CA2011548A1

CA2011548A1 - Process for the production of two-chain t-pa

Info

Publication number: CA2011548A1
Application number: CA002011548A
Authority: CA
Inventors: Hubertus Stockinger; Christine Kionka; Ulrich Weidle
Original assignee: Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1989-03-07
Filing date: 1990-03-06
Publication date: 1990-09-07
Also published as: EP0391080A2; JPH02273180A; EP0391080A3

Abstract

A b s t r a c t In order to produce two-chain t-PA by the culture of eukaryotic cells which are transfected with a vector containing the t-PA gene the culture or culture supernatant is incubated, after reaching the stationary growth phase of the cells, 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 from the medium.

Description

- 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 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 ~

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).

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.

.

T a b l e 3 . _ _.

Fibrinogen Time (hours) (mg/l~) 1 3 5 21 .
t-PA (mg/ml) , _ .
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.

~ .
.,.,.. - .

~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.

Claims

1. Process for the production of two-chain t-PA by culturing eukaryotic cells which are transfected with a vector containing the t-PA gene, wherein after reaching the stationary growth phase of 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 from the medium.

2. Process as claimed in claim 1, wherein the serum or plasma is present in an amount from 0.01 to 20 % during the re-incubation.

3. Process as claimed in claim 1 or 2, wherein cleavage products of fibrinogen are used as the fibrinogen derivative.

4. Process as claimed in claim 3, wherein the cleavage product FCB2 is used.

5. Process as claimed in claim 1, 2 or 4, wherein the fibrinogen derivative is used in an amount from 5 to 100 mg/l.

6. Process as claimed in claim 3, wherein the fibrinogen derivative is used in an amount from 5 to 100 mg/l.

7. Process as claimed in claim 1, 2, 4 or 6, wherein the incubation with the fibrinogen derivative is carried out at 4 to 40°C.

8. Process as claimed in claim 3, wherein the incubation with the fibrinogen derivative is carried out at 4 to 40°C.

9. Process as claimed in claim 5, wherein the incubation with the fibrinogen derivative is carried out at 4 to 40°C.

10. Process as claimed in claim 1, 2, 4, 6, 8 or 9, wherein the same medium is used for culturing the cells and for the re-incubation.

11. Process as claimed in claim 3, wherein the same medium is used for culturing the cells and for the re-incubation.

12. Process as claimed in claim 5, wherein the same medium is used for culturing the cells and for the re-incubation.

13. Process as claimed in claim 7, wherein the same medium is used for culturing the cells and for the re-incubation.

14. Process as claimed in claim 1, 2, 4, 6, 8, 9, 11, 12 or 13, wherein the re-incubation medium and, if desired, also the culture medium contain 0.01 to 10 %
foetal calf serum.

15. Process as claimed in claim 3, wherein the re-incubation medium and, if desired, also the culture medium contain 0.01 to 10 % foetal calf serum.

16. Process as claimed in claim 5, wherein the re-incubation medium and, if desired, also the culture medium contain 0.01 to 10 % foetal calf serum.

17. Process as claimed in claim 7, wherein the re-incubation medium and, if desired, also the culture medium contain 0.01 to 10 % foetal calf serum.

18. Process as claimed in claim 10, wherein the re-incubation medium and, if desired, also the culture medium contain 0.01 to 10 % foetal calf serum.