natural or biotechnological origin,
especially originating from gene manipulation.
Examples of chelating agents which can be used are EDTA,
EGTA, a salt of citric acid or oxalic acid or a combina
tion of the latter.
Examples of ionic detergents which can be used are cholic
acid, taurocholic acid, taurodehydrocholic acid, deoxy
cholic acid, taurodeoxycholic acid or taurochenodeoxy
cholic acid or a salt of the latter or a mixture of the
latter.
It is possible to use as ion exchanger an anion exchan-
ger, preferably DEAE RSepharose, -RSephacel, RFractogel
or Q-RSepharose, particularly preferably DEAE RSepharose.
The chelating agent and the detergent can be removed from
the protein-containing solution after the treatment
according to the invention by dialysis or by
chromatography in a buffer solution of pH 7.4-9.5,
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preferably pH 8.0-9.5, particularly preferably pH 8.0-
9Ø
In one procedure, a solution of the protein which con-
tains a buffer substance, such as tris, glycine, HEPES or
PBS, with a pH of 7.0-10.0, and at least 0.1 mmol/1 of a
chelating reagent such as EDTA, EGTA, of a salt of citric
acid or of oxalic acid or of a combination of these and
at least 0.05 g/1 of an ionic detergent such as Na chol.,
Na Doc., Na Tdoc., Na Tchol., Tcheno-Doc or Tdcho. or of
a mixture of these, is brought into contact with an anion
exchanger, the exchanger is washed with buffer solution,
and the adsorbed protein is eluted with a salt gradient,
for example using LiCl, KC1 or NaCl.
In a preferred procedure, a solution of the protein with
a concentration of 0.01-30 mg/ml, particularly preferably
0.2-5 mg/ml, which contains tris in a concentration of 2-
80 mmol/1 and a pH of 7.0-9.5, particularly preferably
mmol/1 tris/HC1 and a pH of 8.0-9.0, as well as 1-
100 mmol/1 of a chelating reagent, particularly prefer-
20 ably 5-20 mmol/1 EDTA, and 0.2-5 g/1, particularly
preferably 0.8-1.5 g/1, Na chol. or Na Doc. or of a
mixture, is brought into contact with DEAE RSepharose,
RSephacel, RFractogel, or Q RSepharose, particularly
preferably DEAE RSepharose. After the exchanger has been
washed with buffer solution, the adsorbed protein is
eluted with a linear increasing NaCl gradient.
Chelating reagents and detergents can be removed from the
protein-containing column flow-throughs or eluates by
dialysis against a buffer solution composed of tris,
HEPES, glycine or PBS, particularly preferably against a
buffer solution of pH 8.0-9.0, or by a further chromato-
graphic step such as gel permeation chromatography with
AeA 202 or AcA 54.
It is possible, where appropriate, for the preparations
treated in this way to be further purified. The
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following abbreviations have been used for the descrip-
tion:
DEAE: diethylaminoethyl
EDTA: ethylenediaminotetraacetic acid
HEPES: N-2-hydroxyethylpiperazine-N-2-ethane-
sulfonic acid
Na Chol: sodium cholate
Na Doc: sodium deoxycholate
Na Tchol: sodium taurocholate
Na Tdoc: sodium taurodeoxycholate
PBS: sodium or potassium phosphate buffer
rPP4: PP4 prepared by genetically engineered
expression in E. coli
rPP4-X: PP4-X prepared by genetically engineered
expression in E. coli
PAP III: placental anticoagulant protein III
PAGE: polyacrylamide gel electrophoresis
Q: quaternary amine
SDS: sodium dodecyl sulfate
Tcheno-Doc: taurochenodeoxycholic acid
Tdchol: taurodehydrocholic acid
tris: tris(hydroxymethyl)aminomethane
The invention is illustrated by the examples which
follow:
The starting substances employed for the detoxification
were preparations of the proteins PPS, PP4-X, PAP III,
p68, lipocortins I and II from human placenta and of
proteins rPP4 and rPP4-X from transformed E. coli cul-
tures with a purity of greater than 95$ based on the
protein content, in a buffer solution composed of
0.02 mol/1 tris/HC1, pH 8.5, with a protein concentration
of 2.5 mg/ml. These preparations had an evident content
of toxic substances (Table I) as was determined using the
Limulus test (carried out in solutions at pH 7,2) and the
animal model.
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Toxicity tests
1. Limulus test
This test was carried out as described by Concept t~abH
(Heidelberg, Germany): 0.1 ml of the protein-containing
solution to be tested was gently mixed with 0.1 ml of
Limulus amebocyte lysate in a pyrogen-free tube, and the
tube was incubated at 37°C without shaking for 60 min.
After the end of the incubation time, the tube was
examined visually to find whether a solid gel had formed.
The pyrogenicity of the tested substance, expressed in EU
(endotoxin units), was determined using a calibration
plot constructed with the aid of a reference endotoxin
(EC-5).
2. Pyrogen test on rabbits:
The toxicity of the protein samples was determined by
measuring the increase in the body temperatures (rectal)
of rabbits compared with the body temperature determined
in a 90-minute preliminary test. Protein samples were
administered i.v. in a bolus (1 mg/kg of body weight)
into an ear vein of the rabbits, and the body temperature
was recorded for a period of 180 min. The highest value
was used as basis for the evaluation. Samples were
assessed as pyrogen-free if the total of the temperature
differences of 6 animals was less than or equal to 2.2°C.
Example 1
After addition of EDTA to a final concentration of
0.01 mol/1, while checking the pH, and 0.1% Na Doc, the
PP4-, rPP4-, PP4-X-, rPP4-X-, PAP III-, p68- or lipo-
cortin I- or II-containing solutions were brought into
contact with DEAF RSepharose (from Pharmacia, Sweden)
equilibrated with 0.02 M tris/HC1, pH 8.5, 0.01 M EDTA
and O.I% Na Doc. (column buffer) in a column, the gel
material was washed with column buffer, and adsorbed
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proteins were eluted with an NaCl gradient increasing
linearly.
The eluates were extensively dialyzed against a buffer
solution composed of 0.02 mol/1 tris/HC1, pH 8.5, and
subsequently against a buffer solution composed of
0.02 mol/1 tris/HC1, pH 7.2, and the dialyzates were
examined for toxicity both in the Limulus test and in the
pyrogen test on rabbits. The proteins treated in this way
caused only very low or no increases in temperature in
the pyrogen test or scarcely measurable endotoxin con-
tents in the Limulus test (Table I), and it was possible
to assess them as pyrogen-free.
Proteins PP4-X and rPP4-X were not adsorbed onto the gel
material under the said conditions and were found in the
column flow-through, but they were likewise pyrogen-free
after the stated process had been carried out (Table I).
The biological activity, examined using the modified
prothrombin time, based on the protein concentration, was
fully retained by comparison with the starting materials
through this process step. The yields of the proteins
were between 64 and 85$ based on the toxic starting
materials.
Example 2
PP4-, rPP4-, PP4-X-, rPP4-X-, PAP III-, p68- or lipocor-
tin I- or II-containing buffer solutions were mixed with
Na Chol or Na Tchol to a final concentration of 0.5 g/1
in each case, as well as 0.01 mol/1 EDTA, the latter were
brought into contact with Q-RSepharose (from Pharmacia,
Sweden) equilibrated with 0.02 mol/1 PBS, pH 8.5,
0.01 mol/1 EDTA, 0.05 g/1 Na Chol and 0.5 g/1 Na Tchol
(column buffer) in a column, the gel material was washed,
and adsorbed proteins were eluted with an NaCl gradient
increasingly linearly.
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The procedures for further treatment of the protein
solutions and examination thereof for toxicity were as
described in Example 1. The results of these investiga
tions corresponded to those for Example 1 and are listed
in Table I.
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