FI94963B - Process for streamlining the production of t-PA-like proteins - Google Patents

Abstract

A method for increasing the production of protein, especially t-PA, in cultures of cells producing this protein, where a substance which induces the production of the protein is added to the cell culture, is characterised in that thioglycolic acid, thiodiglycolic acid, L-cysteine, glutathione, butyrylcholine bromide, butyrylcholine chloride, nonactic acid, furan fatty acid, ascorbic acid, aphidicolin, 6-hydroxy-4,6-dimethyl-3-hepten-2-one, fusaric acid, mevalonic acid, trans-anhydromevalonic acid, anhydromevalonolactone, cis-anhydromevalonolactone, D- alpha -hydroxy-substituted (C3 or C4) aliphatic mono- or dicarboxylic acid or the salts thereof is used as protein-inducing substance.

Description

94963

Method for enhancing the production of t-PA-like proteins Förfarande för effektivering av framställningen av t-PA-artade proteiner

The invention relates to a method for enhancing the production of t-PA-like protein from a culture of transformed CHO cells.

Plasminogen activators are a class of serine proteases whose individual proteases activate the proenzyme plasminogen to the active enzyme plasmin by cleavage of the peptide bond between amino acid residues Arg-560 and Val 561. Plasmin, in turn, is the final step in the vascular fibrin dissolution system, in which the fibrin network of the thrombus becomes cleaved into soluble peptides.

In pathogenic disorders, such as coronary heart disease, blood clots do not dissolve quickly enough for the tissue to regain enough oxygen. Coronary arteries blocked by a blood clot result in a heart attack, resulting in too little oxygen supply to the heart muscle resulting in necrosis of that tissue.

By rapidly rerouting these blood vessels, myocardial blood flow can be secured most quickly and thus preventing the death of entire myocardial components.

So far, streptokinases and uro kinases have been used in the treatment. However, the properties of t-PA offer some advantages over these known plasminogen activators, which are: fibrin-specific local lysis, no systemic lytic effects due to fibrinogen degradation, high reflux rates achievable, and absence of antibody formation.

2,94963 t-PA is a glycoprotein with a molecular weight of approximately 65,000 daltons, present as both a single and double chain enzyme, consisting of 527 amino acids.

The affinity of t-PA for plasminogen in the presence of fibrin is about 100-fold higher than in its absence. This high affinity of t-PA for plasminogen in the presence of fibrin ensures efficient activation without activation of peripheral free plasminogen.

Human t-PA was isolated pure for the first time from the uterus. Other tissues and cells, e.g., endothelial cells, including arterial and venous endothelial cells, were also able to detect t-PA. However, the amounts of t-PA formed are so low that isolation of t-PA from these for commercial purposes is impossible.

Another natural source of t-PA are cell cultures. Several cell lines were examined for their potential t-PA production capacity (Gronow, M. et al., Trends in Biotechnology 1_ (9883) 26-29).

Larger amounts of t-PA could be isolated from a human cell line, Bowes melanoma cells, for limited clinical trials as well as for structural studies (Wallen et al., European Journal of Biochemistry 132 (1983) 681-686).

For the production of large amounts of t-PA for large-scale clinical use, these yields are far too low. Only genetic engineering allowed the production of large amounts of recombinant t-PA for therapeutic use.

t-PA was cloned in 1983 and its amino acid sequence elucidated (Pennicd, Nature 301 (1983) 214-221).

Attempts to isolate t-PA from E. coli, to which genetic information from t-PA melanoma cells was transferred, were unsuccessful because the resulting protein was not glycosylated (by the cell) and thus did not correspond to native t-PA. .

Therefore, different groups of researchers have screened persistent mammalian cell lines from various sources for the production of human t-PA.

Some of these cell lines are human lines with a somewhat higher t-PA yield. Another cell line consists of ordinary Chinese hamster ovary cells.

EP-A-0093619 describes the production of t-PA from transformed Chinese hamster ovary (CHO) cells. The resulting t-PA (r-tPA) is in no way different from naturally occurring t-PA.

Several patents describe t-PA irons and their preparation, e.g. EP-A Nos. 241,208, 241,209, 241,210, 240,334, 234,051, 233,013, 231,624, 225,286, 213,794, 201,153, 199,574, 196,920 and DE-A: o 3708681.

In this application, all t-PA derivatives are referred to as mutants, regardless of whether they contain one or more amino acids that differ from those contained in the corresponding positions of the naturally occurring t-PA, or "these mutants" do not contain one or more amino acids that: occur in naturally occurring t-PA. A species of t-PA that lacks several of the amino acid residues of naturally occurring t-PA, as described, for example, in ΕΡ-Λ Application 196,920, is often referred to as degenerate species.

For example, EP-A-199,574 describes a t-PA mutant comprising certain amino acid residues at positions 270-279, which, however, differ from the amino acids comprised at the corresponding positions by native t-PA.

4,94963 In DE-A-3708681, the applicant has described certain t-PA species having an amino acid at position 117 which differs from the amino acid comprised at the corresponding position by native t-PA, whereby mutant t-PA, unlike native t-ΡΛ, does not not glycosylated at this position.

Several patents have described the effect of different substances on the ability of cell cultures to produce protein syntheses.

For various cellular systems, data on the interdependence of intracellular c-AMP mirror and t-PA synthesis are presented (see, e.g., Kooistra, et.a 1., Thrombosis and Haemostasis, 54 (1): 1929, Abstract P 1133), for dibutyryl-c-AMP increases the production of t-PA in human endothelial cells 2- / 3-fold, however, it shows no effect on the synthesis of t-PA in Bowes melanoma cells. In renal cells, phosphodiesterase inhibitors increased the rate of t-PA synthesis (Journal of Cell Biology 91 (1981) 195-200). On the other hand, c-AMP inhibits t-PA synthesis in macrophages (Vassalli et.ai., Cells 8 (1 976) 271-281) and has no effect on embryonic cancer cells (Nishimune et.ai., Experimental Cell Research 146 (1983) 439 -444).

Butyric acid induces t-PA synthesis in embryonic cancer cells (Nishimune, supra), however, its effect in renal cancer cells is inhibitory (Nelson et al., Proc. Am. Ass. For Cancer Research 26 (1985) 35) and no effect is seen in embryonic lung cells. , reported by Brouty- tioe et.ai., Biotechnology 1 2 (1 984) 10.

** In human endothelial cells, t-PA synthesis can be stimulated by thrombin (Levin et al., Thrombosis and Haemostasis 56: 2 (1986) 115-116), alcohol (Laug, Jama, 250: 6 (1983 B) 772-776) , Vitamin C and vitamin A (Inada et al., Biochemical and Biophysical Commumincations 1 30: 1 (1 985) 1 82-187).

5,94963

However, in bovine endothelial cells, thrombin inhibits t-PA secretion (Loskutoff, Journal of Clinical Investigations 64 (1979) 329-332) as well as endotoxin (Crutchley et al., Journal of Biological Chemistry 26: 1 (1986) 154-159). ).

In porcine endothelial cells, heparin mediates t-PA secretion (March-wardt et al., Thrombosis Research 8 (1976) 217-223), however, in human embryonic lung cells, the effect of heparin is minimal.

t-PA synthesis was also positively affected by concanavalin A in embryonic lung cells (Brouty-Boye, supra) and epithelial cells (Electricwala et.ai., Thrombosis and Haemostasis 53 (1985) 200-203), 5-azacytidine in epithelial cells ( Elec-tricwala, supra) and melanoma cells (Silagi et.ai., Biological Medicine 57 (1 984) 41 8), titsabrine 11a in melanoma cells (Roba et.ai., Thrombosis and Haemostasis 50: 1 (1 983) 83) and afidicolin in liver tumor cells (Orfanoudakis et.ai., Biological Chemistry, Hoppe-Seyler 336: 9 (985) 832).

EP 0219270 discloses that the combination of heparin and endothelial cell growth factor (ECGF) increases the production of t-PA by normal human diploid lung tissue-forming cells and the production of single-chain urokinase plasminogen activator in serum-free environment.

The effect of butyric acid on Chinese hamster ovary cells has been described by Storrie et al., Journal of Cell Physiology 94 (1978) 69-76 and Wright, Experimental Cell Research 78 (1978) 456-460. However, these studies were performed on non-transfor-. moitella cell1 injoi 1la and overall the results were related to • morphological changes in the cells as well as changes in their growth rate.

The present invention relates to a method for increasing the production of proteins in cultures of cells producing these proteins, comprising adding to the cell culture at a concentration of 0.005 to 500 mM thio-glycolic acid, thiodiglycolic acid, L-cysteine, afuthionic acid, glutathione, butyrylcholine diacid, butyrylcholine diacid, butyryl , 6-hydroxy-4,6-dimethyl-3-hepten-2-one, Da-hydroxy-substituted (C3- or C4-) aliphatic mono- or dicarboxylic acids, C3-C4-aliphatic monocarboxylic acids or their salts.

Suitable proteins are tissue plasminogen activators and mutants thereof.

t-PA mutants are derivatives of t-PA which have one or more amino acid residues which differ from the amino acid residue of the corresponding t-PA in the corresponding position or which do not contain t-PA mutants described in the literature, in particular in the above-mentioned in patent publications and application publications, in particular EP-A No. 199,574 and DE-A No. 3708681.

In addition, we found that C3-C5 aliphatic monocarboxylic acids and their salts were able to increase t-PA production and t-PAm in transformed CHO cell cultures of mutants.

Preferred protein production enhancers are thioglycolic acid, nonactic acid, butyric acid and propionic acid.

Protein production enhancers are commercially available from substances known in the literature or can be prepared according to known methods.

The compounds butyrylcholine bromide and chloride are commercially available, for example, from Sigma Chemical Co., Louis, Missouri, U.S.A.

7 94963

Nonactic acid and methods for its preparation are described, e.g., in Hely.Chim.Acta. Parts XLV (1962), Nos. 15 and 16, pp. 129-138; Tetrahedron, Parts 36 No. 1, pp. 46-49 and J.Orq.Chem. 45 (1980) 4259-4260.

Furan fatty acids are described in various sources, e.g., Lipids 12:10 (1977) 828-836; J.Chem.Soc.Chem.Commun. 16 (1976) 630-631; Lipids 10: 1 (1975) 695-702; and Fetter. Seifen. Anstrich-mittel no. 8 (1986) 290-292.

Useful acids of this type can also be prepared in the conversion reaction according to the following reaction scheme:

3 fU CU

KCH ^ CH

C + BrMg (CH2) n- TOA -► CH, (CH2) „-A V (CH2) - TOA

H O

1 2 3 ch3 ch3 hydrolysis nΛ

3 -► CH3lcna) a —p- (CH2) nCOOH

'4 where m = 2-5, preferably 4, n = 7-12, preferably 8 or 10, and -TOA =: = o - / \ o- 94963

O

b) CHj CH3 ♦ Br (CH2) n- TOA 3 i * ”1 *** 1. 4 c> 5 6

The preparation of compounds 2 and 6 is described in Voss et.ai. . Location.Chim.Acta 66 (1983) 2294.

m is preferably 4, and n is 8 or 10.

The compound 6-hydroxy-4,6-dimethyl-3-hepten-2-one can be prepared by a method known in the art.

It is also a natural substance and can be isolated from the species Capiscum annuns var angulosum, as described in C.A. 97: 159552f and Eiyo & Shokuryo 35:2 (1982) 95-101. This compound is also described in the dissertation "Neue Sekundärstoffe aus Streptomycesten: Isolation und Strukturaufklärung der Colabomycine, Pyrrolame und des Pyridazamycins", Groten, R. Göttingen University 1987, which also describes its preparation in Streptomvces ollvaceus (strain Tu3082) Microorganism of 23 November 1987 under the Budapest Treaty under accession number 4309) by cultivation and further isolation from this. Its NMR spectrum is as follows: il in mm ι ι t ai i 9 94963 C9H16 ° 2 (156.23) EI-MS: m / e = 138 (M + -H2O, high resolution, C9H140, 6%); 123 (M + -CH 5 O, 8%); 98 high resolution CgH10Of 48%); 83 (100%) 1 H-NMR (200 MHz, CDCl 3): δ = 1.27 (s, 7-H 3 and 8-H 3); 1.43 (s, broad, HO, exchange MeOD); 2.21 (s, 1-H3); 2.35 (d, J = 1.3 Hz, 9-H3); 2.32 (s, broad, 5-H2); 6.13 (s, broad, 3H) ppm 13 C-NMR (50.3 MHz, CDCl 3): δ = 22.0 (o, C-9) / 30.0 (o, 2C, C-7 and C- 8); 32.0 (o, C-1); 54.4 (u, C-5); 71.1 (u, C-6); 127.1 (o, C-3); 155.1 (u, C-4), 198.6 (u, C-2) ppm.

Suitable salts of said acids are, in particular, alkali metal salts, preferably sodium and potassium salts.

The method of the invention may be performed in a serum-containing medium or, preferably, in a serum-free environment.

When using the method in a serum-containing environment, it is preferable to use e.g. thioglycolic acid, nonactic acid or furan fatty acids or their salts.

When using the method in a serum-free environment, a C3-C5 aliphatic monocarboxylic acid, in particular butyric or propionic acid, or their salts are preferably used.

We found that compounds that increase protein production have an effect at concentrations of 10 μΜ to 500 mM, e.g. 1 mM to 10 mM. In particular, C3-C5 aliphatic and glutathione and their salts have an effect at concentrations of 1 mM to 10 mM.

10 94963

Protein production enhancers can be added to the culture at the time of preparation, e.g., on day 0, i.e. at the start of cultivation, and from there until the end of the third day of cultivation.

The protein production enhancing agent can also be used in multi-addition form. That is, the cells can be cultured in the presence of an agent, after which the cells are removed from the culture and transferred to another medium and further cultured in the presence of an agent that increases protein production. In the case of this multi-addition form, in particular the C3-C5 aliphatic monocarboxylic acids and their salts, preferably butyric acid or thioglycolic acid or their salts, show an increasing effect.

In the multi-addition form, it is not necessary to use the same substance for all additions, but one substance may be used in one addition and another substance in a subsequent addition. This multi-addition form also produces an addition effect, such as in particular the use of afidicolyl in one addition and the use of another of the substances, e.g. butyric acid, propionic acid, butyrylcholine chloride or butyrylcholine bromide or their salts, in a subsequent addition.

A combination of two or more substances may also be used. Preferably, no more than one anti-cell growth agent is used. For example, a C3-C5 aliphatic monocarboxylic acid, preferably butyric acid or thioglycolic acid, or salts thereof may be used, with the monocarboxylic acid or a salt thereof being preferably added to the cell culture as the 2nd agent.

= '«H <Hill I I t st 11 94963

By transformed Chinese hamster ovary cells are meant CHO cells transformed with a vector encoding a desired protein so that when cultured, they are capable of expressing and synthesizing that protein.

Suitable transformed Chinese hamster ovary cells are, for example, the cells described in EP-A-0093619 and 0199574 and DE-A-3708681. By culturing these transformed CHO cells, t-PA (EP-A 0093619) and certain t-PA mutants (EP-A 0199574 and DE-A 3708681) can be produced. Transformation of CHO cells with vectors, followed by expression of new proteins by them, is performed according to a procedure known from the literature, as described, for example, in the above-mentioned EP and DE publications.

The host cells can be CHO-K1-isolated cells isolated from tissue sample material by Puck in 1957 and added to the American Type Culture Collection (ATCC) under the code CCL-61 in 1970. This cell line has been re-deposited by the ATCC on December 23. 1987 under the Budapest Treaty under the symbol CRL 9618.

A DNA sequence encoding the desired protein can be prepared. according to a procedure already known from the literature, for example as described in several of the above-mentioned patent and application publications. Vectors containing the respective sequence as well as regulatory sequences can be constructed by methods known per se, for example using E. coli strain K-12294 (ATCC 31446) or E. coli strain X-1776 (ATCC 31537), which strains have been reposted by the ATCC storage facility. On December 23, 1,987, according to the Budapest Treaty, accession numbers 11a 53704 and 53705, in which case, for example, the plasmids pETPER and pETPFR described in EP-A-093619 can be constructed. CHO cells can be transformed as described in application 1 2 94963 EP -A U93619, DE-Λ 3708681, ΕΡ-Α 0117059 and ΕΡ-Α 199574 or other methods known from the literature.

The t-PA production vector was integrated into the ul clone CHO-K1-DUX H11 (Urlaub et al. 1, Proceedings of the National Academy of Science, U.S.A. 77 (1980) 4216-4220). This cell line - a mutant lacking dihydrofolate reductase (DHFR) - is suitable for the selection of transformed cell clones for DHFR. The human c-DNA vector may be the familiar plasma vector pBR322 or its progeny in Esherichia coli. The promoter region of this gene, as well as the DHFR gene, is derived from SV40 and the termination region of both genes is from a hepatitis B surface antioene. CHO-K1-DUX-B11 cells transformed with plasmid pETPER or pETPFR were used in the examples (see EP-A 0117059).

One of several known products is suitable as a serum-free medium (see, for example, the BMFT situation seminar 12 November-13 November 985, Bundesministerium fiir Forschung und Technologie, 5300 Bonn 2, pp. 111-120). In the following example, DMEM / llam's F 12 (1: 1) (Gibco) is used as the serum-free medium. Calf serum (FKS) at a concentration of 7.5% or 1-2% is added to the medium.

Cell cultures are incubated at 37 ° C in an incubator and developed. a gas mixture with 7.5% carbon dioxide in the air and a relative humidity of 100% is used. Depending on the culture, different culture vessels are used: shake flasks are used for 100-1000 ml aliquots, 175 cm ^ Roux flasks (Nunc) for 70-100 ml aliquots, 75 cm ^ same aliquots for 30-50 ml aliquots, cm ^ m the same flasks for 7-10 ml aliquots and Costar 2 cm ^ multiculture plates for 1 ml culture aliquots. To maintain the strain, cells are placed in cultures in a layer of 0.2-0.3 x 10 6 cells / ml and subcloned every 2-3 days. day.

1 3 94963

Protein production stimulants can be added in 1: 100 dilutions at any time, i.e. they are first dissolved in medium or, if sparingly soluble, in ethanol and then in medium, after which this batch of medium containing the substance to be added is added to the cell medium in a ratio of 1: 100 (v / v). .

In the following examples, to produce stimulation of t-PA synthesis, cells are taken from an exponentially growing culture and separated by centrifugation in plastic tubes (Falcon) for 10 minutes in a 1000 x g Beckmann T-3-6 centrifuge. The culture supernatant is removed by decantation, after which the cell pellet is resuspended in a new batch of channels.

F12 / DMEM supplemented with 7.5% FKS and gentamicin was used as a new medium to produce t-PA in serum-containing medium. The resulting suspension can then be transferred to 2 cm 2, 24-well plates at a cell concentration of 0.2-0.3 x 10® cells / ml.

To produce t-PA in serum-free medium, cells were initially adapted for 3-4 days in medium containing 1-2% FKS, after which they were centrifuged repeatedly and the cell pellet resuspended in serum-free medium and the resulting suspension was transferred to 2 wells of 24 wells. at a cell concentration of 0.4-0.5 x 10® cells / ml.

Quantitative t-PA assays.

Cell culture supernutants and cell extracts were sampled or prepared at various time points. Samples were measured either immediately or following storage at -20 ° C using the following methods: 1 4 94963

METHODS OF DETERMINATION

1. Direct chromogen assay (DCA) t-PA reacts with p-nitroaniline from Kabi Diagnostica's synthetic peptide substrate S-2288 (D-H-Ile-Pro-Arg-p-nitroaniline) as a reaction product. t-PA activity is proportional to the formation of p-nitroaniline measured at 405 nm at 37 ° C.

Enzyme kinetics were determined spectrophotometrically on an Eppendorf automated ACP-5010 analyzer.

Enzyme activity was calculated from a given standard A standard t-PA concentration plot of 1-15 μm was prepared using a Laboratory standard, with the enzyme used consisting of 72% 1-chain material.

The usefulness of the substrate solution (100 mM Tris / 106 mM NaCl) was 50 mM.

2, ELISA.

Quantitative assays of samples taken from culture supernatants and cell extracts were performed as an ELISA assay ·· (enzyme-linked immunosorbent assay). A graph prepared using a Laboratory standard in the concentration range of 0.038 to 5 ug / ml was used as a standard. The samples were diluted 1:10 to 1: 1,000.

Characterization of transformed CHO cells.

CHO cells were inoculated at a concentration of 0.2-0.3 x 10 6 cells / ml into culture dishes (7.5% FKS in medium), with a very slow increase at first. It was only after the 24-hour lag phase that the logarithmic cell growth phase began. At a concentration of il. m i nm I I «« 1: 15 94963 1.3 ^ 0.04 x 10 ^ cells / ml appeared on day 4 of culturing the first non-eligible cells; their share of the total cell number was 13.2 1, 3%. The maximal cell yield was reached on day 5 and was 1.8 x 0.05 x 10 6 cells / ml (Appendix 1).

After seven days, the proportion of living cells was still 72 3% of the total cell mass.

The post-exponential cell growth phase is followed by several factors leading to the statiouear phase. Due to the high population density and the consumption of essential nutrients, the cells no longer divide. The duration of the stationary phase varies widely and depends on the components contained in the medium such as serum. The accumulation of acids and toxic metabolites in the medium, as well as changes in environmental factors (acidic pH, low partial pressure of oxygen, deficient aeration), also accelerate cell death.

T-PÄ production by CHO cells.

There is a linear relationship between cell growth and t-PA synthesis.

There are 2 phases in t-PA production over time. In the first. in step t, the concentration of t-PA in the culture supernatant increases steadily. In the second step, the enzyme synthesis stops virtually completely and the t-PA concentration in the medium remains approximately constant (Appendix 2A). The maximum t-PA concentration on day 4 according to the ELISA assay is 11.1 +0.7 / ug / ml. The t-PA concentrations measured by the chromogen assays are somewhat higher, presumably due to the presence of 2-chain t-PA, however, when compared to the ELISA assay, the plots according to the assay methods are parallel until day 4.

The intracellular t-PA concentration also follows cell growth, however, 16,94963 is relatively low relative to the total amount of t-PA synthesized. On the fourth day, it accounts for 7% of total activity (Appendix 2B).

As shown in Appendix 3A, t-PA synthesis calculated from cell number is slightly enhanced until day 4; According to the amount of ELI SA, the maximum value is 8.1 ^ 1.2 λιί t-PA per 1 x 10 ^ cells. Intracellular t-PA was determined only by ELISA, as these low levels of t-PA (<1 μg) cannot be determined by DCA assay.

However, the intracellular t-PA concentration per cell number is somewhat higher during the log phase of cell growth, being 500-900 ng t-PA per 1x10 6 cells and from day 4 onwards about 450 ng / 1 x 10 6 cells (Appendix 3B ).

Effect of medium-containing serum and serum factors on CHO cell growth and t-PA production.

Cells were plated at 0.25 x 10 6 cells / ml, and the serum concentration of the medium was varied.

A serum concentration of less than 5% in the medium has a negative effect on cell proliferation, resulting in t-PA production capacity. contrary to the minor.

The effect of medium serum concentration on cell growth and cell t-PA production after culturing the cells for 48 hours is shown in Table 1. Values shown are percentages of maximum values (medium containing 7.5% FKS). The maximum cell number is 0.56 0.03 x ΙΟ ** cells / ml and the t-PA concentration is 5.7 ^ 0.5, υκ / ηιΐ.- PA transformations were performed by the DCA method (mean ^ standard deviation, n = 4).

Il i id i ι, ιΐ! , I, IF), 1 7 94963 TABLE 1.

f! "---

:% FKS Cell number t-PA

40 61.5 + 2.8 59.2 + 3.9 20 79.4 13.8, 81, 2 1 2.4 10 88.6 1 5.3 I 90.3 1 6.7 7.5 100 .0 1 6.4 100.0 1 7.9 5 72.4 14.9 87.0 111.3 1 43.1 1 1, 5 79.2 + 6.7 0 37.5 1 1, 8 75 .4 1 4.6

Cell culture in serum-free medium.

Cells were plated in serum-free medium at a cell concentration of 0.4-0.5 x 10 6 cells / ml. The cells continued to divide until day 4, when the maximum value of 1.0 1 0.2 x 10 6 cells / ml of living cells was also reached on day 4 (li ice 4).

t-PA production in serum-free medium.

The t-PA content of the culture supernatant rises steadily until day 7, reaching a maximum ELISA assay of 13.6 L 0.54 ug / ml. The t-PA values measured by DCA assays on days 1-3 are lower and from day 4 onwards higher than the t-PA values obtained by ELISA assays (Appendix 5).

The t-PA synthesis value per cell in the serum-free environment is approximately as high as in the serum-containing medium and / (7.2 13.6) - (10.3 12.8) / μg / l x 10 6 cells (ELISA) (Appendix 6).

18 94963

In a serum-free environment, the percentage of t-PA in the total protein content of the culture supernatant is / (10.9 1.0) - (12.5 + _ 1.4) /%. The results are shown in Table 2. t-PA values were determined as ELISA assays (mean ^ standard deviation, n = 4).

TABLE 2.

I - "I 'f Day protein .ug / lx10® cells, t-PA v. <</ 1 x 1 0 ^ cells | 1 71.0 + 8.0 7.2 + 3.6 3 81.8+ 9.4 | 9.0 + 2.3 5 82.0 + 12.2 i 9.3 + 0.8 _I__ Effect of aliphatic monocarboxylic acids.

The increase in t-PA production capacity of different aliphatic monocarboxylic acids in CHO cells in medium containing 7.5% FKS is shown in Table 3. Each acid was used at a concentration of 10 mM to 500 mM. The optimum dose for all monocarboxylic acids was 1 to 10 mM. The effect of these acids depends on their chain length.

All active monocarboxylic acids inhibit cell growth. The range of action of propionic, concentrated butyric and isovaleric acids is wider in their concentration, because their concentration-dependent inhibition of primary metabolism leading to inhibition of cell growth occurs simultaneously with the enhancement of t-PA synthesis. The values in Table 3 are calculated per ml of culture supernatant and are expressed as a percentage of control (= 0%). t-PA assays were performed using DCA (mean-> äu-t emi li t iu • · 9 94963 mean standard deviation, n = 3-24). The maximum value is obtained from one single sample. The measurement results obtained from individual samples are shown in parentheses.

TABLE 3.

('; Monocarboxyl- Optimal dose t-PA synthesis meat acid enhancement%

Maximum Average f formic acid 100 / im 7.4 6.5 ^ 0.9 propionic acid 5-10 mM (10 mM) 45.5 28.0 ^ 2.5 butyric acid 1-5 mM (1 mM) 68.3 41.9 + 3.8 isobutyric acid 0 , 6-10 mM (10 mM) 32.9 16.6 + 2.8

isoValerian- J

acid 1-10 mM (5 mM) 39.3 18.7 + 2.4

Mean values have been obtained from several 3-4 day production cultures with monocarboxylic acids added to the culture on days 0-3, as shown in Table 3A below. Data for individual samples are shown in parentheses.

20 94963

TABLE 3A

monocarboxyl-1 addition date sampling acid day n i.

formic acid 10 (0) I 2-3 (2) 4 propionic acid 0-3 (2)! 1-4 (3) 24 butyric acid 1 (3) 2-4 (3) 18 isobutyric acid 0-2 (3) 2-4 (3) 11 isovaleric acid 0-2 (4) 1-4 (4) 19 n = production growth figure

Appendix 7 shows the increase in t-PA production by C 1 -C 5 monocarboxylic acids as a function of time in a medium containing 7.5% FKS. After only 24 hours, a stimulation of t-PA production of 18.1 + can be observed. From 5.7% (C4) to 24.8 to 0.5% (C5). In the case of isobutyric acid, the increase in production rate did not occur until 48 hours later. For C3 and C4 carboxylic acids, the maximum enhancement of t-PA production was reached on day 3 and was 37.2 ^ 5.1% for Nu-butyrate. In the case of butyric and butyric acid, the increase in production is only short-lived and the effect is no longer noticeable on day 4. Propionic acid and isova-i. by contrast, the effect of enhancing the production of leric acid is still present on day 4 and is manifested by a 31.8% increase in t-PA production due to C3-acid and an 8.5% increase in C5-acid.

In addition, the effects depend on the physiological state of the cells. At their most sensitive to stimulation, cells are during their exponential growth.

Table 4 shows the increase in CHO cells and t-PA production by C3-C5 inonocarboxylic acids in medium containing 7.5% FKS as a function of carboxylic acid addition day when cells were grown for 48 hours at 7.5% FKS: in a medium containing The concentration of butyric acid and isovaleric acid was 1 mM and that of propionic acid and isovaleric acid was 5 mM. Values are calculated per 1 ml of culture supernatant and are expressed as a percentage of control (= 0%) (mean ^ standard deviation, n = 3-5). t-PA assays were performed using DCA.

TABLE 4.

date of addition C3 C4 and C4-I C5 0 28.7 + 9.3 33.0+ 5, * 20.1 + 10.7 17.2 + 1.0 1 24.4 + 4.1 40.2 + 12 , 2 25.3 + 5.1 1 9.4 + 1, 5! ______I_i

Effect of butyric acid on t-PA production by CUO cells.

The stimulatory effect can be combined with both an increase in intracellular t-PA and an increase in extracellular t-PA.

As shown in Appendix 8, the increase in intracellular t-PA in the culture supplemented with butyrate compared to the control is greatest after 24 hours of incubation (43.8-9.8%). In culture supernatant, the percentage increase after 24 hours is somewhat lower, averaging 19.2% (DCA and ELISA) of control values. This effect is maintained in the supernatant for 72 hours.

22 94963

Appendix 9A-C shows the t-PA synthesis values calculated per cell number and shows that the stimulation of t-PA synthesis by Na-butyrate is maintained for 7 days despite the anti-cell growth effect. During the first 4 days, enzyme synthesis per cell is steadily enhanced. The concentration of t-PA in the culture supernatant increases from 7.5 μl / μg per 1 x 10 6 cells to 28.9 5.3 μg t-PA as well as per 1 x 10 6 cells (ELISA assay) ( Appendix 8A). According to the DCA method, a maximum t-PA concentration of 47.4 to 7.4 μg of t-PA per 1 x 10 6 cells is obtained (Appendix 9B). At the same time, the intracellular t-PA concentration decreases from 1.5 to 0.2 μg of t-PA per 1 x 10 6 cells until day 5 (Appendix 9C).

Table 5 shows the stimulation of t-PA synthesis per cell number by Na-butyrate in medium containing 7.5% FKS. Na-butyrate was added on day 0 at a concentration of 1 mM. The values for days 1-4 are expressed as a percentage of the control (-0%) and calculated per 1 x 10 6 cells. The t-PA content of the culture supernatant and cell extract was determined by ELISA (mean standard deviation, n = 5-10).

TABLE 5 '; i r day intracellular extracellular concentration concentration 1 90.9 + 9.5 68.5 + 12.4 2! 99.7 + 26.5 118.8 + 16.1 3 93.9 + 31.4 223.4 +77.4 4 209.8 + 41, 4 280.7 + 85.9 23 94963

Optimal production efficiency was achieved when Na-butyrate was added to the cells during their exponential growth. The maximum effect of na-butyrate occurs at the time of addition 1 to days 0-2 up to day 3 (Appendix 10).

By adding Na-butyrate every 2-3. day in the same cell population following the change of this medium, t-PA production was able to maintain elevated levels for extended periods of time.

Table 6 shows the increase in t-PA production by CHO cells when culturing the cells in medium containing 7.5% FKS. Na-butyrate was added at 1 mM for each medium change or only on day 0. Two, four, seven or nine days after the medium change, the values measured are expressed as a percentage of the control (= 0%) and calculated per 1 ml of culture supernatant. t-PA assays were performed using DCA (mean ^ standard deviation, n = 3).

TABLE 6 '-i - “- 1-r tool tray change [date of addition on: j 0, 2, 4, 7 i -1 - --- 2 j 33.0 + 5.4 4 107.4 + 7.6 7 96 , 6 + 5.8 9 62.0 + 4.8

Effect of butyric acid on t-PA production by CHO cells in serum-free medium.

Contrary to the short duration of the butyrate suspension in serum-containing medium, this effect is prolonged in serum-free medium and is 44.1-7.1% of the control 1 day after the addition of Na-butyrate. Maximum recovery was reached 3 days after the addition of Na-butyrate and was 143, 12.8%. The increase in production was still observed 6 days after the addition of Na-butyrate, which was 49.9 7.0% (Appendices 11 and 12).

The level of t-PA synthesis per 1 x 10 6 cells is also higher in the serum-free medium than in the serum-containing medium and increases by 50 μg per 1x10 6 cells (Appendix 13).

Effect of propionic acid.

This C3 carboxylic acid shows very similar effects on t-PA synthesis as butyric acid. However, the percentage enhancement of production remains about 14% lower and the concentrations required for t-PA stimulation are higher (5-10 mM). Furthermore, propionic acid inhibits cell proliferation to a lesser extent and therefore produces longer-term enhancement of t-PA synthesis.

In contrast to the effect of butyric acid, the enhancement of t-PA synthesis by propionic acid appears to be very specific, as in the serum-free medium the extracellular protein concentration increased only slightly compared to the control and the consumption of β-leucine for protein synthesis increased only slightly.

Effect of dicarboxylic acids, hydroxycarboxylic acids and ketocarboxylic acids on t-PA production by CHO cells.

Hydroxycarboxylic acids such as lactic acid, glyceric acid, malic acid and tartaric acid show only a slight enhancement of t-PA synthesis of 6-10%, with t-PA synthesis being best stimulated by tartaric acid at 10 mM (obtained <1 .mu.i Milli). III «I · 25 94963 value 10.3 2.9% of control). Hydroxyl acids do not affect cell division and their effect on t-PA synthesis is still detectable after 96 hours.

Effect of ascorbic acid

The effect of ascorbic acid at 10 mM on t-PA production lasted 72 hours and on day 0, added at 24 hours, produced an increase in t-PA synthesis of 15.2 by 3.1% (Appendix 14).

Effect of long chain fatty acids on t-PA production by CHO cells.

We found that long-chain fatty acids such as nonactic acid comprising 10 carbon atoms as well as furan fatty acids were also able to provide enhanced t-PA production (Table). In this example, furan fatty acid of formula 4 was used. in the formula m = 4 and n = 8.

However, in the serum-free environment, no enhancement of t-PA synthesis or protein synthesis in general was observed.

The enhancement of t-PA production by CHO cells by long-chain fatty acids is shown in Table 8. The cells were cultured in medium containing 7.5% FKS. Concentrations of 10 to 10 mM were tested for acids, with a concentration of 1 mM being optimal. The values obtained are calculated per ml of culture supernatant and are expressed as a percentage of the control (= 0%) (mean ^ standard deviation, n = 4-9). The maximum value obtained is measured from a single sample. t-PA assays were performed using DCA.

TABLE 7 26 94963 f —-- 1 -—-——

% Increase in fatty acid t-syn synthesis

Maximum Average __1

Nonactic acid 58.3 30.3 6.5

Furan fatty acid 32.1 19.0 3, 2

Mean values have been calculated from several 4-day production cultures with acids added on day 0, as shown in Table 8A. Data for individual samples are shown in parentheses.

TABLE 7A

Fatty acid Date of addition Sampling date n

Nonactic acid 0 (0) 1 to 4 (2) 4

Furan fatty acid 0 (0) 1 - 4 (2) 9

Effect of thiols and sulfides on t-PA production by CHO cells.

Four sulfur-containing compounds, which are derivatives of carboxylic acid or their substitution products, showed an increase in t-PA production of 16 to 31.2%, but had no negative effect on cell growth (Table 8). The optimal effect of the substituted carboxylic acids was 27,94963 at the same concentrations (1-10 mM) as the other active caroxylic acids. Only glutathione was effective at lower concentrations, 1-10. Thioglycolic acid proved to be a beneficial agent for stimulating t-PA synthesis in CHO cells.

In this example, cells were cultured in medium containing 7.5% FKS. Each substance was tested at concentrations of 1 μM. The values shown in Table 9 are calculated per 1 ml of culture supernatant and are expressed as a percentage of the control (= 0%) (mean standard deviation, n = 6-19).

The maximum value obtained is measured from a single sample. t-PA assays were performed using DCA. The concentration of the respective substance is indicated in parentheses.

TABLE 8 —-— I ---—--— »

Thiol or sulphide Optimal dose Enhancement of t-PA synthesis%

Maximum Average

Thioglycolic acid 1 mM 66.4 31.2 + 3.2 'Thiodiglycolic acid 1 mM 31.0.0.0.0.0 L-cysteine 1-10 mM (1 mM) 41.4 20.9 +3.4

Glutathione 1-10 μM (10 μM) 30.6 16.0 + 2.0

Mean values have been calculated from several 3-day production determinations, with substances added on days 0-2, as shown in Table 9A. Data for individual samples are shown in parentheses.

«

TABLE 8A

28 94963 -! - ~ —Π — 1-Π- ~ -1

Fatty acid Date of addition Sampling day n!

Thioglycolic acid 0-2 (1) 1-3 (2) 19

Thiodiglycolic acid 1 (1) 2-3 (3) 9 L-cysteine 0-1 (0) 1-3 (2) 11

Glutathione 0 (0) 2-3 (2) 6

The advantage of thiocarboxylic acids over monocarboxylic acids is that they do not have an inhibitory effect on cell proliferation, although even these substances did not cause t-PA production to rise further from the level reached with butyric acid. Likewise, the potentiating effect is short-lived and the maximum value is reached after 24 hours for thioglycolic acid and after 48 hours for cysteine. After 4 days of incubation of the cells, the effect on t-PA production is still negligible (Appendix 15).

However, in serum-free medium, the stimulatory effect on t-PA synthesis is less than in serum-containing medium, since all these substances inhibit cell proliferation; of which 10-20%.

Effect of thioglycolic acid.

Following the addition of thioglycolic acid, t-PA production remains elevated for 72 hours regardless of the date of addition. However, the percentage increase in enzyme synthesis is at its highest after 24 hours and decreases continuously from this time onwards.

Table 9 shows the t-0 of CHO cells as a function of the day of addition

Il: lll.L lllli 1.1 * M.

29 94963 Increase in PA production when culturing cells for 24-96 hours in medium containing 7.5% FKS and 1 mM thioglycolic acid.

Values are calculated per ml of culture supernatant and are expressed as a percentage of control (= 0%) (mean 1 standard deviation, n = 4-6). t-PA assays were performed using DCA.

TABLE 9 ——, '1 1 - - I —— r - "1'}

Time (hours). Date of addition i

i I

-1 --- i 0 1 2 24 27.6 + 5.6 34.7 + 10.9 34.5 + 7.5 48 16.9 12.3 17.1 1 3.6 16.3 + 8 .6 72 14.7 11.0 10.2 1 2.7 9.4 11.7 96 3.413.0 2.710.8 0.511.0 t-PA production can be increased by adding thioglycolic acid instead of once in two different additions (Appendix 16). ).

Annex 17A (DCA) and Annex 17B (ELISA) are presented. Progression of t-PA synthesis per 1x10® cell.

t-PA synthesis is enhanced by the action of 1 mM thioglycolic acid in the first 3 days (DCA) compared to the control. In contrast, t-PA values obtained by ELISA remain elevated throughout the period. The rate of rise on day 4 is still about 35%. Thioglycolic acid likewise increases intracellular as well as cell-calculated t-PA. The intracellular maximum t-PA concentration is 1, 1 1 0.1, μg 1 x 10® per cell after 24 hours and decreases in 96 hours to about half of the initial concentration (Appendix 17C).

30 94963

The extracellular t-PA concentration on day 5 is increased by 10.7% compared to the control. The percentage enhancement of t-PA synthesis produced by thioglycolic acid is somewhat lower in serum-free medium, as cell proliferation is somewhat inhibited (10-20%).

Effect of monocarboxylic acid derivatives on t-PA production by CHO cells.

Additional compounds were found in the group of C4 carboxylic acid derivatives that show an effect on t-PA synthesis in CHO cells. These compounds primarily include butyrylcholine bromide (BCB) and chloride (BCC), which increase t-PA production by 30-40%. These C4 derivatives show the same effects as Na-butyrate in all respects and have the effect at the same concentrations.

Table 10 shows the enhancement of t-PA production by CHO cells produced by the two aforementioned derivatives. Cells were cultured in medium containing 7.5% FKS. All substances were tested at concentrations of 10 μM to 10 mM. Result values are expressed as a percentage of control (= 0%) and are calculated per ml of culture supernatant (mean ^ standard deviation, n = 4-15). Values obtained by one individual sample are indicated in parentheses. The maximum value obtained is obtained from a single sample. t-PA assays were performed using DCA.

TABLE 1 0 31 94963

Monocarboxyl- Optimum- t-lih synthesis meat acid derivative enhancement% female __

Maximum Mean n BCC 5-10 mM (10 mM) 63.9 38.8 + 4.4 15 BCB 5-10 mM (2.5 mM) 49.6 29.1 + 4.4 9

Mean values were obtained from several 4-day production cultures as shown in Table 10A. Values for individual samples are indicated in parentheses.

TABLE 10A

Substance Date of addition Sampling date n BCC 1 (1) 2-4 (4) 15 BCB 1 (1) 2-4 (4) 9 When a combination of substances is used, at least one substance should be non-cell growth inhibiting.

Improved yields were achieved e.g. by using thioglycol ·. a combination of acid and butyric acid. The difference between the times of addition of the individual substances may then play a significant role, which is why, for example, butyric acid was always added as the 2nd substance in order to reduce its inhibitory effect on cell growth.

32 94963

The addition of these substances at the same time on day O does not improve the effect obtained with butyric acid.

The effect of the combination of butyric acid and thioglycolic acid is additive. The efficiency effect was at best 70%.

In the serum-free medium, the effect of butyric acid could not be increased by the use of another substance.

Table 11 shows the increase in t-PA production by CHO cells when the cells were cultured for 3 days in medium containing 7.5% FKS. A combination of 1 mM thioglycolic acid (TG) and 1 mM butyric acid was added to the cells at various times. Values are calculated per 1 ml of culture supernatant and are expressed as a percentage of control (= 0%) (mean standard deviation, n = 4-8). t-PA assays were performed using DCA.

TABLE 11

Date of addition Butyric acid 0 1 2 0 31.3 +1.7 67.9 +5.2 30.3 +3.2 1 68.8 + 4.4 31.4 + 5.5 2 41.4 + 5, 5

Effect of aphidicoline.

Afidicoline, a diterpene from Cephalosporium aphidicola, specifically inhibits DNA alpha polymerase and stimulates at a concentration of 1-10 μm. T-PA synthesis in CHO cells in 24 hours, t-PA yield is increased by 44.9 + 13.9%.

Aphidicoline showed stimulation of t-PA synthesis in CHO cells.

It may reversibly inhibit DNA synthesis, resulting in no effects on RNA and protein synthesis. Thus, aphidicoline is suitable for stimulation of t-PA synthesis on the one hand and for cell synchronization on the other hand. The effect of afidicoline on t-PA synthesis when cells are incubated for a prolonged period of time in medium containing afidicoline has been described above. This section deals with experiments on cells that were exposed to afidiol for 24 hours and then further cultured in medium that did not contain afidicoline. The various stages of the cell growth cycle as well as the migration of β-thymidine into the synthesized proteins were analyzed during and after the 24-hour exposure period to monitor the inhibition and resumption of DNA synthesis following the removal of afidicolin. RNA, protein and t-PA synthesis levels were further determined.

Cell growth.

Afidicoline (1 / mO slightly inhibited cell growth after 2 h of exposure (5-10% inhibitory effect). However, when the medium was changed, the culture cells containing afidicolin were placed in new cultures at the same cell concentration as the control cultures.

t-PA synthesis.

't-PA synthesis stimulated! At a concentration of 1 mM aphidicoline in synchronized cells. The effect was still noticeable 96 hours after the change of medium.

34 94963

Table 12 shows the increase in t-PA production in CHO cells exposed to a αf-dicoline concentration of 1 μM cultured in medium containing 7.5% FKS. Values are calculated per ml of culture supernatant and are expressed as a percentage of control (= 0%) (mean ^ standard deviation, n = 4). t-PA assays were performed using DCA.

TABLE 12

Time (hours of medium-t-PA synthesis.

% of efficiency gains 24 40.2 + 6.7 48 59.9 + 11.1 72 32.8 + 6.4 96 14.7 + 1.3

Appendix 18 shows the enhancement of t-PA synthesis in serum-free medium as a function of time. The effect is maximal 48 hours after changing the medium.

V. The t-PA content of the serum-free medium was determined after 96, 120 and 144 hours as ELISA assays, the results of which were substantially higher than the values measured by DCA compared to the control values. The results are shown in Table 13. The experiment was performed on CHO cells exposed to an aphidicolin concentration of 1 μM and cultured in serum-free medium. Values are calculated per 1 ml of culture supernatant and are expressed as a percentage of control (= 0%) (mean + _ standard deviation, n = 4). t-PA assays were performed as ELISA assays.

. * here i dj »l i 1 st; TABLE 13 35 94963

Time (hours after change of medium-t-PA synthesis)% increase 96 36.7 + 6.2 120 42.0 + 8.8 144 51.3 + 10.2

Elevated intracellular t-PA was only detectable at 24-hour afidicolin exposure and was approximately 30% elevated.

Butyric acid, propionic acid, butyrylcholine chloride and bromide were able to further increase t-PA production in this system. Stimulation occurred in both serum-free and serum-containing medium, however, in serum-free medium, an additional elevating effect was achieved only by the addition of these substances after 24 hours of adaptation of the cells to that medium.

Table 14 shows the percentage increase in t-PA production in cultures exposed to aphidicolin following the addition of Na-butyrate to the culture. Na-butyrate further increases the degree of t-PA synthesis by 14% after 96 hours.

The experiment was performed on CHO cells exposed to an aphidicoline concentration of 1 μM, cultured in the presence or absence of Na-butyrate as serum-free cultures. Values are calculated per 1 ml of culture supernatant and are expressed as a percentage of control (= 0%) (mean standard deviation, n = 4). t-PA assays were performed as ELISA assays.

TABLE 14 36 94963

Time (hours of growth- Afidico- + Na-butyrate: vessel exchange from time of addition) ____ point (hours) _ ___0_24_ 96 36.7 +6.2 34.1 + 9.7 50.6 +11.0 120 42, Effect of 0 +8.8 31.8 +10.2 49.7 + 4.3 6-Hydroxy-4,6-dimethyl-3-hepten-2-one (DHO).

t-PA production is at an elevated level 144 hours after exposure of the culture to the title compound. The stimulatory effect on t-PA synthesis was manifested at micromolar concentrations of compounds, e.g. 0.005 - 100 μM.

Appendix 19 shows the increase in t-PA production in CHO cells cultured for up to 168 hours in serum-free medium with a DHO concentration of> 7 μM to 7 mM. Values are determined using DCA. The corresponding values obtained as ELISA assays are given in the Annex.

Cell cultures prepared according to the methods described above can be further processed in a manner known per se to isolate t-PA, e.g. at the end of the production step the medium is separated from the cell mass and the culture supernatant is purified by ultrafiltration and chromatographic procedures.

The isolated t-PA can then be formulated into pharmaceutical preparations, e.g., as a solution or lyophilized preparation.

The foregoing examples can be performed by replacing t-PA-producing CHO cells with transformed CHO cells capable of expressing other proteins, e.g., t-PA mutants or other pharmacologically active proteins, e.g., as described in the aforementioned EP- and DE patent and application applications, in particular CHO cells in EP-A 199574 and 196920 and DE-A 3708681.

4

Claims (8)

94963 38 A method for enhancing the production of t-PA-like proteins from a culture of transformed CHO cells, characterized in that thioglycolic acid, thiodiglycolic acid, L-cysteine, glutathione, butyrylcholinacol chloride, butyl chloride, butyl chloride, butyl, are added to the culture at a concentration of 0.005 to 500 mM. ascorbic acid, aphidicolin, 6-hydroxy-4,6-dimethyl-3-hepten-2-one, Da-hydroxy-substituted (C3- or C4-) aliphatic mono- or dicarboxylic acids, C3-C4-aliphatic monocarboxylic acids or their salts. Method according to claim 1, characterized in that the protein is t-Pa or a mutant thereof. Process according to Claim 1 or 2, characterized in that butyric acid or a salt thereof is used as the protein inducing agent. Method according to one of Claims 1 to 3, characterized in that the culture is carried out in a serum-containing medium. A method according to any one of claims 1 to 4,. characterized in that the C3-C4 aliphatic monocarboxylic acid or its salt is added in concentrations of 1 mM to 10 mM. Process according to one of Claims 1 to 5, characterized in that the cells are cultured in the presence of one of the following substances: thioglycolic acid, thiodiglycolic acid, L-cysteine, glutathione, butylcholine bromide, butyrylcholine chloride, nonactic acid, furan fatty acid, furanic fatty acid nitric acid, aphidiccholine, 6-hydroxy-4,6-dimethyl-3-hepten-2-one, Da-hydroxy-substituted (C3- or C4-) aliphatic mono- or dicarboxylic acid or their salts, the cells are separated from the culture and then added to a new culture interval - • I td; » dill I I I n I 39 94963 and cultured in the presence of a C3 or C4 aliphatic monocarboxylic acid or a salt thereof. Process according to Claim 6, characterized in that the aphidicoline is the first substance and the butyric acid, propionic acid or C 3 or C 4 aliphatic monocarboxylic acid or a salt thereof is the second substance. Process according to Claim 6, characterized in that butyric acid or thioglycolic acid or a salt thereof is used. «40 94963