Note: Descriptions are shown in the official language in which they were submitted.
~024701
BEHRINGWERRE AKTIENGESELLSCHAET HOE 89/B 038 - Ma 779
Dr. Ha/Bi
Description
A process for the purification of plasminogen activator
inhibitor 2 (PAI-2)
The invention relates to a process for the purification
of plasminogen activator inhibitor 2 (PAI-2), wherein
impurities are precipitated with an acridine or quinoline
base.
Plasminogen activators (PA) are serine proteases which
activate the fibrinolytic system. They convert the
inactive proenzyme plasminogen into the active enzyme
plasmin which degrades fibrin and fibrinogen. In the body
there are two PA, the tissue plasminogen activator ~t-PA)
and the PA which is identical with urokinase (u-PA) and
which is produced by renal cells but also other normal or
malignant cells. The t-PA possesses particular importance
in the prevention of thromboses. u-PA prevents formation
of fibrin clots in the urinary tract but also contributes
to the healing of wounds and has been found where there
is neoplastic growth.
The concentration and activity of the PA are regulated by
their synthesis but also by inhibitors. Two specific PA
inhibitors (PAI) which do not inhibit plasmin or other
proteases are known. One of them is produced in endothe-
lial cells (PAI-l~, the other one found in placenta (PAI-
2) has also been called "minactivin".
Starting from an extract from placenta a 120-fold concen-
tration of PAI-2 can be achieved according to the state
of the art by an ammonium sulfate fractionation, adsorp-
tion of impurities on CM-Sephadex C-50, a gel filtration
and hydroxyapatite chromatography. This process results
in two forms of PAI-2 with molecular weights of 70 and
43 kDa. Immunoaffinity chromatography and a combination
20~7Gl
-- 2 --
of immunoaffinity chromatography and FPLC have also been
used for purification. A combination of 8 process fiteps
comprising a precipitation with neutral salts, chromato-
graphies on CM- and DEAE- RSepharose and hydroxyapatite,
and a preparative electrophoresis led, starting from
placenta, to a pure PAI-2 with a molecular weight of
47 kDa. It has also been known that chromatographic
purification steps can be influenced advantageously by
the addition of reducing agents of the dithiothreitol
type.
The object of the invention is to find a simpler process
which can be used on the production scale for the isola-
tion of PAI-2.
Surprisingly it has been that impurities can be precipit-
ated from a solution of PAI-2 using 2-ethoxy-6,9-diamino-
acridine lactate while the PAI-2 itself r0mains in
solution in the supernatant.
A preincubation with dithiothreitol (DTT) proved to be
advantageous.
The invention relates to a process for the purification
of plasminogen act~vator inhibitor 2 (PAI-2), which
comprises a PAI-2-containing solution being preincubated
with an agent cleaving disulfide linkages, preferably
dithiothreitol (DTT), and being mixed with ~ suitable,
water-soluble acridine or quinoline base, preferably 2-
ethoxy-6,9-diaminoacridine lactate, in such an amount
that the precipitate formed does not contain more than a
little PAI-2, this precipitate being separated off, and
the PAI-2 being obtained from the supernatant and, where
appropriate, further purified using known processes.
PAI-2 will also precipitate if small amounts of acridine
or quinoline base in relation to the amount of proteins
in solution are added. An appropriate concentration of a
water-soluble salt of the base, preferably 2-ethoxy-6,9-
202~7~
-- 3 --
diaminoacridine lactate, is from 200 mg to 2 g/g ofprotein in solution (PAI-2 remains substantially in
solution). The process is carried out at a pH of 5.5-8.5.
The PAI-2-containing solution may have been pasteurized
beforehand, where appropriate with the addition of
stabilizers, preferably glycine and/or ~ucrose.
The PAI-2-containing solution is preferably an extract of
placenta or a solution containing genetically engineered
PAI-2.
The disulfide-cleaving agent is used in a concentration
of from 10 mmol/l to 250 mmol/l, preferably 100 mmol/l.
Preincubation is carried out for from 15 min to 3 hours,
preferably for 1 hour, at 10 to 40C, preferably about
37C.
85 - 90 % of the employed PAI-2 activity, but only 5 -
8 % of the employed amount of protein, are present in the
supernatant when the process is carried out as described.
After the precipitation the acridine base can be salted
out of the supernatant, preferably using NaCl, preferably
5 %, and the PAI-2 can be concentrated by an ammonium
sulfate precipitation, for example by 80 ~ saturation of
the solution with ammonium sulfate, which yields an addi-
tional purification in combination with a preliminary
precipitation at 30 ~ ~aturation of the solution with
ammonium sulfate. ~ince about 90 ~ of the employed
protein are removed by the Rivanol and ammonium sulfate
precipitation, the small residual amount can then be sub-
jected to purification processes which are su~ect to
volume limitation: for example a chromatography on DEAE-
RAffigel Blue, a chromatography gel with bifunctionalaffinity which i8 composed of diethylaminoethyl groups
and RCibacron Blue F3GA dye covalently bonded to agarose,
and on hydroxyapatite. Naterial of this degree of purity
should already be suitable for therapeutic use. Ultra-
2 ~ 2 ~ r~ o ~
-- 4 --
purification can be carried out by hydrophobic chroma-
tography on a phenylalanine column but is associated with
substantial losses.
The PAI-2 which is obtained may additionally be pas-
teurized, if this has not been done beforehand.
The process according to the invention is described
hereinafter:
An amidolytic method was used in combination with the
chromogenic substrate S-2444 (Glu-Gly-Arg-pNA) from Rabi
for monitoring the isolation of PAI-2.
For the determination, 50 ~1 of urokinase (u-PA)
(1000 U/ml) were incubated with 100 ~1 of PAI-2-contain-
ing sample for 4 min at room temperature, and 80 ~1 of
this mixture were transferred into a plastic cuvette
which had been prewarmed to 37C. 50 ~1 of buffer A and
20 ~1 of S-2444 (6 mM) were then added. The increase in
absorption at 405 nm was determined in a Cobas Bio
centrifugal analyzer. The measurements were evaluated by
comparison with a dilution plot which had been con-
structed via serial dilution of an in-house PAI-2
standard.
Materials which were used for the isolation of PAI-2:
- 2-Ethoxy-6,9-diaminoacridine lactate (6,9-diamino-
2-ethoxyacridine lactate): Sigma Chemie GmbH, D-6100
Darmstadt; a 2.5 % (w/v) strength solution in
12.5 mM tris, pH 6.8, was used for the fractional
precipitation.
- Dithiothreitol (DTT): Serva Feinbiochemika GmbH
Co./ D-6900 Heidelberg
- Hydroxyapatite: RBioRad, Richmond, CA, USA
- Phenylalanine-RSepharose and CNBr-activated
2~2~701
-- 5 --
RSepharose 4B: Deutsche Pharmacia GmbH, D-69~0
Freiburg
- S-2444: Rabi Vitrum, S-11287 Stockholm, Sweden
- Tris(hydroxymethyl)aminomethane (tris): E. Merck,
D-6100 Darmstadt
- Urokinase (RActosolv): Behringwerke AG, D-3550
Marburg
- Buffer A: 50 mM tris, pH 8.4, 1 % polygeline, 100 mM
NaCl, 0.01 % ~Triton X 100 and 0.01 ~ NaN3
- Buffer B: 20 mM tris, p~ 7.5, 20 mM DTT
- Buffer C: 20 mM Na2HPO4, pH 6.8, 20 mM DTT
- Buffer D: 20 mM tris, pH 7.5, 20 mM DTT, 30 %
saturation with ammonium sulfate
- Buffer E: 20 mM tris, pH 7.5, 100 mM NaCl
- Buffer F: 20 mM tris, pH 6.8
Example
Frozen human placenta which had been washed free from
blood was used as raw material for the preparation of
PAI-2; the placenta had been chopped up in a cutter and
then extracted with 0.5 % NaCl and 3 mM EDTA. The rem-
nants of cells were removed by centrifugation and the
supernatant was precipitated with 8 % Rivanol, and the
precipitate was dissolved and sub~ected to a fractional
precipitation with ammonium sulfate. The precipitate
which contained the PAI-2 was dissolved in buffer F and
dialyzed against it. The concentrated extract of placenta
contained 4,615 U of P~I-2Jml with a specific activity
of 120 U/mg. This material was used for the
ultrapurification.
202~
-- 6 --
- Rivanol and ammonium sulfate precipitation
500 ml of the starting material described above (Tab. 1)
were incubated with 7.7 g of DTT (final concentration
100 mM) at 37C for 1 hour. 1,520 ml of a 2.5 % strength
Rivanol solution were added dropwise to this solution
while continuously stirring cautiously, thus reaching a
final concentration corresponding to 200 %. The precipi-
tate resulting from the precipitation was removed by
centrifugation, and excess Rivanol was removed from the
supernatant by addition of 100 g of solid NaCl to final
concentration of 5 %. 343 g of solid ammonium sulfate
were then added to the supernatant until a final con-
centration corresponding to 30 ~ saturation was reached.
The precipitate was removed by centrifugation (20 min,
3000 rpm), and 694 g of solid ammonium sulfate were then
added to a final concentration of 80 % saturation. The
precipitate resulting from this was dissolved buffer B in
high concentration and dialyzed. This resulted in 80 ml
of dissolved and dialyzed ammonium sulfate residue.
- DEAE-~Affigel Blue chromatography
80 ml of the ammonium sulfate precipitate were applied to
a DEAE-Affigel Blue column (17.5 x 4.4 cm, 260 ml gel
bed) which had been equilibrated with buffer B. The
column was eluted using an NaCl gradient in buffer B
(2 x 10~0 ml) which covered a range from 0 to 200 mM NaCl
at a flow rate of 90 ml/h. The protein concentration was
continuously measured via the O~ at 280 nm and the PAI-2
activity was determined as described.
- Hydroxyapatite chromatography
472 ml of the PAI-2-containing fractions from the previ-
ous step were collected, dialyzed against buffer C and
applied to a hydroxyapatite column (15 x 3.2 cm, 56 ml
gel bed) (Table 1) which had been equilibrated with the
same buffer. The column was eluted at a flow rate of
202~7Gl
-- 7 --
same buffer. The column was elu~ed at a flow rate of
50 ml/h and using a salt gradient of sodium phosphate
(0.02 to 0.3 M; 2 x 250 ml) in buffer C. The protein
concentration and PAI-2 activity of the eluate were
determined continuously.
- Phenylalanine-RSepharose
The PAI-2-containing fractions after hydroxyapatite
chromatography were collected, and 13.2 g of solid ammo-
nium sulfate were added to 120 ml of thi6 material to
reach 30 ~ saturation. Thi6 solution was applied to a
phenylalanine-RSepharose column which had been equili-
brated with buffer D. The resin was eluted at a flow rate
of 20 ml/h and using a gradient formed from buffer D and
B (2 x 100 ml). The PAI-2-containing fractions were col-
lected and characterized. The purest material had aspecific activity of 60,000 U (= units)/mg and migrated
in SDS polyacrylamide gel electrophoresis as one band
which had a molecular weight of 43 kDa and could be
demonstrated in an immunoblot with a specific monoclonal
anti~ody.
202470 ~
~,
, o ~ ~ t` , U~
a~ o OD U~
dP
-
o
, ~
~ ,
~,
.,, 4
~1 C
~ O ~
P~
~ ~ o ~ o o
p ~ ~ r o o
O U~ 0 -- N 11
J~
.,1
~ O O O O O O
.,_1 ~
~ X X X X X X
O I -1 0 0 U~ O O O
H ~ -- ~ CO _I
0 '¢ U p
~ P~ 0 -- N r~
.
U
0 ~ .
~1 o ~ o
O_I O U~_t
O -- o~ c~
t5~ 0 ~ t~t '
O S~ O
.,1
~n
0 a~
_I~ O O O ~C:~ O
Q
O
_1 a
n~
O ~ O
0 0 ~ ~ 0 a~
0 ~ 0 ~ 0
o ~ I o
R ~ ~ 0 a~ a~
m
