CA1341400C - Process for preparing proteins - Google Patents

Abstract

A method of increasing the production of a protein by cells in culture, which method includes the step of adding to the culture medium an effective amount of an inducer of protein production selected from thioglycolic acid, thiodiglycolic acid, L-cysteine, glutathione, butyryl choline bromide, butyryl choline 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, anhydromevalonic acid lactone, cis-anhydromevalonic acid lactone, D-.alpha.-hydroxy-substituted (C3 or C4) aliphatic mono-or dicarboxylic acid or salts thereof.

Description

~ ~~~ goo _1_ Method for preparing proteins This invention relates to a method For preparing proteins, particularly plasminogen activators suc'n as t-PA (tissue p'Lasminogen activator) and the mutants thereof, in general, the i.nwention provides a method of increasing the nroductiwity of cell cultures {e.g. C~i~ ce~_Is) For t~.'ie s~ynthes;_s of proteins.
i?lasminogen activators are a c:,la.,, :p serine proteases which activate the proen~;y;n~~ pl:-~~--;mirlagen :»r cleaving the peptide bone between Arg-o6~ an:~ teal-5~1_ to produce the active enz°,rme pla~~~nin. Plasmin, in turn, is the last stage of the ~~i'~rinolvsis system of the blood stream which splits the fi'orin structure of a clot into soluble pepti<~es.
In pathogenetic disorders such as coronary disease, blood clot's are not brcoken up fast fanough to ensure that the tissue receives an adequate supply of oxygen. The consequent=a of occlusion of_ the coronary arteries by a blood clot is an infarction, whilst the deficiency in the supply of oxygen to the heart muscle res~_zlts in necrasi.s of the affected tissue.
Rapid reopening of the blood ~leSS2ls can ensure that there is a blood supply to the heart muscle in a very short time and thus pe~event whole sections of the heart muscle from dying off.
Up till now, streptokinase and t:rrokinase have been used in therapy. However, the properties of t-PA

_~_ have some advantages over these known plasminogen activators: the fibrin--specific loc~~7_ lysis, the absence of systemic lyt~ic eFfect:s caused by fibrinogen breakdown, high rates of reperfusiorn and the absence of antibody formation.
t-PA is a glyco«rotein with a molecular caeight of about 65,000 nalton:~ which or.cur:, as a single-c:inaitned and t;ao-rhai.ne~l en:~ymt~ :end is na;~e up of 1!7 5'?'7 amino aai<1 s.
The a~ ~- ini':y ov i:-~r, r:~or plasrn ir-u~ge:1 i:~ ab«~.zt 1.90 ti.~nfes cheater in tlne :~r-t~:~~~nce ofi ti.°~Ain than in the .~'.~sence of- i-iorin. ~Chi.s h:i.t~h a~'lvinity oi. t-PA
7_5 for ~ol.a:~~ni.noc~erl in tine p.re:~r~rl:~~p nF ~,-it~rin en:,ures E?~~f?,';i'.l',7F? c'~Ctl.V;~tLt~1"t W;. I:'r'lOtli; ~i;'.t.l',7~'~.1.t1~ ctW7 i r;-'_C~
~La;~;ni noge~n i n flue mer iplnery.
H~_zman t-PA was Firat obtained pure form from in 20the uterus , t-PA has ~~l so been eta~cted in other :~

tissues and cells, e.g. endothel.i.al cells, including arterial and venous en~ot'nelial ells. Ftowever, c the arnot.znts of t-PA formed are s nail that it ~~a is impossible to obtain them on commercial scale.
a Another natural so~_zrce of t-PA cell cczlture.
is A number of cell Lines leave been investi.c7ated for possible t-PA orodr.zctican (A:~rono~ar~t, et al. , Trends in T3iotechno.logy l: 26--2~3, ~ai.rly large 1983? .

30quantities of_ t-PA were obtained for limited clinical studies and for ingestigati.on t!n~~ir_ structure ofv from a human cell. line, namely we~ wel,~noma cells Fa~o (wallen et al., European ;Tourna3.of Hiochernistry 132: G81-686, 1983).

However, these yields are too small for large scale production of t-PA for wide ran<~ing clinical user.

~ X41 x+00 -Only by genetic engineering has it been possible to produce larger quantities of recombinant t-PA
for therapeutic use.
In 1983, t-PA was cloned and the amino acid sequence was determined (Pennica, nature 301: 214-221, 1983).
Attempts to ontai.n t:-P?~ t=torn ~; -_coli in which the genetic inraYmation Eor t=-P:~, C-'r-orn :nelanorna cell=>
had been intP~7_r.~~-e;i were unsuccessful_ since the resulting) roroteilv i.~~ not c-~lycc~~~°,,~~ate<~ and conseqrzently dOeS nOt !~OC"C~~s:)_irl~) t0 tilE-? F1,~~_3 ~"'<? t_pT~.
COr thlS CE'~.~'.;On, V-~Y' LOlIS re'>eaL"C:',f1 =~rOtl~7.':p lOO~Cl?1C~
into the br_oc7uct.iorl c~F hv.~marz t--'"?;~ yaave sought out permanent vn~~m~nal.an c-el_ i. lrie~~ ~~rom various sources.
Some of the:;e ;~.el.l 1_ines .ire hnroar~ cell lines with an appac-entl~y im~oroved yield of t-PA. Another cell Line i_s the n~~rrna7_ ~'.hine~e hamsl:er ovary cell.
EP-A-93,519 descrihes the nreba~~ation of t-PA from transformed ''hinese hamster ovary cells (CHO cells).
The resulting t-1?A (r-tPA) does not differ from naturally occurrin.3 t-PA in any way.
Several publication: descritae ml.~tants of t-PA and the preparation thea-eof, e.g. ~~i?-A ?_41,208, 241,209, 247_,210, 240,334, 234,051, 233,()1.3, 231,624, 225,286, 2.13,794, ?_01,153, 1_'x9,574, 196,920 and D~-A 3,708,681.
The present application refers to all t-PA derivatives as mutants, regardless of whether they contain one or more amino acids which differ from the amino acid in the same position i.n the naturally occurring t-PA or wherein one or more amino aphids which occur in the natural t-PA da not appear in these "mutants". A t:-PA in which a number of_ amino acid groups c?F the natexrally occurring t-PA are absent, as in F;P-A-1.96,920, for example, is sometimes ~ 341 400 referred to as a "degraded species".
EP-A 199,574, for example, describes a t-PA mutant which has, in posit.ion~ 27c) to 279, certain amino acids which differ from t:hf~ amina acids in the corresponding positions of the natural t-PA.
In DE-A 3,08,681, we prepared some t-PAs whi<-h have an amino aci~~ i.n position 1.17 ~~hich di.f_fe!-s l_0 from the amino acir~ in the cor-ra;;;~onuinq no:~ition of the natural t-PA, bet iznli"e the natural_ t-''A
the mutant t-PA is not ~~L.yc<>syl~te:~i :~t this position.
A number of ou'~Lications ?~avf> :~~~-~-r hoed t!~~~ in'iuence of various suhstanr.es on t'~~~ r-~r,-~bW_ic~:i~~itv of cel_1 cultures for the synthc~:~i:~ <v>f- ~,r:~te i.ns.
In various cell syste~n:~, it is Ye~o~-ted that there is a connection between intr:arell.~zl<~r c-AMP levels and t-PA synthesis (sec> for exarr~,a>1e F;ooistra et al. in Thr_omhosis and liaemost~asi.s, 54 (l_1 : 1_92, Abstract P 1133) and it is re~~orted that dibutyryl c-AMP increases the prodr.rct i..on r~~ f t ~ PA i n human endothelial. cells f->y a factor of '<? to 3, hut has no affect on the synthesis of t--PA in Bower melanoma cells. In kidney cells=a t:~h~~ synt',~ps ~s of t-PA was increased by means of phosoho~:li~asterase inhibitors (,lournal of Cell Biology y 91: 1_9~~--20f), 1981) . On the other hand, c-AMP inhihit~ t-P:A :synthesis in macrophages (Vassalli tit al., CE:lls, 8: ?71-281, 1976) and i.s inert in teratocaroinorna cells (Nis'nimune et al. , Experimental C~~17. Resaar-c~h 1_.:~6: 439-444, 1983) .
Butyric acid induces the synthesis of t-PA in terato-carcinoma cells (Nishimune - lor.cit.) but has an inhibitory effect in renal tumour_ cells (Nelson et al., Proc. zlm. Ass. for Cancwr Research, 26:35, 1985) and F3routy-Boye ~~t al., report in Biotechnology ~ ~4~ goo - 5 _ 12: 10, 1984 that it has a neutral effect i.n embryonic lung cells.
In human endothelial cells, t-PA synthesis can be stimulated by thromioin (T~evin et al. , Thombosis and Haemostasis, 56 (2) : 715-119, 19136) , Alcohol (Lang, ,journal ~f the American Medic-~.al Association, 250 (6) : 772-776, 1983 ~:3) , z.7itamin ~~ and T7itamin (Inada et al. , Bi.ochemica7. anc3 Biophysical rommuni-canons ? 30 (1) : 182-18-', 19F; >) , However, t.-PA
secretion in booine endothelial celi.s is in'~ibited '~°~ t'~ro~nhin (Lar7~;utaf~', ~'Iournal_ of (''finical Investi-gations, 64: 323-332, 1.979 and Enc3<:~toxin (Crutci~ley et al.., ,7ourna7_ of Biological Chemi:=try, 261 (11 t5 154-159, 1.986).
The secretion of t-PA from the oig endothelium is aF~ected by means of~ her~arin (Mar~chwardt. et al. , Thrombosis Research, f3: 217-22'3, 1_976) , whereas in human embryonic lung cells hepar~.n has a minimal effect.
The synthesis of t-PA is also positively affected by concanavali.n A in em'urycanic lung r_ells (Brouty-Boye - loc.cit.l and e~~ithelial cells (Fl.ectricwala et al., Thrombosis and Fiaemostasis, 53: 200-203, 1985) , 5-azacyti~~ine ire epithelial cells (r~_,lectricwala - loc.cit. ) and rnel_anoma cells (.~il<xgi et al. , Biological ~~Iedicirle, 57: 41_3, 1984) , Tizabrine in melanoma cells (Roba et al. , "i°hr<ambosis and Haemostasis 50 (1) : 83, 1983) and aptn idicoli.n i_n hepatoma cells (Orf-anoudak:is et al., Biological Chemistry, Hoppe-~eyler, 336(9): 83'?, 1985).
It is stated in EP-A-219,270 that a combination of heparin and endothelial cell growth factor (ECGF) increases the production of t-PA and single -chained urokinase ~lasminogen activator from normal human diploid lung fibroblast: cells in serum-free medium.

1 34~ 4p0 _~_ The influence of butyric acid on Chinese hamster ovary cells was reported by Starrie et al., Journal of Cell Physiology, 94: 69-76, 1.978, and Wright, Experimental tell Rese~~rr_h 78: 486-460, 19'78.
S However, these investigations were carried out with untransformed cell lines anti the results were by and large concerned with the changes in the morphology and growth rate of_ the cells.
1.0 Thus while extensive wcar_k has °~ec~n reported on method of increasing nroi:.ein orc~duction i_n cell cultures , ~:he r_ exults '~~ave i n ciener ~a_ been -specafic, variable or are negative. There is a continuing need for a ~rc~cess fi>rv increasing protein l.~ production i.n cell cultures.
Accordingly, one aspect of our i_n~~ention r~rovides a oethod of increasing t'~e produ.ction of a or_otei_n by cells in culture, wnic~h rnethc;~c3 includes the 20 step of ad,-sing to the ~-ult~are vnc~c3ium an efFective amount of an anducer of protean t~rod~~ction selected from thioglycolic acid, t~hiodigl.ycolic acid, T~-cysteane, glutathione, biatyryl rhol.ine bromide, butyrvl choline chloride, nonact.ic arid, furan 25 fatty acid, ascorbic acid, aphiriicolin, 6-hydroxy-4,6-dimethyl-3-hepten-2-one, fu-,ar~ic acid, meval.onic acid, tries-anhyciromevalonic aci.~3, anhydromevalonic acid lactone, cis-anhydromevalanic acid lactone, D-cx-hydroxy-substita~te~i (t'.3 or ~~4) aliphatic mono-30 or dicarboxylic acid or salts t'~ereof_.
The T~roteins i.n question <~re preferably the plasminogen activators, particularly t-PA and the mutants thereof. However, the eFfect of the method 35 is independent of the proteins <3nd the ~'~A coding for these proteins and can therefore be applied to e.g. CHO cells which are transformed in order_ to express any foreign protein.

_ ., Mutant t-PAs are derivatives of t-PA which have one or more amino acid groups which differ from the amino acid groups in the corresponding position of natural t-PA, or in which one or more amino acid groups of natural t-PA dc~ not occur. Some t-PA mutants of this kind are described in the literature, especially in the patent applications mentioned above, more particularly in EP-A 199,574 and DE-A 3,108,681.
The method. of the invention is preferably carried out with transformed CHO <:ells.
It has also been found that the C3-C5 aliphatic monocarboxylic acids anal t:he salts thereof are capable of increasing the production of t-FA and the mutants thereof in cultures of transformed CHO cells.
Thus another aspect of our :i.nvention provides a method of increasing the production of a protein from cultures of transformed CHO cells, which comprl.ses adding to the culture medium an effective amount of a C'3-C5, preferably C3-C4 aliphatic monocarboxylic acid ox° a salt thereof as an inducer of protein synthesis.
Preferred protein productiar~--increasing substances are thioglycolic acid, nor~actic acid, butyric acid and propionic acid.
The protein production-increasing substances are commercially available products or are known from the literature or may be prepared by known methods.
",,e 27169-151 ~ 341 400 7a 'The compounds butyryl czhol ~_ne bromide and chloride, fusar:ic acid, mavaloz~.irw ac::i.d 1 actone are commercially available px-oduct_.:~ and rnay be obtained, for example, from Sigma Chemical Co., ;7t:.. Louis, Missouri, USA.
According to a further aspect of the present invention, there is prov_i.ded a method of increasing the production of a r-tPA or mutarnt: t.hezwF:~of f room culture: of transformed CHO cells, wr=er~:ai.n <~ C:,-C:',, a:i iphatic monocarboxylic acid or a sa:Lt t.hereaf_ is added to the culture, and cuL.tivation is carried c.ut in serum-free medium.

_~-Nonactic acid and processes for the preparation thereof are described 'for example in Helv. Chim.
Acta, Vol. XLV, (1962), No. 15-1.6, n. 129-138;
Tetrahedron, Vol. 36, No. 1, x~. 46-49 and ,7. Org.
Chem., 1980, 45, p. 42~>9-4260.
Various publications describe f.uran fatty acids, such as Lipids, 197?, 1_2 (1.0) , 82,8-36, J. Chem.
Soc. :them. ~omrnun. , 1~~?6, (1 a) , ?~. 630-l, Lipids, 1975, 10(1_7.1, n. 595-7~)2, and Fette, Seifen, Anstr_ich-mitt~el, Vo. 8, 1986, ~. 29!7-292.
UseFul acids of= this t~~~pe may also lop prepared by carrying out a r_eactic~n accor_uin~7 to the following 1..'~ .~!~eme CH3 CHI CH3 CH3.
CH3(LH ) ~-__ ~~-C +. 3rMg(~~H ) - '~C~A --~ Ci-i (r?i ) ~ ~~(CH_) .-! C~:
2 m ~ 0/~ 2 ~ z 3 2 ~ -~ C ~

__. ._ C~33 C H; __ /'' Hydrolysis ______..... , yH~(Ci-i.;)~... ~ _._..,~,.., (;;-?~)n-.C~~:'t?
'+
wherein m = 2 to o, ~reFerahly 4, n - 7 to 7.?, preferably 8 or i_0, ;~ __~/
aryj _mp.~, _ _ _ 3 5 ,~;?

b) ~-iyc~ro 1 ys is CH3(CH2)m- ~~--~,i + ~r(CH2;n-TOA....;. j .__________,_.~-y q 1 ~4? 400 The preparation of Compounds 2 and 6 is described in Voss et al., Helv. Chim. Acta, 1.983, 66, 2294..
Preferably, m is 4 and n i.s 8 or 10.
The preparation of_ t.rans-anhydrc~mavalonic acid is described by H. nieckmann in °'~ie Isoli<~rung and Darstellung von traps-5-hydroxy-3-methyl-pentan-2-sure", ~rchiv fClr ~Ii.cr;rohic~l.::~<~ie, 62, 322-327 1_0 (1986) . The compounds ar~l.-r~~drotztz~~,~al.roroi.c~ acid lactone and cis-anhydromavalonic acid 1_.~c~tooe an:l the prepa-ration ther_eof_ were de~cri.l~ed t~~.~ ~~e.~:~r eL al. , in "i7ie 8ildung von ,/?-~Anhydronrr~~~v~tons~urel_actern lurch versc:hiedene Tai lie" , ~~3iocl:mm Z<a i r_schr i. fit, l5 341, 378-386 (19651.
The compound 6-hydrc:>xv-4,6-dimet:hyl-3--heyteiz-?-one may be prepared by methc~d~, r;:nou,~n f_-r.«m the art.
It is also a natural sr~h:;t:ance ~:~rld cao '~e isolated 20 from Ca:~iscum annunC var . angnl.cosum, as de scr ibed in C.A. 97: 159552f an.3 rivo to ~~hokaryo 1982, 35(2) 95-101.. This compound i ~ also described in dissertation "Neu~e ~ekund~rstofFe aus Strepto-mycesten: Isolierun<a and ~trukt°.iraufkl~rung der 25 Colabomycine, Pyrrol_ame and des ;~yridazornycins"
by R. Grote, ~lniver>ity of Ct~tt i.ngen, 1987, as well as its preparation loy the wulti.vation of_ Strepto-myces olivaceus (strain Tt1 3082r deposited at ~eutschen Sammlr~ng far mikroorganism on <'?3rd November 1987 30 in accordance with t:he Treaty o(- Budapest under No. 4309) and its i=~olation. It: has the following NMR spectrum:
C9H1602 (156.23) EI-MS: m/e - 138 (M -H?O, high resolution, C9H140, 6g); 123 (M~-CH5f7, 8~); 98 (high resolution, ~ 34~ 400 - 1.0 -C6H100, 48~) , 83 (100'0 1H-NMR (200 MHz, CDCl~l: ~ = 1..27 (s, 7-H.~ u.
8-H3) ; 1..43 (s, broad, f~A7, c.~xchanges with Me(~n) ; 2,21. {s, 1.-I-I~} ; ?..35 (d. J --- 1.3 Hz, 9-Fi.~) ; 2.3'..~ {s, broad, w-H?) ; 6.13 (s, broad, 3-r~) ppm 13C-NMR (50.3 MHz, CDC13): ~ = 22.0 (o, C-9); 30.0 (o, 2C, C-7 u. C-8); 32.0 (o, C-1); 54.4 (u, C-5) ; 71 .1 (u, r-6) , I_2 7.2 (o, C-3) ;
155.1 {u, ~'-4) ; 198.6 (~.2, c'-_?) pom Examples of thc~ salts ~>f_ the ~~i~i~.5 ~>mer_iFied include, in particular, salts of~ the al.'<ai i. 'petals, preferably the sodium and c,~otassium salts.
1_ 5 The method according t~~ them invent,'_~~n may he carried out in a serum-<yontaining mef~i.u~r~ or :~reFeT_ably in a serum-free medium.
If the method is carried out i.n a s!~rum-containing medium, it is preferab7_e to use, for exambl.e, thio-glycolic acid, nonactir. acid or Furan fatty acid or the salts thereof.
If the method is carried out in a serum--free medium, it is preferable to use a c'3~5 aliphatic monocarboxylic acid, more particularly butyric or s>ropioni.c acid, or the salts thereof.
It has been found that the compound which increases protein production exhibits effecti~reness at concen-trations o~ from 0.005 mcM to 500 mM, preferably from 10 ~~to 500 mM, e.g. from I_ mM to 10 mM.
(C3-5) aliphatic monocarboxylic aci~:3, thioglycolic acid, thiodiglycolic acid, L,-cystei~ne and glutathione or the salts thereof are particularly effective at 1 mM to 10 mM.

- 11. -The substances which increase protein production may be added on the corresponding day of cultivation, e.g. on day 0, i.e. at the start. of_ cultivation, up to the end of_ the third day of cultivation.
The inducer substances which increase protein production may also be used in several stages.
Thus, the cells may be cultivated in the presence of a first one or mare inducers, the cells are then removed from the culture, :laced in fr_es?~
cultivation mec~iurn and cul.ti.vat~=girl i.n t'ne presence of a second one car more i.n:~rlcer7 whi~-h increacns protein oraduction. In this mufti-stage process, C3-5 aliphatic rnonocarboxylic ac;i.ds or toe salts thereof, nrefer_ably bu~~vr:~ic ac.~.i~~ and t:hi.og~L°~colic acid or their salts, in parti.-:n).ar, show an enhancing effect.
With mufti-stage use. it is not necessary to use the same inducer for all stage ; rather, ape of.
the inducers may oe usea in one stare and another inducer in another stage. This mufti-stage process also shows an enhancing effect, sc.zch as, in particular, the use of_ aphidicolin in one stage and ape of the other inducers e.g. butyric acid, prapionic acid, butyryl choli.ne chloride or. ~~utyryl choline bromide or the salts of any of t:hesL, in another stage.
Combinations of two or more substan~~es may !~e used.
Preferably, not more than ape substance is used which inhibits r_el.l_ growth. Ta'or exarnble, a aliphatic monocarbaxylic acid, preferably butyric acid and thioglycol.ic acid or the salts thereof may be used, the manocarboxylic acid or the salt:
thereof preferably being added to the cell culture as the second substance.

~ 3~~~ goo - 7_2 -Transformed Chinese hamster ovary cells a.re CHO
cells which are transformed with a vector coding for a desired protein, these cells being capable of synthesising and expressing t:he protein under cultivation.
The transformed Chinese hamster ovary cells used may be, for example, the cells described in FP-A 93,619 and FP-A-199574 and in DF-A-3,708,681.
By cultivating theses trap:>formed Cr-IO r_ells it ins possible to prepare t-PA (FP-;'~ 03,619) and certain t-"A rnutants (EP-A 7 99, 0'74 anr~ roe-A. 3, 708, 581) .
The transformation c>f '~Ht) cells ~a.ith vectors whilst the cells express ot:her_ proteins is effected by metho<3s ~;nown from t:he literat~.zr~e, P.g. in the Furo:~ean arid ~;er_man patent ap~?7..i.c~.ations referred her_einhefor_e.
The host cell used rnay bc~ the cell known by the name CHO-K1_ which was isolated i.n 7957 from biopsy material by Puck and was included in 1970 in the' American Tvae Culture ~~o:llection (ATCC) under the name CCr_,-61. This cell Line war. deposited again 'oy the ATCC' on 23rd December 1987 in accordance with the Treaty of Hudapest under_ '~o. CRL 9618.
The DNA sequence coding for the desired protein may be r~repared by t:he method known from the liter-ature, e.g. in some of the above-mentioned patent applications. Vectors which contain this sequence and controLlin9 ~reqrlence5 as wel_1. may be constructed by methods known er- se, e.g. ~.~:~ing ~'. coli K 1:?294 (ATCC 37_446) and E. coli X 1776 (A~'CC 31537), which were re-der~osited by the ATCC on 23rd December 1987 in accordance with the Treaty of Budapest under Nos. 53704 and 53705; examples of_ such vectors include the plasmids pETPER anc3 pF~~PFR, which ar_e - 1.3 -described in EP-A 93,619. The CHO cells may be transformed by the methods known from EP-A 93,619, DE-A 3,708,681, FP-A 11_7,059 and EP--A 199,574 or by other methods known from the literature.
The vector for the t-P7~ production ~.aas integrated in a subclone, CHO-Kl-I7tlX B11 (Ilrlaub et al., Proceed-ings of the National Academy of ~cic~nce, USA, 77:
4216-4270, 1980) . '1''~i~~ ceLt line, a c~i.hydrof_olate reductase negati ve ~n~at~~nr~ (DHE'R-) , is sui table f_or a D'3FR-deperWc~nt: selection c-~f the transfected cell r. lone,. T~~m ~.~r:c:t~.~r F~~~r lvunv,~n 1.-DNA man he a known r~tasrni~~3 vt~c;t-:or_ n:~R-322 or the ~scherich.ia coli der ivative t:hE~reo~r 'The oromot~er region for the gene in que~~ t i~~n arld for the nHl~~~~ gene comes From ;;~T 47 and t'he t:.~-yrr~~i.nati.on ~:eyio~n for bot'.~
genes cc~me:~ '~rorn a hea,-~titi.s Fi marface antigen.
In the '~xamplF s, c'T-IO-T<'-I)r1'~ R1_1 c:el a_s are used which are t.ransCormed tw tie plasmiais pFTPER or ~EPEF~ (see rP-;~ 11.7,0 >9) .
various known products are available for use as the serum-free medium see f_or exam~~le BMFT-Status Seminar 1.2, held on l3t:h November 1985, Federal Ministry of Resear_,~b and 'Technology, 5300 Bonn 2, page 111-1201. Tn the foll.owi.ng Example, a DMEM/Ham's F 12 Medium (1.:1) !'Gibcot is used as the serum-free rne~~:ium. As the addit=ive, foetal calves' serum (~'c'S) is used in ;~ concentrat:ion of_ 7. 5° or_ 1-?.° .
The cell cultures are incubated at i7°C in a Iiereaus incubator and supplied with a mi.xtur_e of_ carbon dioxide and air containing 7. So C02 with a relative humidity of 1000. l7epe~nding on the batch, different culture vessels are used: spinner vessels for 100-1000 ml of medium, Roux dishes (Nuns) 175 cm2 (70-100 ml of medium), 75 cm2 X30-50 ml of medium), 25cm'~ (7-10 ml of medium) and multiple culture dishes made by Costar with 2 cm2 for 1 ml cultures. For maintaining ~,he strain, tlxe cells are used in a density of 0.2-0.3 x 10~ cellslml and subcloned every 2-3 days.
The substances which stimulate protein production may be added in a 1:100 dilution at the various stages, i.e. they are first dissolved in medium or, if they do not dissolve readily, they are first dissolved in ethyl alcohol and then added to the medium and then the medium containing this substance is added to the cell-containing medium in the ratio 1:100 vlv.
The invention is further illustrated in the following examples and the accompanying drawings. Of the drawings:
Figure 1 is a graph showing replication of transformed CHO cells;
Figures 2A and 3A are graphs showing extracellular concentration of t-PA during growth a transformed CH0 cells cultivated in medium containing 7,5% FCS;
Figures 2H arid 3B are graphs showing intracellular growth of t-PA during growth of transformed CHO cells cultivated in medium containing 7.5% FCS;
Figure 4 is a graph showing replication of transformed CHO cells in a serum-free medium;
Figure 5 is a graph showing t--P,A cancentrati.on in the supernatant from cultivating transformed CHO cells in a serum-free medium;
Figure 6 is a graph showing t-PA synthesis per cell in the supernatant from cultivating transformed CHO cells in a serum-~ 34~ 400 free medium;
Figure 7 shows the time curve for t-PA production increase in medium containing 7.5~ FCS brought about by means of C3-C5-monocarboxylic acids;
Figure 8 shows the time curve for increase in intracellular t-PA content in Na butyrate-treated cultures;
Figure 9A shows the extracellular concentration of t-PA
per cell, determined by '4CA, in Na butyrate-treated cultures;
Figure 98 shows the extrace:~.lular concentration of t-PA
per cell, determined by ELISA, in Na 1;>utyrate-treated cultures;
Figure 9C shGws the intracellular concentration of t-PA
per cell, determined by EbISA, in Na tmtyrate-treated cultures;
Figure 10 is a bar chart showing the effect of addition of Na-butyrate at different numbers of days after initiation of the culture;
Figure 11, which appears on the same page as Figure 8, is a graph showing percentage increase of t-PA over time in a serum-free culture with addition of Na-butyrate;
Figure 12 is a graph shr~winc~ concentration of t-PA over 24 time in a serum-free culture with ada~ t.xos~ of Na-~butyrate;
Figure 1:3 is a graph showing prcaduction of t-PA per cell in a serum-free culture with addition of Na-butyrate;
Figure 14 is a graph showing production of t-PA per cell in a culture containing 7.5% FCS and ascorbic acid;
Figure 15 is a graph showing percentage increase in t-PA
production over time with thioglycol.ic acid, thiodiglycolic acid, 'b-cysteine and glutathione;
14a ~ 3~~ 4oa Figure :16 is a bar chart showing the effect of addition of thioglycolic acid at different numbers of days after initiation of the culture;
Figure 17A is a graph showing extracellular production of t-PA per cell, determined by DCA in the presence of thioglycolic acid;
Figure 17B is a graph showing extracellular production of t-PA per cell, determined by ELISA in the presence of thioglycolic acid;
Figure 17C is a graph showing intracellular production of t-PA per cell,, determined by ELISA in the presence of thioglycolic acid;
Figure 18 shows the time curve for the increase in t-PA
synthesis in 1 mcM aphidicolin-synchronized CHO cells cultivated in serum-free medium;
Figure 19 is a bar chart showing the increase in t-PA
production in CHO cells cultivated in serum-free medium and in the presence of 6-hydraxy-4,6-dimethyl-3-heptem-2-one (DHO), determined by DCA; and Figure 20 is a bar chart showing the increase in t-PA
production in CHO cells cultivated in serum-free medium and in the presence of 6-hydroxy-4,6-dimethyl-3-hepten-2-one (DHO), determined by ELISA.
In the examples which follow, cells were taken from an exponentially growing culture i,n order to stimulate the synthesis of t-PA and centrifuged in plastic test tubes (Falcon) for 10 7.4b 13414n0 minutes at 1000 x g in a Beckmann* T ~-6 centrifuge. After the supernatant had been decanted the cell pellet was resuspended with fresh medium.
For the production of t-PA in serum-containing medium, F12/DMEM + 7.5% FCS + gentamycin was used as the fresh medium.
The resulting suspension may be prepared in 2 cm2 2~4-well dishes at a cell density of 0.2 to 0.3 x 106 cel.ls/ml.
For the production of t-PA in serum-free medium, the cells were first adapted fax' 3 to 4 d~~ys in a medium of 1 to 2%
foetal calves' serum, centrifuged again a.nd the cell pellet was resuspended in serum-free medium and then transferred into 2 cm2 24-well dishes in a ce:l l density caf 0. ~-(:1 . 5 x 106 cel.ls/ml .
*Trade Mark 14c i, -15- ~~41400 Quantitative t-PA determinations 1 ml Aliquots of_ cell supernatants and cell extracts as appropriate were taken or prepared at various times.
o increases given below and in the figures are compared with the control (04). The sam,ra7_es were measured either immediately or after storage at -20°C using the following methods:
METHOD OF I)ETFRMINA'I'IO'V
1 - Direct chramogenic a:~sa~:- (t>'A) p-Ni.troaniline is Eormee~ as the reaction yr-r~r3ue~t: from the synt'nei~ic peptic~le sr.i'.~,trate v-??.8t3 ('7-H-Ile--Pro-Arg-p-ni.troani:Line) cof: "~W _~~;~w~, t;,~hi ~ia~3nost:ica using t-PA. The ac t:ivity of t--''3,~, i s pr_aportional to the formation of p-nitrrraniline, m;~;=rsurerj at 405 mn~ 37°~'.
Spectroohotometric determination of the enzyme r;inetics was car_riec9 prat automatically with an ACP-5010 analyser made by Messrs. I'ppendorf.
The activity of the enzyme was t~al~,°ulated using this given standard. ~ t:-PA standarr series af_ 1-15 mcg/ml was prepared using a laboratory ,7tandard consisting of 72°s of 1.-chained material.
The substrate salution (100 mM tri:~s,/1_0& mid NaCl) was used in a concentration c~f 'p0 rroM.
Gr rcr Using an ELISA (Enzyme linked immuno-sarbent assay) the t-PA samples of the supernatant an~i the cell extract were quantitatively determined. The standard used was a labor-atory standard in a cancentration queries of 0.038-5 mcg/ml.
The samples were diluted beforehan<i to di.lutions of 1:10 to 1:1000.

Characterisation of the transformed CHO cells Cell growth The CHO cells are inoculated with 0.2-0.3 x 10&
cells/ml in culture dishes (in medium containing 7.5o FCS) and initially replicate very slowly.
Only after a dormant phase lasting '.i4 hours does logarithmic cell. ~3rowt'~ l-~egin. With a cell density of 1.3 + 0.04 x 1.!7~' ce'Ll_s,/ml., the first non-vital cells appear in the cul_t~ura on the fourth day;
their proportion i;~ tep°rns of the total number of cells is 1?~.2 _+ 1.3~~. The maximazm cell density is reached on the ~th day at. 1.f3 + 0.05 x 1.0~ cells/ml LS (rig. 1_) .
The number of living cf~lls is still 72 + 3~ of the total cell rnas > after 7 days.
The stationary pha se which i=oI_lows the exponential cell growth i s initiatk~d by various factors. As a result of a high population density and the using up of essential m.atrients, the cells no longer divide. Tree clurati.on (of the stationary phase varies considerably an~~l depends on components of the medium, such as serum. An acc~~mulation of acids and toxic metabolic products and changes in factors of the medium (aci.d pH, low 0.~ partial pressure, inadequate ventilation) also e~ausE~ t:he cells to die off_.
t-PA production in CFO r_ells There is a linear relationship between cell growth and t-PA synthesis for O_~i0 cells cultivated in medium containing 7.5~ FC'S.
The pattern of t-PA pre~duction i.n time shows two phases. In the first phase, the t-PA concentration in the supernatant increases constantly. In the second phase, enzyme synthesis c,~ames to a virtual standstill and the t-Pz~ c:oncentr-ati.an in the medium remains substantially canstant (F'ig. 2.~.. Values given relate to 1. ml of cell. suyernatant). The maximum t-PA concentratian on the 4th day is 11.1 + 0.7 mcg/ml in the Elisa. The t-PA crancentratians measured are somewhat higher in tk~e chrvamaqenic assay, presumably caused by ?-chained t-nA, b!..vt with toe F~;lisa rnet:hod the curves run parallel i.n ~at:!~~ methods of_- dete~tion up to the 6th day.
The intracellular t-PA concentrr~tic-an al:;o runs parallel to the cell. growth and i.s relati.vely law compared wi_t'n the total ~rmautit~:~ ofi t-P~ synt'ne=;ised.
On the 4th dap, the brc~~ar~rti.o~v ~:li~~re~W L:, 7'=>> of the total activity (Ficl. '~'.~3. ~7~-~7._nzPS :li~,~on r_elat:e to 1 ml of cell suspen~ic:>nl .
As can be seen Pram Fig. 3~, the t-PA synt'nesis increases sliqhtl.y i:p to the 4tku day, based on the m.zmber of cells; with the ~~;~f i.sa methad, a maximum of 8.1_ + 1.2 mcg t-PA/1. :s 1()6 ccvr:Ll.s i.s deterrn.ined.
Cellularly bound t-T~A wa~~ detc~rrni:ned only by the Elisa methad since these law t-PA cancentr_ations (less than 1 mcg) cannot be detcarmined by DO.A.
The intracellular t--PA content, based on the cell number, is somewhat higher dur.i.r~ck the logarithmic phase of cell grawtrr and i_s between p00 and 900 ng of t-PA,/1_ x 106 r_ell.s, and from i~he 4th <:3ay the t-PA value is about 450 ng,/7_ x I_~)F cells (Fig. 3B) .
Influence of serum and serum factors in the medium on cell growth and t:he t-PA procluct.ion in CHO cells The cells were prepared in quantities of 0.25 x 106 cells/ml and at different serum concentrations in the medium.

A serum concentration of below 'y~ in the medium also has a negative effect on cel_1 =division, but.
the productivity if affected only s:Lightly.
The influence of_ the serum concentration in the medium on cell growth and the t-PA production after 48 hours' incubation of t=he cc~l~.s is shown in Table 1.
The values are given in ,r~ercerrt c~f the maximum values (7.5o FCC in the medium). The maximum value f_or the cel_1 number is 0 * 56 + 0. (')3 ~~ 3 06 cells and , for the t-F~ c:~once~ntar at i<7n , . '7 + 0 * 5 rncg/ml .
The t-PA determinat.i.on4a ;aert~ v.arr ie.r o~.zt by means of DCA (average + ~~,; , ,:1 4y , TABT~E 1 o FCS ~lurnber of cells t-PA
40 61.5 + 2.8 r>9.2 + 3.9 20 79.4 + 3.8 81.2 + 2.4 10 863.6 + 5.3 90.3 + 6.7 7. 5 l_00. 0 + 6 * 4 1()0 . 0 + 7.

5 72.4 + 4.9 87.0 + 11.3 1 43.1 + 7_.5 79.~ + 6.7 0 37.5 + 1.8 75.4 + 4.6 Cell culture in a serum-free medium The cells were prepared in. serum free medium in a density of 0.4 to 0. '3 x 1()6 ce~l.ls/ml. The cells divide until the 4th day and the maximum number of living cells is alsco achieved on the 4th da_y at 1.0 + 0.2 x 106 cells~'ml (~'ic~. 4) .

- 7_ 9 -t-PA production in serum-free medium The t-PA concentration in the supernatant increases constantly until the 7th day and, by the Elisa method, reaches a maximum of 1'3.6 ~ 0.54 mcg/m1"
The t-PA values determined try f)CA are below the t-PA values determined by Eli.sa on the 1st to 3rd days and from the 4t:h clay onwarr'3s they are above these t-PA values (~'ig. 5) .
The t-PA synthesis per cell in serum 'ree me:ii~.im is approxi~natel_y t!nra same as i.n ~nec7ium containing serum and is between 7.2 + X3.6 ~_~n~.7 1Ø3 + 2.8 mcg/1 x 106 cells (Flisa) (I'i.g. 6) .
The percentage of t-PA in serunn ~r~3e medium iaased on the total quanti.t:y of x~rotei.n i.n the su~ernat:ant is between 7Ø9 + :1..0 anc7 1_?.5 -i- 1..4~. The res;.alts are shown :in Table f'. The t-PA va7.ues were determined by Elisa (<average + SF, n - 4).

Day - - Protein mcg,/lxl~)6 cell.:; t-PA mcg/lx).06 cells 1 7L.0 + 8,f~ '~".2 +
3.6 3 81.8 + 9.4 9.0 +
2.3 5 82.0 + L?.. ' ~~. 3 +
0.8 Effect of_ aliphatic monocarboxyl_ic acids The increase in t~-PA proc9uction in CHO cells in medium containing 7.5~ FCS as a result of different aliphatic monocarboxylic acids is indicated in _ ~~ _ Table 3. All the acids were used i.n a concentration range of from 7.0 mM to 500 mM. The optimum dosage range for_ all monocarboxy.lic acids is between 1 and 10 mM. The effectiveness of the acids depends on the chain length.
All effective monocarboxylic acids inhibit cell growth. Pronionic, butyric, isr~but.yric and isovaleric acids are ef_f_ective in a wider concentration range since the concent:.r_at:ion-depenr3ent inhibition of the arimary metabolism w1W eh re~~ults in inhibition of_ cell growth coincides with the increase in t-PA
synthesis.
The values given in Table= 3 r~=7..~ate to 1 ml of cell supernatant and are given in '~:o~~y:~ar-ed with th~~
control (0°). The t-P~ was det:carmined by DCA (average + SE, n _ 3-24) . The maxa..mnm v<-tlue relates to an individual sample. rata for the individual samples are shown iru hrac~kets.

Monocarbox~~l.ic acidOptimum ~30<.-,age'~ increase t-PA
in range synthesis Maximum Average Formic acid 100 mc~1 7.4 6.5 + 0.9 Propionic acid 5-1(7 (1s7 45.5 28.0 + 2.5 mr~t mM) Butyric acid 1-5 mM ( ~_ 68.3 41.9 + 3.8 mM) Isobutyric acid 0.6-10 (T_0 32.9 16.6 + 2.8 mM mM) Isovaleric acid t-i?() ( 5 mM) 39.3 18.7 + 2.4 mM

The average values relate to several production runs lasting 3 to 4 days, the monocarboxylic acids having been added on day 0 to clay ~~ of cultivation, as shown in Table 3A below. rata for the individual 134140~

samples are shown in brackets.
mTar~ '~T
Monocarboxylic acid Days of addition Sample days n Formic acid 10 {0) 2-3 (2) 4 Propionic acid 0-3 (2'? 1-4 {3) 24 Buty.r_ic 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 = number of production runs Fig. 7 shows the time curve for the t-PA production (determined by 17CA) increase i.n medium containing 7.5s FCS brought about by .means of ~~'3-C5 monocarboxylic acids. They concentration of butyric acid and isobutyric acid was 1mM and the concentration of proprionic acid and isovaleric acid was 'imM. After 24 hours it is already possible to detect a stimulation of t-PA production from i.8.1 -~- '':>.7~ (C4) to 24.8 +
0.5°~ (C5) . In the case o.f isot~utyric acid, there was no increase in production until 48 hours had elapsed. A maximum increase a.n t-PA production was achieved for the C3 and C4 carboxylic acid on the 3rd dayr with 3'7.2 + 5.1.r> for ~1a-butyrate.
The increase in oroductian is only short-lived for butyric: and isobut~~r_x.c acid and an the 4th day the effect can no longer he observed. The increasing effect of. propianic: acid and isovaleric acid, on the other hand, is sti~.~_ present an the 4th day an~9 manifests itself in an increase in t-PA production of 31.8 for C3 and 8.5~ for C5.
The ef..fects are also dependent ran the physiological state of the cells. 'the cells are most sensitive 13414pp to stimulation during exponentia:L growth.
Table 4 shows the increase in t~--PA production in CHO cells in medium coat<~ining ;~.5~ FCS by means of C3-C5 monocarboxylic acids depending on the day of appLi.cation c>f the caroo~:ylic acids are 48 hours' <growth of the cells i.n medium containing 7.5o FCS. The concentration was 7. m'~ f_or butyric and isovaler is acid and ~i mM for.- propionic and 1.0 isovaler_ is acid. The val~.aes r_~~t-E~r to 1 ml of_ cell supernat;ant_ ~~nd are given in ~ cvom;:aared with the' control f0'~l (average +. A.~t~, 3 ._ 3_~;) , mtle t-PA
was deter_rnir~e~~ by rnf~ans cW ~)~,~.

Day of appl i.-canon C3 ~'4 C4-I C5 0 28.7 -~- 9.3 33.0 + 5.4 20.1. + 10.7 17.2 + 1.0 1 ?.4..4 + 4. 1 40.2 + 7_2.2 2 5.3 + 5.1 19.4 + 1. 5 Effects of hut~ric acid on t-PA .production in CFiO
cells The stimulating effect can '~e mc~nit:ored by an increase both in the intrace7..lular~ t-P:'~ cvr>ncentr_ation anc3 also in the extracel.7.ular t-PA t-:onrentr_ation.
As is clear_ from Fic~. 8, the inc:r_ease in intracellular t-PA content i.n Na-Y~utyrate-tr.e~~ted .ctaltures is greatest aFter 24 hours inc~ibat: i.c~n compared with the control (4'3.8 + 9.8~;) . Tn the <=>~.rpernatant, the percentage increase is somewhat: less after 24 ho~zrs, on average 19.2 (DCA and Flisa) compared with the control values. This effect last s for °,~:2 hours in the 134140p supernatant. The Na-butyrate was added to a medium containing 7.5 ~ FCC: at a c~ancentrat.ion of lmM.
Fig. 9A-C shows the synthesis of t-PA based on the cell number and show:a that the stimulation of t-PA synthesis by Na-lutyrate lasts for more than 7 days in spite of the inhi.t~itian of_ cell growth. As above, the medium contained 7.5o FCS
and 1mM Na-butyrate. ~7ur:i.n~ t~?ue First ~ d<~ys, enzyme synthesis per cell incre<~ses ~onstantly.
The t-PA cancentrat ion i r~c~rna~e<From 7. 5 -+- 1, 1. rncg to 28.9 + 5..3 mcg t-PA./7_ x 1 0~' c~ell.s (Tlisa ~ in the supernatant (rid. t3A) . ~Jvi.r~:-~ t;~e ~!',~1 rnethod, a maximum t-PA content of~:~ 4''.~l '- 7.4 mcq t-P:~; 1_ x 10~' cells is deterrninec3 (Fig. 9~'~) . ~t t?oe same time the intracell~.ilar t-PA cc:ant~n?: i n,~re<~sc~, and Fa:l.ls from 1_. 5 + 0.2 mcg of t~-1?A,/1. v 1 ~5 cel3.s on the 5th day (Fi.g. 9C) .
Table 5 shows the stim~~lat:ian c~fv t-!?A synthesis by Na-butyrate in medium containing 7.5°s FCS based on the cell_ number. Na-but~Jrate was added on day 0 in a conr_entr,ation of 1. mM. The values on days 1-4 are given in o compared wi_t:.t°a. the control (0~) and relate to 1 x 106 cells. Tt~e t-1?A content in the supernatant and in the cel_1 extra~:.t was determined by rlisa (average ~- '"1, n - 5-1!1) .

Day Intracellular Fxtracellular 1 90.9 + 9.5 68.5 + 12.~

2 99.7 + 25.'3 118.8 -~- 16.1 3 93.9 ~~ 3L.~ 223.4 + 77.4 4 209. 8 + 41_. ~ 2.80. 7 + 85. 9 An optimum increase in production was achieved when Na-butyrate was added to the cells during exponential. growth. The maximum effect of Na-butyrate always occurs on the 3rd day when there is an appli-cation interval from day 0 to 2 (rig. 10).
By applying Na-butyrate every 2-3 days after changing the medium in the same cell pcapu.~.ation, it was possible to rnainta:in t~~e t-i?A i~codu-:tion at a higher level for a fairly lon<~ time.
Table 6 shows thf~ :i.ncrc~ase in t---T~.~1 production in CBO cells which were culti.vate<9 in medium with 7.5$ FCC. 1.. m~~I of Na-b~.~tyrG3tr.~ ~:~.~r=~s ~.r3ded each time the medium was changed or ~~nZ~ ~n dr~~.~ 0. The values of 2, 4, 7 or 9 days, respectively, after t'ne medium had been changed are ga.ven in 'c~om~aared with the control (0~) anc~ r.elat~~ to 1 rn1 c~f_ .ell supernatant.
The t-PA determination was effected by means of DCA (average + ~E, n = 3).

Change of medium Application Day Day ~'a , '~ , 4 . 7 2 33.0 + 5.4 4 1. 0 ? . 4 ~ 7 . k~
7 96.6 + 5.8 9 6 2 . 0 ~+- 4 . 8 Effects of butyric acid on the t-PA production in CHO cells in a serum free medium By contrast with the short-term effect of. butyrate-stimulated t-PA synthesis in medium containing serum, the effect in serum-free medium lasts longer and, 1 day after the application of Na-butyrate (at a concentration of 1mM), it is 44.1 + 7.1~
compared with the control. The maximum increase was achieved 3 days after the application of Na~-butyrate, at 143.3 -+- 32.8. The increasing effect can still be detected 6 days after the addition of Na-butyrate, at 49.9 + 7.0~ (F'ic,~s. l1 and 12) .
1_ 0 The t-PA synthesis, based on 1 ;c 1. f)6 cells, is also higher in serum-free medium than in serum-containing medil.am aTld rises to ~nos~e p0 mcg,/1 x 106 cells (Fig. 13). Na-butyrate was added to give 1.5 a concentration of ?.mM after 24 homers cultivation in serum-free medium.
rff-_ects of propionic: acid 20 C3-carboxylic acid exhibits very similar effects on t-PA synthesis to those produced by butyric acid. However, the percentage ~.ncrease is about 14~ less and higher concentrations (5-10 mM) are needed in order to stimulate t-~?~. Furthermore, 25 propionic acid inhibits cell. grtawth to a lesser extent and therefore leads to a langer-lasting increase in the t-PA synthesis, Stimulation of t-PA synthesis by propionic acid 30 appears to be very speciFic, in contrast to butyric acid, since in serum-free medi~z~rl the extracellular protein content was increased only slightly with the contro:L and the incorporaticvn of C3H]-leucine was increased on7_y slightly.

Effects of dicarboxylic acids L hydro carboxylic acids and ketocarboxylic acids on t-PA production in CHO cells Hydroxycarboxylic acids such as lactic, glyceric, malic and tartaric aci~~ show only a slight increase in t-PA synthesis of between 6 and 10$, whilst tartaric acid in a conrver~trat:i_ar~ of 1.0 mM stimulated the t-PA synthesis best (1Ø3 -~- 2.9o compared with the control) . Th.e ~°el.l c~i.vi.sio~~ i;~ not affected by hydroxycarboxylir_ a~~i.c~s and the eFfect on t-PA
synthesis can still be c~etec;tea aFter 96 hours.
r,f fects of ascor3aic ac id The effect of. ascor'~ic acid s i.n a concentration of 10 mM on t-PA or_oduction lasts f_d.or 72 hours and when ar>plied on da~:~ 0 it re>~lts in a 15.2 +. 3.1°
increase in t-PA synthesis after ?~ hours (Fig. 14).
The medium contained 7.57 F~~'S.
Effects of long-chaine<i fatty acids on t-PA production in CHI cell.s It was found that 1_ong~-~chaineo!1 fatty acids such as nonactic acid with 1.0 carbon atoms and furan fatty acids were at~le ~~o ca~xs<~ ~~n increase (Table 7) .
In this examp7_er a Fur<3n fatty acid according to formula 4 was used wherein m = 4 and n = 8.
However, in serum-free medium there was no evidence of an increase in t-PA synthesis; or protein synthesis.
The increase in t-PA production in CHO cells caused by long-chained fatty acids is shown in Table 8.
The cells were cultivated in medium containing 7.5~ FCS. The acids wer.er tested in a concentration range from 10 mcM to 10 mM, a concentration of.

~ 34~ 400 1 mM being optimum. The val..ues relate to :L ml of cell supernatant and are gi.v~~>n as a percentage compred with the control (0'~) average + Sr, n ~ 4-9).
The maximum value refers to an ~.ndividual sample.
The t-PA was determined toy r~CA, Fatty acir~ -_.~._._._.__ _._ _~_-~n ,~~,;~as,e inlt-PA._svnthesis Ma:~.inusv Average Nonactic acid 53. 3 - .. _._______._'30.3 _ ~ _~. 5 .__ _ Furan fatty aci<~ 32.1 '_9.r) + 3.2 The average values relate to sea~~era1 production runs of 4 days, the acids bFaing added an day 0, as shown in Taole 8A. Oata Fcar the individual samples are shaven in brackets.

Fatty acids -_~~Y_~~__additian.___~.nay_af samplp.__ n____ Nonacti.c acid 0 t0~ -_--.__i!~__~4~ _______ Furan fatty acid 0 (0" L_4 ~2) 9 _ _ _ ~.___ _~.______ _ ______________~______ Effects of thiols and~hides,on t-_PA,production in CHO cells For sulphur-containing campounds which are derivatives or substitution products of carL:~axylic acids showed an increase in t-PA prcaduction c.~F between 16~ and 31.2, without having a negative effect on cell -28- ~~41400 growth (Table 8). 'fhe substituted carboxylic acids show their optimum activity in the same concentration range (1-10 mM) as t:he other active carboxylic acids. Only glutathione was ,~ffect:ive in a lower concentration range of 1--1_0 mcP,~. 'fhioglycolic acid proved to be tree c~referred stimulator of t-~PA
synthesis in CHO cells.
In this Example, the cel:Ls were cultivated in medium containing 7.5°s F'CS. All. the :=>~..rbstances were tested in a concentration range of from 1 mcM to 10 mM.
The val~zes given in Table 9 rel.at~e to 1 ml of- cell supernatant and are given i.n > c°ompar_ed faith they control (0'-s) (avera<le val~.ze + c~ta;, n -- 6-19) .
The maxirnurn value relates to an i ndividual aample.
The t-PA was determined t->y nCA. The conr_entrati.on of the sub~~tance is shown i.n br~~c:kets.

Thiols and Optimum dc~sarle s increase in t:-PA
sulphides range synthesis ?. 5 Max imum Average Tr110g1yC011C
acid 1 mM 56.4 31.?. + 3.2 Th iod iglycol. is acid 1. mM 31.0 18.0 + 3.8 L-cysteine 1-1.0 mM ( 1mM) 41.4 20.9 + 3.4 Glutathione 1-1.0 uM (lOuM) 30.5 16.0 + 2.0 The average values refer to several production runs of 3 days, the substances flaying been added from day 0 to day 2 as shown in Table 9A. Data for the individual samples are shown in brackets.

Thiols and Day of addition Day of sarnple n sulphides Thioglycolic acid ()-2 (1) 1-3 (2) 19 Thiodiglycolic acid l ( l_1 2-3 (3) 9 L-cysteine C)-1 (i)) 1-3 (2) 11 Clutathione 0 (~)~ 2-3 (2) 6 One advantage of thi.ocarboxylic acids over rnonocarboxylic acids is the absence of ,.~n~r i.nhibitory effect on cell proliferation, but the pror-iuction of_ t-PA
co~~ld not be increa4sed fcartner with these substances than with butyric acid. The r~~.~r-ation of_ the increasing effect is also short: and attain=~ its maximum value after 24 hours fc3r t:hioglycnlic acid and after 48 hours for cystei.ne. after 4 days incubation of_ the cells the effect c7n t-PA production is still small (Fig. 15) .
However, in serum-free medium, the stimulating effect on t-PA synthesis is less than in medium containing serum since all the r~~ubstances inhibit cell division betweE>n l_0'~ and ?i)2~»
Effects of thioglycolic acid t-PA production i.s i.ncreased fcar 7? hours after treatment with thioglyco:tic ac~.d and is independent of the day of ar~plication. The percentage increase in the enzyme synthesis is at it4~ maximum after 24 hours and then falls canti.nuc~usl.y»

~ ~~~ goo Table 9 shows the increase in t-PA production in CHO cells cultivated for 24 to '~6 hours in medium containing 7.5$ FCS and 1 mM thiogLycolic acid, depending on the day of application. The~values relate to 1 ml of cell suriernat<~nt and are given in ~ compared with the control (0$) (average +
SE, n = 4-6) . The t:.-PA was determined by DCA.

Tine (hl t)ay ,oF apb~Lication 1. 2 -__._.____.____.____._.___._.___._ _.____ __._____.____.__._____ ..__.._...__._._ 24 27.6 + 5.fs 34.7 -t- 10.9 34.5 -~ 7.5 48 16.9 + ?.~~ 1.7.1. E.. 3r6 16.3 + 8.5 72 14.7 + 1..0 10.2 + 2.7 9.4 + 1.7 96 3.4 + 1.~1 ?_.7 -~- 0.8 0.5 +1.0 t-PA production may be in~:reasecl by the application of thioglycolic arid (1mM) twice rather than once (Fig. 16).
In Fig. 17A (DCA) and Fig. 17B (Eli.sa) the curve of t-PA synthesis is shown oasec~ on 1 x 106 cells.
The t-PA synthesis is inc:r.ease~3 compared with the control by means of 1 mM th.ioglycolic acid for the f first 3 days (DC°.A) . T3y con~_rast, the t-PA
values determined by Elisa are increased for the entire period. The rate of incs~ease is still about 35g on the 4th day. The intracellular t-PA content per cell is also increased by means of thioglycolic acid. The maximum intracellu.lax~ t-PA concentration is 1.1 + 0.7. mcg,/1 x 10~ cells after 24 hours and falls within 96 hours to approxirnately half the original concentration (1a'ig. 17C') .

~ 3~~ 400 _ ~1 _ The extracellular t-PA concentration is increased by 10.7 compared with the control on the 5th day.
The percentage increase in t-PA synthesis by means of thioglycolic acid i.s somewhat :Lower in serum~-free medium since cells division is slightly inhibited (10-20~).
Effects of derivatives of monocarboxylic acids on t-PA production in C~iC cell's V7ithin the group of C4 carboxya.ic acid derivatives, other substances were found which have an effect on t-PA synthesis irz CT~O cPlLs. These substances include, primarily, butyryl choline bromide (RCl3) and chloride (r-_3CC.') which inc~reat~P t--P~ production by between 30 anti 40~. These ~'4 derivatives show the same effects as Na-butyrate in every respect and are effective in the sane concentrations.
Table 10 shows the increase i.n tv~-P~ production in CHO cells achieved by means of the two above-mentioned ;~eriva~:ives of_ monocarboxylic acids.
The cells were c~zlti.vated in a medium containing 7.5% ECS. All the ~~ubstances ~rere tested at concen-trations ranging from 1.0 mcM to l0 mM. The values are given in ~ compared with the control (0$) and relate to 1 ml of cell supernak:ant (average ~ SE, n = 4-15). The values which relate to the individual samples are given in brackets. The maximum value refers to an individual sample. The t-PA was determined by DCA.

~ 341 40Q
32 _ Derivatives Optimum dosage ~ increase in t-PA
of mono- range synthesis carboxylic Maacimum Average n acid BCC 5-10 mM ( l0 mM) 6'x.9 38.8 + 4.4 15 BC13 5--10 m1~ (2. 5 mM) 4ia.6 29.1 + 4.4 9 The average values relate to several production runs of 4 days a s shown in Tabl.fa lf9A. '~lalues for the individual samples are shown in brackets.

Substance Day of_ addition Sample days n BCC 1 (1.) 2-4 (4) 15 BCB 1 (1) 2--4 (4) 9 Combined effects of different t-PA synthesis stimulators When combinations of substances are used at least one substance shoulc9 have the property of not inhibiting cell growth.
An increased yield was achieved, For example, with a combination of thioglycolic acid and butyric acid. The application interval of_ the individual substances rnay play an important role, with butyric acid, for example, always being added as the second substance in order to reduce the inhibitory effect on cell growth.
When the substances are administered simultaneously on day 0 the effect of. the t~utyric acid is not increased.
The effect is cumulative for a combination of butyric and thioglycalic acid, A maxi.m~:~m increase of 70~
may be achieved, In serum-free medium the effects of butyric acid could not be increarted any fucvt~~er b~.~ the addition of another substance.
The increase in t-rA proc~uctinn in -H0 cells which are cultivated far 3 d3vs in rne~3iurn containing 7.5o I'CS i~~ shown i..n Tak~le 1.1. 1 mM of_ thioglycolic acid (TG) and 1 m~i of o utyric acid were added to the cells in combination at various times. The values relate to 1 ml of cell s~.ac~ernatant and are given as a percentage comparer~ with the control (0~) (average + SE, n - 4-8). The t-PA was determined by DCA.

Day of Application TG Butyric acid 0 ~ 2 0 31.3 + 1.7 57.9 + 5.2 X0.3 + 3.2 1 68.3 + 4.4 31.4 + 5.5 2 41.4 + 5.5 - 34 _ ~ X41 400 Effects of aphidicolin Aphidicolin, a diterpene obtained from Ce halos~orium aphidicola, specifir_ally inhibits IaNA alpha-polymerase and stimulates t-PA synthesis in CF-~O cells in a concentration of from 1_ to 10 mcM. After 24 hours the t-PA yield has been increascy:~ r:~y 44.9 + 13.~~~.
Aphidicolin was found to stimulate t-PA synthesis in CHO cells. It may inhibit nNA synthesis in reversible manner f~~.~t the R'~~A <;~~~id protein synthesis is unaFfected. Aphir3icolin is thus suitable on the one hand For stim~zlata.n:~ t-°'~,~ syntheais and on the other hand for sync:hroni. ~sing the cetls.
The e.f.fect of aphidi.coli.n on t-RA synthe.is when the cells are inr...u'~ate-~ For a frairly long time in medium c:ontaininc~ ant~idicalir~ is descrioed herein-before. r~'his passage re7_ates t_:o studies with CHO cells which have been exposed to aphidicolin for 24 hours and sut7se~:~uentl.y cta:Ltivated furt'ner in the medium Taithout aphidicolin. The phases of the cell cycle and the incor,rvoration of_ ~3H~-.
thymidine during 24 hour labell_inQ and thereafter were analysed in order tc~ mon:itc>r the inhibition of DNA synthesis and resumption of synthesis after removal of_ the abhir~ic~~li.n. ~'urtherrnore, the RNA, protein and t-PA synthesis were determined.
Cell growth The cell growth was inhibited slightly (S-10~) by aphidicolin (1 mcM) after 24 hours' treatment.
However, when the rnedit~m was changed, the aphidicolin-treated cultures were returned to the same cell number as the control ~,ul.tures.

- 35 _ 1 341 400 t-PA ~nthesis The t-PA synthesis was stimulated in 1 mcM aphidicolin-synchronised cells. An ef_f_ect roul.d still be seen 96 hours after the r_hanging of the medium.
The increase in t-PA production in 1 mcM aphidicolin-treated CHO cells cultivated in medium containing 7.5o FCS is shown in Tab'1e L.2. ~fhe values relat;e to 1 ml of_ cell supernatant and are given as a percentage compared with 2;he control. t~~? (average + SF, n - 4) . The t:-PA was det;s~rrmi ned ,.zsing nCA.
'I'ABLF 7_ 2 Time (h after changing ~ incr~e~ase in t-P,~ synthesis of medium) 24 40.2 + 6.7 48 59.9 + 11.1 72 32.8 + 6.4 96 14.7 + 1.3 Fig. 18 shows the time curve foa- the increa se in.
t-PA synthesis in lmcM aphidicholin-synchronised CHO cells cultivated io serum--Free medium. The effect reaches its maximum 48 haurs after the changing of the medium.
The t-PA concentration in serum-free medium was determined after 96, 1:?0 and 144 h using Elisa, whilst the values obta:~ned compared with the control values were substantially higher than the values determined by DCA. The results are shown in Table 13.
The test was carried orat with 1 mcM aphidicolin-treated CHO cells which were c~zltivated in serum-free medium.

~3414~~

The values relate to 1 ml of cell supernatant and are given as a percentage compared with the control (0o) (average + SE, n -- ~) . ~!'h~~ t-PA was determined by means of- F;lisa.

Time (h after c'nanc~ing ~ ~.nr,:rease in t-PA synthesis of medium) _9 6 ___ _ _ _. ._ _ _ _ __._ _ __ _ _ _ __.._..___..______.~; .~ ___ ___._._~
_._ - .7 +- .2 12 0 ~1 2 . 0 -+- 8 . 2 14 4 ~~ 1_ . ~' -~- t f) . 2 An increased intrac~ell~.xlar t-PA concentration could only be observed dGZr in~~ 24 ho~zr i.nc~.zhation with aphidicolin and was inr~r.eased bte ab~:~ut 300.
Butyric acid, proaionic acid, but:yryl r_holine chloride and bromide were able to increase t-PA production still further in this system. stimulation was successful both in ser~..zm-free an<~ serum-rich medium, whilst an additional increasing effect in serum-free medium was only ac..hieved when the substances were applied after 24 hours adaptation of. the cells to this condition of- the medic.zm.
Table 14 shows the pert-entage increase in t-PA
production in anhi~3ico:Lin-treated c~.zltures after the application oF_ ~Ia-butyrate. Na-butyrate increases t-PA synthesis by a further 14g a.f_ter 96 hours.
The test was carried oEzt with 1 mcM arzhidicolin-treated CHC:~ cells, which were culti~~ated with and without Na-butyrate, in serum-free culture. The 1 34~ 400 values relate to 1 ml of cell supernatant and are given as a o compared with the control (0~) (average + SE, n = 4). The f.-PA was determined by means of Elisa.
mrtzr t~ 1 A
Time (h after Aphidicolin + Na-butyrate changing of Time of application (h) medium) 96 36 . 7 + fa . 2 34 . 1. + 9 . 7 50 . F~ + 11. 0 120 42.0 + 8.$ 3I.8 + 10..'? 49.T + 4.3 Effects of 6-hydroxy-4,6-dimethyl-3-heoten-2-one (DHO) The t-PA production is increased after treatment with above compound after 144 hours. For stimulating t-PA synthesis, concentrations i.n micromolar range were effective, e.g. 0.005 to 1.00 mcM.
Fig. 7.9 shows the increase in t-LEA production in CHO cells which were cultivated up to 168 hours in serum-free medium and 7 mM tr,m 7 mcM nli0. The values were determined by f~CA. Fig. 20 shows the corresponding values ootained by Elisa.
Cell cultures prepared by the method described above may be worked over in known manner to isolate the t-PA, for example after the production phase the culture medium is ~~eparated from the cell mass and the cell supernatant is purified by ultra.fil.tration and chromatogra;ohic procedures.

The isolated t-PA can then be f<:>rmulated into pharma-ceutical preparations, e.g. in dissolved or lyophilised Corm.
The above Examples may be repeated, replacing the t-PA producing CHO cells by transformed CHO cells which produce other proteins e.<1, t-PA mutants or other_ pharmacologically effective proteins, as in the above-mentioned E~~rop~~an and German patent applir_ations, particularly the ~'HO cells in EP-A
199,574 and 196,920 and in 1~E-A 3,708,681.
It should be noted that, although the invention has been illustrated above with reference to a variety of inducers, the compr».ands f-usaric acids mevalonic acid, trans-anhydromevalonic acid, anhydro-mevalonic acid lactone and cis-anhydro-mevalonic acid lactone can ail. be used in like manner as inducers.

Claims (6)

1. A method of increasing the production of a r-tPA
or a mutant thereof from cultures of transformed CHO cells, wherein a C3-C4 aliphatic monocarboxylic acid or a salt thereof is added to the culture, and cultivation is carried out in serum-free medium.

2. The method according to claim 1, wherein the C3-C4 aliphatic monocarboxylic acid or a salt thereof is added in a concentration of from 1mM to 10mM.

3. The method according to claim 1 or 2, wherein the cells are cultivated in the presence of a substance selected from thioglycolic acid, thodiglycolic acid, L-cysteine, gluthathione, butyryl choline bromide, butyryl choline chloride, nonactic acid, furan fatty acid, ascorbic acid, aphidicolin, 6-hydroxy-4,6-dimethyl-3-hepten-2-one, D-.alpha.-hydroxy-substituted (C3 or C4) alphatic mono- or dicarboxylic acid and salts thereof, the cells are removed form the culture and then placed in new serum-free culture medium and cultivated in the presence of the C3-C4 aliphatic monocarboxylic acid or salt thereof.

4. The method according to claim 3, wherein the substance that the cells are cultivated in the presence of is aphidicolin or a salt thereof and the cells are then placed in new serum-free culture medium wherein the C3-C4 aliphatic monocarboxylic acid or salt thereof is selected from butyric acid, propionic acid, and salts of butyric acid and propionic acid.

5. The method according to claim 3, wherein the substance that the cells are cultivated in the presence of is thioglycolic acid or a salt thereof and the cells are then placed in new serum-free medium wherein the C3-C4 monocarboxylic acid or salt thereof is butyric acid or a salt of butyric acid.

6. ~The method of any ore of claims 1 to 4, wherein the C3-C4 aliphatic monocarboxylic acid or salt thereof is butyric acid or a salt of butyric acid.