Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
- l -
D e s c r i p t i o n
'1'he invention concerns a process for the production of
two-chain t-P~ by culturing eukaryotic cells which are
trans~ected with a vec-~or ~ontaininy the t-PA gene.
The tissue-type plasminogen activator t-PA is a serine
protease wikh a molecular weiyht of 68000 Dalkons. Its
~unction is to convert plasmilloyell to plasmin, which is
also a ~erine protease with a wicle range oE activities.
Like other serine proteases, t-PA is also synthesi~ed as
a sinyle polypepticle chain and is then conver-ted into a
two-chain form by cleavaye at position 257/258. Both
chains of the two-chain ~orm are hsld together by a
clisulphide bridge. The liyhter chain contains the
enzymatically active part, while the heavier chain
contains the so-called finyer, EGF and kringle domains.
In investiyations in whi~h the aryinine at the amino
acid position 257 was eliminated, it was estab~ished
that cleavage at this site is essential for the
enzymatic activity (~ol. Biol. Meth. 3 (l986~ 449-457).
In the methods of preparation known up to now a mixture
of sinyle and two-chain t-PA is produced. Ik is,
howeYer, advantageous for a therapeutic application to
be able to prepare t-PA in an uncomplicated manner in a
pure single or two-chain form which is Lree of
degradation products.
Processes ~or the production oE sinc~le-chain t-PA are
known for example Erom European appllcation EP 151 996
A2, published on Auyust 2L, 1985, and Biochem. J. 226
(1985) 631-636 in whlch recombinan-t single-chain t-PA
is prepared in eukaryotic cells in the presence oE
aprotinin. In these processes the t-PA ob-tained is
al.most entirely sirlyle-chain. It is also known from
EP 151 996 A2 that t-PA can be obtained almost
complet~ly in a two-chain form. For this, the culture
super~atan-t ~rom eukaryo~ic cells ln whlch recombln~nt
DNA is produced, is subjected to an enzyme treatmen~
i.e. treatment with plasmin, kallikrein, trypsin,
actlvated factor XI or XII. The amounts of contamination
by single-chain t-PA are so low that the purity is
su~ficient ~or mally applications.
~owever, Eor a therapeutic application it is absolutely
essential that t-PA can be prepared either in a pure
single-chain form or in a pure two-chain form. The
prep~ra~lorl oE ~-PA in ~ pure single-chairl ~orm i~
described in German application DE 38 29 244, published
on March 1, 1990. The object of the present inven-tion was
to enable the production of two-chain t-PA in a pure form.
This objec-t is achieved according to the present
invention by a process for the production of two-chain
t-PA by culturing eukaryotic cells which are transfected
with a vector containing the t-PA yene, which is
characterized in that aEter reaching the stationary
yrowth phase o~ the cells, the culture or culture
supernatant is incubated with a re-incubation medium
which contains serum or plasma from the horse, ox or
foetal calf and a fibrinogen derivative and the t-PA is
isolated Erom the medium.
The invention is based on the suprising observa-tion tha-t
two-chain t-PA can be produced in pure form after
incubation by addition o~ serum or plasma from the ox,
horse or foetal calf and a fibrinoyen derivative to the
culture after the stationary growth phase has been
reached or to the culture supernatant containinCJ a
mixture oE sinyle and two-chain t-PA. It is also
possible within the scope of the invention to convert
pure sinyle-chain t-PA, which has been prepared e.g.
according to DE 3~ 29 244, in-to the pure two-chain form
by ~reatment with serum or plasma from th2 ox, hors~ or
foetal calf and with a ~ibrinogen derivative. Within the
scope o~ the inventioll -the ~erm "cul~ure supernatant"
therefore also inclu~es pure sinyle~ch~in t-PA. In
addition within the scope of the invention the term t-PA
is understood to lnclude muteins or derlvatives of the
protein. For the production of buch muteins or
derivatives, a correspondiny~ yene which encodes them is
then trans~ec~ed in tlle process accordiny to the present
invention.
Within the scope o~ the invention fibrinogen derivatives
are understood as cleavage products of fibrinogen which
are described for example in Eur. J. ~iochem. 172
(1988), 399-404, J. Biochem. 262 (1987) 5944-5946 and
Eur. J. Biochem. 166 (1987) 393-397 or BBA 748 (1983)
~-92. In a particularly pr~Eerred embodiment of the
invention a ~ibrinogen derivative is used which is
designated FCB2 in the above-mentioned references.
All media containing serum or which are free of serum
and are suitable ~or the culture of eukaryotes, e . g.
RPMI 1640 (M.J. Morton, In Vitro 6 (1970) 89), DMEM
(Dulbecco's modified minimal - essenkial medium, J.W.
Gooding, J. Immunol. Methods 39 (1980) 235) or F12
~R. G. Ham, Proc. Natl. Acad. Sci. USA 53 (1965). 288)
or a medium accordiny to German application DE 38 01 236,
published on July 27, 1939 (with or without serum) can be
used as media within the scope oE the inven-tion Eor the
culture of the eukaryotic cells.
It is particularly preferably within the scope of the
invention, to use a mixture of DMEM and F12 medium in a
ratio of about 1 : 1. It is especially pre~erred to use
.1~31
-- 4
the medium described in the German Patent Application
DE 38 01 236 by the same appl cant.
In order to maintain the stability of the plasmid
transformation of the cells, a vector is preferably used
which contains a gene capable of selection in addition
to the t-PA gene and a corresponding selection agent for
the vector is added to the medium. Suitable selection
agents are for example neomycin, hygromycin,
mycophenolic acid, hypoxanth`ine, xanthine, aminopterin
or, alternatively, methotrexate and derivatives thereof.
The medium used according to the present invention
preferably contains no aprotinin since it was found that
aprotinin, in contrast to that which is taught by the
state of the art, has a detrimental effect on the
ability to stimulate the t-PA formed. Change of medium
and conditions for the culture are known to the expert
and can be carried out in the usual way.
After reaching the stationary growth phase, i.e. when
the maximum cell density is reached, the culture or
culture supernatant is incubated with a re-incubation
medium which contains serum or plasma from the ox, horse
or foetal calf as well as a fibrinogen derivative.
Preferably foetal calf serum is used. The serum or
plasma is added to the culture or culture supernatant
preferably in an amount from 0.01 to 20 %.
Within the scope of the invention the fibrinogen
derivative is added in an amount from 5 to 100 mg/l.
Treatment with the fibrinogen derivative is carried out
preferably at 4 to 40C. In this process, it is
preferable to use a lower temperature for larger amounts
of fibrinogen derivative and in contrast a higher
temperature for small amounts of fibrinogen derivative.
2 0 ~
-- 5 --
The duration of the incubation is also dependent on the
temperature and amount of serum or plasma and fibrinogen
derivative added. The amount of t-PA in the two-chain
form can be checked by the well-known immuno-blotting
techniquss and the optimal incubation period can be
determined accordingly.
In a preferred embodiment a medium which is identical
with the re-incubation medium is used for the culture of
the eukaryotic cells in the process according to the
present invention.
The re-incubation medium, and also the culture medium in
the case of the embodiment just mentioned, preferably
contains foetal calf serum, in particular in an amount
from 0.01 to 20 %, preferably in an amount from 0.01 to
10 %.
The conditions preferably used apply to t-PA
concentrations in the culture or in the culture
supernatant from 5 to 20 mg/l. If the t-PA concentration
increases and the above mentioned conditions are
retained, then the content of single-chain t-PA
increases simultaneously.
All eu~aryotic cells which can be grown in culture and
which can produce t-PA come into consideration as cells
within the scope of the invention. Preferred examples
for this are CHO cells (Chinese Hamster Ovary Cells).
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The invention is elucidated further by the following
Example.
E x a m p l e
a) Transfection of a vector containing the t-PA
gene in CHO cells.
CH0 dhfr~ cells (ECACC 88072103) are transfected
as described by R. Kaufmann and P. Sharp,
J. Mol. Biol. 15 (1982) 601-621, with the
plasmid pSVtPA~dhfr (Gene 66, 193-203 (1988))
and stable transformants are isolated.
b) Cell culture
The isolated colonies were incubated in a
conventional medium (DMEM/F12 in a ratio 1:1)
containing foetal calf serum (FCS, 0.01 to 5 %)
or also in a medium free of serum. The duration
of the incubation was so chosen that t-PA was
present in quantitatively detectable amounts
which was as a rule, first after 24 hours up to
a theoretically unlimited time. The cells
producing t-PA and the medium containing t-PA
were separated from one another in order to
isolate t-PA. Subsequently foetal calf serum
(FCS) was added to the medium containing t-PA
and this was incubated for a certain period of
time in the absence of cells. The incubation was
carried out for 20 hours; the yields of single
and two-chain t-PA are shown in Table 1. A
Western blot with immuno-staining was carried
out for this. It is apparent that already a
2 ~
~ small concentration of foetal calf serum
(0.01 %) is sufficient to induce two-chain t-PA.
The ratio of single and two-chain t-PA remains
relatively constant under these conditions.
T a b 1 e
% FCS t-PA
single-chain two-chain
O -~ _
0.01 + +
0.05 + +
O.1 ~ +
0.5 + +
1.0 + +
2.5 + +
5.0 + +
5 ~ FCS and in~reasing concentrations of BrCN
cleavage products of fibrinogen were added
subse~uently to a 48-hour culture supernatant of
CHO t-PA cells which had been cultured free of
serum and incubated at 22C. The t-PA present in
the culture supernatant was tested for the two-
chain form by immuno staining after a Western
blot. The results are shown in Table 2 in
relation to the incubation time. It is apparent
from these data that the formation of almost
exclusively two-chain t-PA is induced by
addition of fibrinogen derivative to the culture
supernatant containing serum.
T a b 1 e 2
Time (hours~
fibrinogen 1 3 5 22
derivative
mg/l ) one two one two one two one two
, . .- - -;
O + -~ ++ + .~(+)
+ + _+ _ + _ +
(+) + - + _ ~ _ +
- + - + - + - +
D . _ A _ _ _ . _ .. _ _ . _ _ _ __ , ____ __ __ _ __ __ _
Finally, it was examined whether addition of
fibrinogen and FCS and subsequent incubation at
22C results in a detectable loss of t-PA~ For
this purpose the amount of t-PA in supernatants
containing fibrinogen was tested by ELISA after
the incubation. The results are shown in
Table 3.
.
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T a b l e 3
. _ _.
Fibrinogen Time (hours)
(mg/l~) 1 3 5 21
.
t-PA (mg/ml)
0 8 - 7 7
9 7 8 6
9 8 8 8
8 8 7
100 9 7 10 7
, _ .
It is clear from these results that the
incubation of t-PA in the presence of fibrinogen
derivatives and FCS did not lead to serious
differences in the t-PA concentration.
E x a m p l e 2
The main culture was carried out analogous to Example 1;
cell supernatant was isolated after reaching the
stationary phase and this culture supernatant was
incubated at 37C in the presence of increasing amounts
of plasmin or in the presence of 100 mg/l BrCN cleavage
products of fibrinogen and 5 % by volume FCS.
Table 4 shows that t-PA is not degraded by addition of
fibrinogen cleavage products and FCS, and thus
conversion of single-chain into two-chain t-PA is
possible without loss. In contrast t-PA is degraded when
plasmin is used for the conversion.
~ .
.,.,.. - .
- 10 --
~r a b l e
~ .
t-PA to-tal activity (U/ml~
after an incubatioll time oE (h)
Addition of O 4 8
_ _ .
- 2~1 293 285
Pla~min 1.0 U/ml 281 194 151
Plasmin 0.1 U/ml 2~0 267 249
Plasmin 0.01 U/ml281 273 235
Plasmin 0.001 U/ml 279 271 257
.
5 % FCS -~ 285 279 281
100 mg/l fibrinogen
cleavag~ products
E x a m p l e 3
a) Transfection of C~IO cells Wit}l a vector which
contains a t-PA mutein.
Plasmld pePal44 (prepared according -to Example 2 of
European application EP A 0 242 836, published on
October 28, 1987) is in-troduced into CHO cells as
described in Example la.
The cell culture is carried out as described in
Example lb). Two-chain t-PA mutein is likewise
obtained almost exclusively.
E x a m p 1 e 4
a) Transfection of CH0 dhfr~ cells with a vector
which contains a t-PA mutein gene.
~2 and ~(K2-~EGF) which are described in
DE 38 25 253 Al were used as t-PA muteins.
CH0 dhfr cells, EC~CC 88072103, were grown and
cultured as described by Urlaub and Chasin,
Proc. Natl. Acad. Sci. USA 77 (1980), 4216-4220.
Calcium phosphate precipitates containing 20 ~g
pSV (aK2)-dhfr (DSM 4721) and pSV ~(K2~EGF)-dhfr
(DSM 4720) were prepared in a volume of 4 ml ~or
the DNA transfection as described by Graham and
Van der Eb, Virology 52 (1973), 456-467. 1 ml of
the precipitate was added to 3x105 to 1x106
cells in 10 ml medium in 9 cm culture plates.
The cells were incubated for 8 to 16 hours with
the precipitate, the medium was then removed and
the cells were washed with 10 ml TBS (25 mmol/l
Tris-XCl, pH 7.4, 137 mmol/l NaCl, 5 mmol/l KCl,
0.6 mmol/l Na2HP0~).
The cell culture was carried out as described in
Example lb). Two-chain t-PA muteins were
likewise obtained practically exclusively.
