DK173693B1 - Preparing purified, biologically active, bacterially produced human recombinant CSF-1 - Google Patents

Description

i DK 173693 B1in DK 173693 B1

Fremstilling af renset, biologisk aktivt, bakterielt fremstillet human-rekombinant-CSF-1.Preparation of purified, biologically active, bacterially prepared human recombinant CSF-1.

Teknisk område 5Technical area 5

Opfindelsen angår fremgangsmåder til rensning og genfoldning af bakterielt fremstillede, rekombinerede proteiner til former, som har en høj specifik biologisk aktivitet. Opfindelsen angår nærmere bestemt fremgangsmåder, som mu-10 liggør fremstillingen af biologisk aktive, dimere former af CSF-1 fra bakterieværter, som udtrykker gener, der koder for monomeren.The invention relates to methods for purifying and refolding bacterially produced, recombined proteins into forms having a high specific biological activity. More particularly, the invention relates to methods which enable the production of biologically active, dimeric forms of CSF-1 from bacterial hosts expressing genes encoding the monomer.

Opfindelsens baggrund 15Background of the Invention 15

Kolonistimulerende faktor-1 (CSF-1) er et af adskillige proteiner, som er i stand til at stimulere kolonidannelsen af benmarvsceller, der er udpladet i halvfast dyrkningsmedium. CSF-1 skelnes fra andre kolonistimulerende 20 faktorer ved dens evne til at stimulere disse celler til i det væsentlige at blive makrofagkolonier. Andre CSF-l'er stimulerer frembringelsen af kolonier, som består af neutrofile granulocytter og makrofager, hovedsagelig neu-trofile granulocytter eller neutrofile og eosinofile gra-25 nulocytter og makrofager. En oversigt over disse CSF'er er blevet publiceret af D., T.M., Nature (1984) 309:746 og af Vadas, M.A., Immunol. (1983) 130:793. For tiden er der ingen in vivo rutineanalyse, som vides at være specifik for CSF-l-aktivitet.Colony Stimulating Factor-1 (CSF-1) is one of several proteins capable of stimulating colony formation of bone marrow cells plated in semi-solid culture medium. CSF-1 is distinguished from other colony-stimulating factors by its ability to stimulate these cells to essentially become macrophage colonies. Other CSF-1s stimulate the production of colonies consisting of neutrophilic granulocytes and macrophages, mainly neutrophilic granulocytes or neutrophilic and eosinophilic granulocytes and macrophages. An overview of these CSFs has been published by D., T.M., Nature (1984) 309: 746 and by Vadas, M.A., Immunol. (1983) 130: 793. Currently, there is no in vivo routine assay known to be specific for CSF-1 activity.

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De særlige kendetegn for nativ human-CSF-1 er. komplekse, og det er faktisk ikke klart, hvilken form af CSF-1, som er aktiv i menneskets legeme. Opløselige former af naturligt frembragte CSF-1 er blevet oprenset i forskellig ud-35 strækning fra human-urin, muse-L-celler, dyrkede human-pancreascarcinomaceller (MIA PaCa-celler) og også fra - .............j---------- DK 173693 B1 2 forskellige human- og muselungecellekonditionerede medier, fra human-T-lymfoblastceller og fra human-placentakonditioneret medium. Mange, hvis ikke alle, éif de isolerede native CSF-l-proteiner synes at være glyco-5 sylerede dimerer uanset kilden. Der er en betragtelig va-rietet i de molekylevægte, som udvises af de monomere komponenter af CSF-1, hvilket tilsyneladende skyldes variationer i C-terminal processering og/eller glycosylo-ringsgraden. For eksempel viser Western analyse, at CSF-10 1, som secerneres af MIA PaCa-cellelinien, indeholder mo nomerer med en molekylevægt på ca. 26 og 30 kd, såvel som former med molekylevægte på 40, 48 og 70 kd. Der er blevet rapporteret andre CSF-l-molekylevægte. For eksempel har den monomere, reducerede form af CSF-1, som er isole-15 ret fra human-urin, en forholdsvis lav molekylevægt på :>5 kd, når den er isoleret, og 14-17 kd, når den i udstrakt grad er deglycosyleret in vitro (Das, S. og Stanley, E.R., J.Biol.Chem. (1982) 257:13679).The special characteristics of native human CSF-1 are. complex, and it is actually not clear which form of CSF-1 is active in the human body. Soluble forms of naturally produced CSF-1 have been purified to various extents from human urine, mouse L cells, cultured human pancreatic carcinoma cells (MIA PaCa cells) and also from - ........ ..... j ---------- DK 173693 B1 2 different human and mouse lung cell conditioned media, from human T lymphoblast cells and from human placental conditioned medium. Many, if not all, of the isolated native CSF-1 proteins appear to be glycosylated dimers regardless of the source. There is a considerable variation in the molecular weights exhibited by the monomeric components of CSF-1, which is apparently due to variations in C-terminal processing and / or glycosylation degree. For example, Western analysis shows that CSF-10 1, secreted by the MIA PaCa cell line, contains monomers having a molecular weight of approx. 26 and 30 kd, as well as shapes with molecular weights of 40, 48 and 70 kd. Other CSF-1 molecular weights have been reported. For example, the monomeric reduced form of CSF-1, isolated from human urine, has a relatively low molecular weight of:> 5 kd when isolated and 14-17 kd when extensively is deglycosylated in vitro (Das, S. and Stanley, ER, J. Biol. Chem. (1982) 257: 13679).

20 Ekstistensen af "nativlignende" CSF-l-referenceproteinar er vigtig, fordi disse proteiner tilvejebringer standarder, med hvilke kvaliteten og den biologiske aktivitet af genfoldede, rekombinerede former af CSF-1 kan sammenlig nes. Hertil har vi anvendt den opløselige CSF-1, som 25 frembringes af MIA PaCa-cellelinien, såvel som egenskabar af andre højt rensede CSF-l-molekyler, som er blevet beskrevet i litteraturen. Den specifikke aktivitet af disse rensede, "nativlignende" referenceproteiner er typisk faldet i området fra 4 til 10 x 107 enheder pr. mg (s?m 30 målt ved in vitro musebenmarvskolonidannende analyser).The existence of "native-like" CSF-1 reference proteins is important because these proteins provide standards by which the quality and biological activity of refolded, recombined forms of CSF-1 can be compared. For this, we have used the soluble CSF-1 produced by the MIA PaCa cell line, as well as properties of other highly purified CSF-1 molecules, which have been described in the literature. The specific activity of these purified "native-like" reference proteins is typically decreased in the range of 4 to 10 x 10 mg (as measured by in vitro mouse bone marrow colony-forming assays).

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Der er også blevet fremstillet CSF-1 ud fra rekombinait DNA under anvendelse af to tilsyneladende beslægtede cDNA-kloner: (1) en "kort" form, som koder for en medde- 35 lelse, der, når den translateres, frembringer et monomert protein med 224 aminosyrer, hvor der foran er en 32- DK 173693 B1 3 aminosyre-lang signalsekvens (Kawasaki, E.S. et al., Science (1985) 230:292-296 og PCT W086/04607, som begge er medtaget her ved denne henvisning), og (2) en "lang" form, der koder for et monomert protein med 522 aminosy-5 rer, som også har en foranstillet 32-aminosyre-lang signalsekvens . Den lange form er blevet klonet og udtrykt af to grupper, hvilket er omtalt i Ladner, M.B. et al., The EMBO. J. (1987) 6(9):2693-2698 og Wong, G. et al., Sci ence (1987) 235:1504-1509, som begge er medtaget her ved 10 denne henvisning. (DNA- og aminosyresekvenserne for både den "korte" og den "lange" form er vist i figurerne 5 ' henholdsvis 6; den 32-aminosyre-lange signalsekvens er imidlertid ufuldstændig, hvilket er illustreret i figur 6) .CSF-1 has also been prepared from recombinant DNA using two apparently related cDNA clones: (1) a "short" form encoding a message that, when translated, produces a monomeric protein with 224 amino acids, in front of which is a 32-amino acid long signal sequence (Kawasaki, ES et al., Science (1985) 230: 292-296 and PCT W086 / 04607, both of which are incorporated herein by reference. ), and (2) a "long" form encoding a 522 amino acid monomeric protein which also has a precursor 32 amino acid long signal sequence. The long form has been cloned and expressed by two groups, as discussed in Ladner, M.B. et al., The EMBO. J. (1987) 6 (9): 2693-2698 and Wong, G. et al., Science (1987) 235: 1504-1509, both of which are incorporated herein by this reference. (The DNA and amino acid sequences for both the "short" and "long" forms are shown in Figures 5 'and 6, respectively; however, the 32-amino acid long signal sequence is incomplete, as illustrated in Figure 6).

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De lange og korte former af det CSF-l-kodende DNA synes at hidrøre fra et variabelt splejsningspunkt ved opstrømsdelen af exon 6 i det genomiske, CSF-l-kodende DNA.The long and short forms of the CSF-1 coding DNA appear to originate from a variable splicing point at the upstream portion of exon 6 in the genomic CSF-1 coding DNA.

Når CSF-1 i visse eucaryotiske celler udtrykkes fra enten 20 de lange eller korte cDNA-former, synes det at blive variabelt processeret i den C-terminale ende og/eller variabelt glycosyleret. Som følge heraf finder man CSF-1-proteiner med varierende molekylevægte, når den reducerede, monomere form analyseres ved Western analyse.When in some eucaryotic cells, CSF-1 is expressed from either the long or short cDNA forms, it appears to be variably processed at the C-terminal end and / or variably glycosylated. As a result, CSF-1 proteins of varying molecular weights are found when the reduced monomeric form is analyzed by Western analysis.

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Aminosyresekvenserne for den lange form og den korte form, som forudsagt ud fra DNA-sekvensen i de isolerede kloner og ved relationen til den genomiske sekvens, er identiske med hensyn til de første 149 aminosyrer i den 30 N-terminale ende i det modne protein og divergerer derefter ved indføjelsen af en yderligere 894 bp lang indføjelse, som koder for 298 yderligere aminosyrer efter glu-tamin 149, i den længere klon. Både den kortere og den længere form af genet muliggør ekspression af proteiner 35 med sekvenser, som indeholder identiske områder i den C-terminale ende såvel som i den N-terminale ende. Der blev DK 17)3693 B1 4 udvundet biologisk aktiv CSF-1, når cDNA, som koder for de første 150 eller 158 aminosyrer af den korte form eller for de første 221 aminosyrer af den lange form, udtrykkes i eucaryotiske celler.The long and short form amino acid sequences predicted from the DNA sequence of the isolated clones and by relation to the genomic sequence are identical with respect to the first 149 amino acids at the 30 N-terminal end of the mature protein and then diverges upon the insertion of an additional 894 bp insert, which encodes 298 additional amino acids after glutamine 149, into the longer clone. Both the shorter and the longer form of the gene allow expression of proteins 35 with sequences containing identical regions at the C-terminal end as well as at the N-terminal end. Biologically active CSF-1 was recovered when cDNA encoding the first 150 or 158 amino acids of the short form or of the first 221 amino acids of the long form is expressed in eucaryotic cells.

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Eftersom de fleste, hvis ikke alle native, secernerede CSF-l-molekyler er glycosylerede og dimere, sker der tilsyneladende en væsentlig posttranslationel processerirg in vivo. På grund af det native CSF-l-molekyles komplek-10 sisitet har det været antaget at være hensigtsmæssigt ét udtrykke CSF-l-genet i celler, som hidrører fra højeie organismer. Det forekom usandsynligt, at man ville oprå aktivt protein, når genet blev udtrykt i mere bekvemme bakterieværter, såsom E. coli. Bakterieværter har ikke 15 evnen til at glycosylere proteiner og deres intracellula:-re miljø bidrager ikke til den genfoldning, dannelse af disulfidbinding og disulfidstabiliseret dimerisering, sera synes at være essentiel for fuld CSF-l-aktivitet. Forsøgsvis fremstilling af rekombinant-CSF-1 i E. coli her 20 således forud for den foreliggende opfindelse resulteret i et protein med en meget lav aktivitet, omend dets identitet som monomert CSF-1 let kunne bekræftes ved immunoé-nalyse, N-terminal sekventering og aminosyreanalyse.Since most, if not all native, CSF-1 secreted molecules are glycosylated and dimer, a substantial post-translational process appears to occur in vivo. Due to the complexity of the native CSF-1 molecule, it has been considered appropriate to express one the CSF-1 gene in cells derived from high organisms. It appeared unlikely that active protein would be generated when the gene was expressed in more convenient bacterial hosts, such as E. coli. Bacterial hosts do not have the ability to glycosylate proteins and their intracellula: - their environment does not contribute to the refolding, disulfide bond formation and disulfide stabilized dimerization, sera appear to be essential for full CSF-1 activity. Experimental preparation of recombinant CSF-1 in E. coli here, thus, prior to the present invention, resulted in a very low activity protein, although its identity as monomeric CSF-1 could be readily confirmed by immunoassay, N-terminal sequencing. and amino acid analysis.

25 Det er nu accepteret, at inaktive former af rekombinere-de, fremmede proteiner, som er frembragt i bakterier, ken kræve yderligere "genfoldningstrin" for at gøre dem bruc-bare til de tilsigtede formål. Som et dimert protein, der indeholder et stort antal cysteiner og disulfidbindinger, 30 der er nødvendige for aktivitet, udgør CSF-1 en specielt vanskelig udfordring ved fremstilling ud fra bakterielle systemer. Ofte findes rekombinerede proteiner, som frembringes i E. coli, herunder det således frembragte CSF-3, i form af meget uopløselige, intracellulære proteinpræc:.-35 pitater, der kaldes inklusionslegemer eller lysbrydencle legemer. Disse inklusioner kan let adskilles fra de oplo- DK 173693 B1 5 selige bakterieproteiner, men de må derefter opløseliggøres under betingelser, som resulterer i stort set fuldstændig denaturering af proteinet. Selv fra bakteriekilder secernerede proteiner kan, omend de ikke nødvendigvis 5 giver de samme opløselighedsproblemer, kræve betragtelig manipulering for at genoprette aktivitet. Hvert forskelligt protein kan kræve en forskellig genfoldningsprotokol til opnåelse af fuld biologisk aktivitet.It is now accepted that inactive forms of recombinant foreign proteins produced in bacteria may require additional "refolding steps" to make them usable for the intended purposes. As a dimeric protein containing a large number of cysteines and disulfide bonds required for activity, CSF-1 presents a particularly difficult challenge in preparation from bacterial systems. Often, recombined proteins produced in E. coli, including the CSF-3 thus produced, are found in the form of highly insoluble, intracellular protein precursors called inclusion bodies or refractory bodies. These inclusions can be easily separated from the soluble bacterial proteins, but they must then be solubilized under conditions that result in almost complete denaturation of the protein. Even from bacterial sources, secreted proteins, although not necessarily the same solubility problems, may require considerable manipulation to restore activity. Each different protein may require a different refolding protocol to achieve full biological activity.

10 Der er fremkommet en række artikler, hvori omtales genfoldningsforsøg af individuelle proteiner frembragt i bakterieværter eller som på anden måde findes i denatureret eller ikke-nativ form. En repræsentativt udvalg følger herefter.A number of articles have appeared which refer to refolding experiments of individual proteins produced in bacterial hosts or otherwise found in denatured or non-native form. A representative committee follows.

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Weber, K. el al., omtaler i J. Biol. Chem. (1971) 246:4504-4509 gendannelsen af et oligomert enzym efter denaturering med natriumdodecylsulfat (SDS). Denne fremgangsmåde blev betragtet som en løsning af et problem 20 skabt ved proteiners binding til SDS, og ved fremgangsmåden anvendtes fjernelse af det denaturerede protein fra SDS i nærværelse af 6 M urinstof, anionbytning til fjernelse af SDS, efterfulgt af fortynding af urinstoffet, hvor alle trin blev udført i nærværelse af reducerende 25 midler. Proteinerne, som i det mindste var delvis genfoldede, omfattede: aspartattranscarbamylase, β- galactosidase, kaninmuskelaldolase og kappeprotein fra bakteriofag R-17.Weber, K. et al., Cited in J. Biol. Chem. (1971) 246: 4504-4509 the recovery of an oligomeric enzyme after denaturing with sodium dodecyl sulfate (SDS). This method was considered a solution to a problem 20 created by protein binding to SDS, and the method used to remove the denatured protein from SDS in the presence of 6 M urea, anion exchange to remove SDS, followed by dilution of the urea where all steps were performed in the presence of reducing agents. The proteins, which were at least partially refolded, included: aspartate transcarbamylase, β-galactosidase, rabbit muscle aldolase, and bacteriophage R-17 coat protein.

30 Light, A. beskriver i Biotechniques (1985) 3:298-306 en række forsøg på at genfolde et stort antal proteiner. Det fremgår af beskrivelsen i denne reference, at de metoder, som kan anvendes, er meget individuelle for det bestemte protein, som det drejer sig om. I nogle tilfælde har gen-35 foldning af væsentlige mængder af bestemte proteiner faktisk ikke været mulig, og resultaterne er ganske uforud- DK 17[3693 B1 6 sigelige. Endvidere blev genfoldningsprocedurer for τφ-kombinant urokinase frembragt i E. coli beskrevet af Winkler, M.E. i Biotechnology (1985) 3:990-999. I det:e tilfælde blev materialet opløst i 8 M urinstof eller 5 M 5 guanidinhydrochlorid, og omlejringen af disulfider blev lettet ved anvendelse af en puffer, som indeholdt et glu-tathionredoxsystem. Rekombinant human—iiranuninterferon, som ikke indeholder disulfidbindinger, er blevet genfoL-det til frembringelse af en mere aktiv præparation und ar 10 anvendelse af chaotropiske midler i nærværelse af thioL-disulfidudskiftningsreagenser (PCT ansøgning WO 86/06385). I et andet eksempel blev også bakterielt syntetiseret granulocytmakrofagkolonistimulerende fakt ar (GM-CSF), et medlem af CSF-gruppen, produceret i E. coli 15 og genfoldet efter opløseliggørelse i 6 M urinstof. Denne CSF er ubeslægtet med CSF-1, eftersom GM-CSF har en særskilt aminosyresekvens og også er monomer.30 Light, A. in Biotechniques (1985) 3: 298-306 describes a series of attempts to refold a large number of proteins. It is apparent from the description in this reference that the methods that can be used are very individual for the particular protein in question. Indeed, in some cases, refolding of substantial amounts of certain proteins has not been possible, and the results are quite unpredictable. Furthermore, refolding procedures for τφ combinant urokinase were produced in E. coli described by Winkler, M.E. in Biotechnology (1985) 3: 990-999. In the first case, the material was dissolved in 8 M urea or 5 M 5 guanidine hydrochloride, and the rearrangement of disulfides was facilitated using a buffer containing a glutathione redox system. Recombinant human urine interferon, which does not contain disulfide bonds, has been recovered to produce a more active preparation and the use of chaotropic agents in the presence of thioL disulfide replacement reagents (PCT application WO 86/06385). In another example, bacterially synthesized granulocyte macrophage colony-stimulating factor (GM-CSF), a member of the CSF group, was also produced in E. coli 15 and refolded after solubilization in 6 M urea. This CSF is unrelated to CSF-1 since GM-CSF has a distinct amino acid sequence and is also monomeric.

Anvendelsen af genfoldningsprocedurer til opnåelse af re-20 konstituering af aktivitet i multimere proteiner er også blevet beskrevet af Herman, R.H. et al. i Biochemistry (1985) 24:1817-1821, for phosphoglyceratmutase, og af Ca-billy, S. i Proc. Natl. Acad. Sci. USA (1984) 82:3273-77, for immunoglobuliner. En yderligere fremgangsmåde til 25 gensamling af immunoglobulin er beskrevet af Boss, M.A. et al, i Nucleic Acids Research (1984) 12:3791-3806. Ved disse fremgangsmåder anvendtes i alle tilfælde denaturering og hensigtsmæssige oxiderende og reducerende midler eller sulfitolysereagenser. Ved en beslægtet fremgangsraå-30 de anvendes katalysatoren thioredoxin, og den omtales af Pigiet, V.P. i Proc. Natl. Acad. Sci. USA (1986) 83:7643-7647.The use of refolding procedures to achieve the reconstitution of activity in multimeric proteins has also been described by Herman, R.H. et al. in Biochemistry (1985) 24: 1817-1821, for phosphoglycerate mutase, and by Ca-billy, S. in Proc. Natl. Acad. Sci. USA (1984) 82: 3273-77, for immunoglobulins. A further method for immunoglobulin gene collection is described by Boss, M.A. et al, in Nucleic Acids Research (1984) 12: 3791-3806. In these processes, denaturation and appropriate oxidizing and reducing agents or sulfitolysis reagents were used in all cases. In a related process, the catalyst thioredoxin is used and it is referred to by Pigiet, V.P. in Proc. Natl. Acad. Sci. USA (1986) 83: 7643-7647.

Visse aspekter af opløseliggørelse, rensning og genfold-35 ning af visse rekombinerede proteiner, der er frembragt som lysbrydende legemer i bakterier, omtales også i U.S.Certain aspects of solubilization, purification and refolding of certain recombined proteins produced as light refracting bodies in bacteria are also discussed in U.S. Pat.

DK 173693 B1 7 patenterne nr. 4.511.562, 4.511.503, 4.512.912 og 4.518.526 samt i EPO publikation nr. 114.506 (Genentech).DK 173693 B1 7 patents Nos. 4,511,562, 4,511,503, 4,512,912 and 4,518,526 as well as in EPO Publication No. 114,506 (Genentech).

De ovennævnte referencer er kun repræsentative for en 5 stor mængde litteratur, som, når den sammenholdes, viser individuelle trin i protokoller, som kan modificeres og kombineres i forskellige sekvenser til opnåelse af individuelt tilpassede procedurer for bestemte proteiner, som er frembragt i overensstemmelse med bestemte ekspressi-10 onssystemer. Det er klart, at en tilpasning af de samlede procedurer til et specifikt tilfælde er et krav for at kunne frembringe genfoldede produkter med fuld biologisk aktivitet i brugbare mængder, 15 I en række af de publicerede procedurer beskrives f.eks. et trin til en vellykket genfoldning af det rekombinerede protein. Det fremgår ikke tydeligt fra disse referencer, men det er velkendt inden for fagområdet, at udgangsmaterialet til genfoldningen kan foreligge i en række former, 20 hvilket afhænger af naturen af det anvendte ekspressionssystem. I tilfældet med bakteriel ekspression er det imidlertid klart, at produktet ikke er glycosyleret, og at frembringelsen af et intracellulært, disulfidbundet, dimert produkt endvidere forhindres af det reducerende 25 miljø i bakterieceller.The above references are only representative of a large body of literature which, when compared, shows individual steps in protocols that can be modified and combined in different sequences to obtain individually tailored procedures for particular proteins produced in accordance with particular proteins. expression systems. It is clear that adapting the overall procedures to a specific case is a requirement for being able to produce refolded products with full biological activity in usable quantities. a step towards successful refolding of the recombined protein. It is not clear from these references, but it is well known in the art that the starting material for the refolding can be in a variety of forms, depending on the nature of the expression system used. However, in the case of bacterial expression, it is clear that the product is not glycosylated and that the production of an intracellular, disulfide-linked dimeric product is further inhibited by the reducing environment in bacterial cells.

For tiden er den mest almindelige form af et udgangsmateriale til et rekombineret protein til genfoldning et intracellulært, uopløseligt protein, som frembringes ved 30 ekspression af et gen for et modent eller bakterielt fusionsprotein, som mangler en funktionel signalsekvens, under styring af bakterielle standardpromotorer, såsom trp eller PL. Eftersom produkter frembragt ved rekombination i bakterier frembringes i høje koncentrationer i et 35 reducerende miljø, og fordi konstruktionerne typisk ikke sætter bakterierne i stand til at secernere det rekombi- DK 173693 B1 8 nerede protein, observeres det ofte, at disse fremmedø proteiner danner uopløselige inklusionslegemer.Currently, the most common form of a recombined protein for refolding is an intracellular, insoluble protein produced by expression of a gene for a mature or bacterial fusion protein lacking a functional signal sequence under the control of standard bacterial promoters, such as trp or PL. Since products produced by recombination in bacteria are produced at high concentrations in a reducing environment, and because the constructs typically do not enable the bacteria to secrete the recombined protein, it is often observed that these foreign-made proteins form insoluble inclusion bodies. .

Der kendes imidlertid signalsekvenser, som fungerer ji 5 bakterier, herunder E. coli penicillinasesekvensen, sojn er omtalt af Gilbert et al. i U.S. patenterne nr.However, signal sequences that function in bacteria, including the E. coli penicillinase sequence, are known, and are disclosed by Gilbert et al. in the U.S. the patents no.

4.411.994 og 4.338.397, B. licheniformis penP-sekvenserne, som omtales af Chang i U.S. patenterne nr. 4.711.843 og 4.711.844 samt phosphatase A-signalsekvensen 10 (phoA) , som omtales af Chang et al. i europæisk patentpublikation nr. 196.864, publiceret 8. oktober 1986 og medtaget her ved denne henvisning. Secernering kan effektueres i nogle stammer. Hvis der imidlertid anvendes Gram-negative værter, sker der måske ikke fuldstændig se-15 cernering, og proteinet kan forblive i det periplasma- tiske rum. Det er ikke desto mindre meget mere sandsyn ligt, at proteiner, som udtrykkes under styring af promotorer og signalsekvenser, såsom phoA, vil blive frembragt i opløselig form, hvis de er i stand til at genfolde cg 20 danne nødvendige disulfidbindinger i det extracellulære miljø. De metoder, som omtales her, forventes at være af værdi for både intracellulære og secernerede produkter, hvor genfoldning er nødvendig.4,41,1994 and 4,338,397, B. licheniformis penP sequences, cited by Chang of U.S. Pat. patents Nos. 4,711,843 and 4,711,844 as well as the phosphatase A signal sequence 10 (phoA) disclosed by Chang et al. in European Patent Publication No. 196,864, published October 8, 1986 and incorporated herein by reference. Secretion can be performed in some strains. However, if Gram-negative hosts are used, complete secretion may not occur and the protein may remain in the periplasmic compartment. Nevertheless, proteins expressed under the control of promoters and signal sequences, such as phoA, are much more likely to be produced in soluble form if they are able to fold and form necessary disulfide bonds in the extracellular environment. The methods discussed herein are expected to be of value for both intracellular and secreted products where refolding is necessary.

25 I litteraturen findes der ingen specifik fremgangsmåde til fremstilling af biologisk aktiv, dimer CSF-1 ud fra bakterier. Med den foreliggende opfindelse beskrives adskillige genfoldningsprocedurer, som involverer CSF-1-proteiner med forskellige, primære strukturer. De frem-30 komne, genfoldede CSF-l-proteiner er helt aktive og ople-selige, og de forskellige molekyler adskiller sig tilstrækkeligt med hensyn til fysiske egenskaber til, at ce kan forventes at udvise en række farmakokinetiske og/eller farmakologiske egenskaber, når de anvendes tera-35 peutisk in vivo.In the literature, there is no specific method for producing biologically active dimeric CSF-1 from bacteria. The present invention describes several refolding procedures involving CSF-1 proteins with various primary structures. The resulting refolded CSF-1 proteins are fully active and detectable, and the various molecules differ sufficiently in physical properties to be expected to exhibit a variety of pharmacokinetic and / or pharmacological properties when they are used therapeutically in vivo.

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Forklaring af opfindelsenExplanation of the Invention

Opfindelsen angår en fremgangsmåde til samling af biologisk aktiv, dimer CSF-1 under anvendelse af det monomere 5 udgangsmateriale, som frembringes af bakterieceller, der er blevet transformeret med egnede CSF-1-genkonstruktioner. Ved fremgangsmåden udnyttes de forskellige egenskaber af den monomere og dimere form af proteinet til frembringelse af brugbare rensningsmetoder, 10 og der anvendes egnede reagenser til at omdanne den uopløselige, monomere form til en aktiv, opløselig, dimer form.The invention relates to a method for assembling biologically active dimer CSF-1 using the monomeric starting material produced by bacterial cells that have been transformed with suitable CSF-1 gene constructs. The process utilizes the various properties of the monomeric and dimeric form of the protein to produce useful purification methods, and suitable reagents are used to convert the insoluble monomeric form into an active, soluble, dimeric form.

I ét aspekt angår opfindelsen således en fremgangsmåde 15 til opnåelse af renset, biologisk aktiv CSF-l-dimer ud fra det reducerede, monomere produkt fra det CSF-l-gen, som udtrykkes rekombineret i bakterier. Den halvrensede, opløseliggjorte, reducerede monomer holdes først under reducerende betingelser for at sikre et ensartet ud-20 gangsmateriale. Dernæst udføres genfoldningen af monomeren og associeringen til dimer under egnede genfoldningsbetingelser. For det tredje renses den genfoldede, dimere CSF-1 til fjernelse af kontaminerende proteiner og endo-toxiner, hvilket giver et produkt, der er egnet til kli-25 nisk brug. I én udførelsesform omfatter fremgangsmåden, at det rekombinant fremstillede CSF-1 opløseliggøres; i andre udførelsesformer udsættes det opløseliggjorte CSF-1 for et rensningstrin før genfoldning.Thus, in one aspect, the invention relates to a method 15 for obtaining purified, biologically active CSF-1 dimer from the reduced, monomeric product of the CSF-1 gene expressed recombined in bacteria. The semi-purified, solubilized reduced monomer is first kept under reducing conditions to ensure a uniform starting material. Next, the refolding of the monomer and the association to dimer is carried out under suitable refolding conditions. Third, the refolded dimeric CSF-1 is purified to remove contaminating proteins and endotoxins, yielding a product suitable for clinical use. In one embodiment, the method comprises solubilizing the recombinantly produced CSF-1; In other embodiments, the solubilized CSF-1 is subjected to a purification step prior to refolding.

30 I et andet aspekt angår opfindelsen en fremgangsmåde til dannelse af heterodimere CSF-l-proteiner ud fra en blanding af forskellige, monomere former. Monomererne kan blandes direkte eller kan være blandede på grund af, at de frembringes af den samme rekombinant-celle. Sådanne 35 heterodimerer kan muliggøre fremstillingen af CSF-l-pro-dukter med forbedret anvendelse in vivo.In another aspect, the invention relates to a method for forming heterodimeric CSF-1 proteins from a mixture of various monomeric forms. The monomers can be directly mixed or may be mixed because they are produced by the same recombinant cell. Such heterodimers can enable the production of CSF-1 products with improved in vivo use.

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Endnu et andet aspekt omfatter om nødvendigt det yderligere trin at genopløse den resterende, uopløselige CSF-l, som er til stede ved slutningen af genfoldningsprocessep, 5 og forøge udbyttet ved recirkulering.Yet another aspect, if necessary, includes the additional step of redissolving the residual insoluble CSF-1 present at the end of refolding process step 5 and increasing the yield by recycling.

I endnu et andet aspekt angår opfindelsen en fremgangsmåde til opnåelse af klinisk rent, dimert CSF-l-protei:i, hvilket omfatter, at det genfoldede, dimere protein u:v-10 derkastes kromatografi under anvendelse af et hydrofobt bæremateriale, såsom ved phenyl-Sepharose- eller phenyL-TSK-HPLC.In yet another aspect, the invention relates to a process for obtaining clinically pure dimeric CSF-1 protein: i, which comprises refolding the dimeric protein u: v-10 using chromatography using a hydrophobic support material such as phenyl -Sepharose or phenyl-TSK-HPLC.

Kort beskrivelse af tegningen 15Brief Description of the Drawing 15

Figur 1 viser den delvise oprensning af én type monomer CSF-1 under anvendelse af molekylesigtekromatografi.Figure 1 shows the partial purification of one type of monomer CSF-1 using molecular sieve chromatography.

Figur 2 viser dimeriseringsgraden analyseret ved moleky-20 lesigtekromatografi.Figure 2 shows the degree of dimerization analyzed by molecular sieve chromatography.

Figur 3 viser RP-HPLC-analyse af én type denatureret bg genfoldet, rekombinant E. coli CSF-1.Figure 3 shows RP-HPLC analysis of one type of denatured bg refolded, recombinant E. coli CSF-1.

25 Figur 4 viser en spektral analyse til bestemmelse af opløseligheden af én type denatureret og genfoldet, rekon-binant E. coli CSF-1.Figure 4 shows a spectral analysis to determine the solubility of one type of denatured and refolded, recombinant E. coli CSF-1.

Figur 5 viser cDNA'et og den deducerede aminosyresekvens 30 for en cDNA-klon, som koder for en ’'kort" form af humajn-CSF-1, kaldet pcCSF-17.Figure 5 shows the cDNA and deduced amino acid sequence 30 for a cDNA clone encoding a "short" form of human CSF-1, called pcCSF-17.

Figur 6 viser cDNA'et og den deducerede aminosyresekvens for en cDNA-klon, som koder for en "lang" form af humajn-35 CSF-1, kaldet pcCSF-4.Figure 6 shows the cDNA and deduced amino acid sequence of a cDNA clone encoding a "long" form of human CSF-1 called PCCSF-4.

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Figur 7 viser resultaterne fra en reducerende og ikke-reducerende SDS-PAGE-analyse af dimer asp59SCSF/C@150 CSF-1.Figure 7 shows the results of a reducing and non-reducing SDS-PAGE analysis of dimer asp59SCSF / C @ 150 CSF-1.

5 Udførelsesformer for opfindelsen A. Definitioner:Embodiments of the Invention A. Definitions:

Som anvendt heri refererer udtrykket "chaotropisk miljø" 10 til et miljø, der indeholder hensigtsmæssige, chaotro-piske midler, såsom urinstof, i en koncentration, som er tilstrækkelig til at bryde proteinernes tertiære struktur, eller et miljø, som holdes ved en temperatur eller i en anden tilstand, som fremkalder en sådan brydning af 15 den tertiære struktur.As used herein, the term "chaotropic environment" 10 refers to an environment containing appropriate chaotropic agents, such as urea, at a concentration sufficient to break the tertiary structure of the proteins, or an environment maintained at a temperature or in another state which induces such a rupture of the tertiary structure.

Chaotropiske midler eller tilstande, såsom temperatur og pH, kan bryde strukturen på en række måder, herunder brydningen af hydrogenbindinger. Egnede chaotropiske mil-20 jøer omfatter 2-8 M urinstof, 4-7 M guanidin, detergenter, såsom SDS, ved koncentrationer på omkring 0,1 vægtprocent, og syrer, såsom eddikesyre, ved koncentrationer på ca. 1 M, basiske betingelser på f.eks. pH 11 og derover samt hævede temperaturer. Når proteiner anbringes i 25 et chaotropisk miljø, kan proteinernes normale fysiologiske konformation blive reversibelt såvel som irreversibelt ændret, og den primære struktur kan blive "foldet ud" i varierende grad, hvilket afhænger af kon centrationen af det chaotropiske middel, samt hvor stren-30 ge de andre chaotropiske betingelser er. Det skal forstås, at midler og/eller betingelser, som frembringer chaotropiske miljøer, kan anvendes i kombination eller i sekvens. Der kan f.eks. anvendes blandinger af chaotropiske midler, eller CSF-1'en kan først anbringes i et 35 chaotropisk miljø, som er frembragt af ét chaotropisk middel, og derefter udsættes for et andet chaotropisk DK 173693 B1 12 miljø, som er frembragt af et andet middel eller som følge af temperaturen.Chaotropic agents or conditions, such as temperature and pH, can break the structure in a number of ways, including the breaking of hydrogen bonds. Suitable chaotropic environments include 2-8 M urea, 4-7 M guanidine, detergents such as SDS at concentrations of about 0.1% by weight, and acids, such as acetic acid, at concentrations of approx. 1 M, basic conditions of e.g. pH 11 and above as well as raised temperatures. When proteins are placed in a chaotropic environment, the normal physiological conformation of the proteins can be reversibly as well as irreversibly altered, and the primary structure can be "unfolded" to varying degrees, depending on the concentration of the chaotropic agent and the degree of stress. ge the other chaotropic conditions are. It is to be understood that agents and / or conditions which produce chaotropic environments can be used in combination or in sequence. For example, mixtures of chaotropic agents are used or the CSF-1 may first be placed in a chaotropic environment produced by one chaotropic agent and then exposed to another chaotropic environment produced by another agent or as a result of the temperature.

iin

Det heri anvendte udtryk "reducerende middel" refererer 5 specifikt til et reducerende middel, der er i stand til at reducere disulfidbindinger til sulfhydrylgrupper. Der kan fås en række mildt reducerende materialer, som er i stand til at udføre denne omdannelse, men de mest almindelige omfatter thiolholdige enheder, såsom 10 mercaptoethanol eller dithiothreitol. Yderligere funktionelt reducerende midler omfatter reduceret glutathion og fri cystein i sig selv. Skønt thiolholdige forbindelser fremhæves, inkluderer denne definition ethvert material«:, som er i stand til at omdanne disulfid til thiol ud4n 15 uønskede sidereaktioner.The term "reducing agent" used herein refers specifically to a reducing agent capable of reducing disulfide bonds to sulfhydryl groups. A variety of mild reducing materials are available which are capable of effecting this conversion, but the most common ones include thiol-containing units such as mercaptoethanol or dithiothreitol. Additional functional reducing agents include reduced glutathione and free cysteine per se. Although thiol-containing compounds are highlighted, this definition includes any material capable of converting disulfide to thiol in 15 undesirable side reactions.

"Reducerende betingelser" refererer til betingelser, som holder eller bringer, som det passer i det foreliggende tilfælde, CSF-l-proteinet i den monomere, reducerede 20 form. Hvis CSF-1 frembringes i et miljø, som i begyndelsen bringer det i en reduceret form (dvs. at cysteinerne er i denne form, ikke cystin), kan det være tilstrækkeligt med mildere betingelser end det ville være nødvendigt, hvis proteinet i begyndelsen var i oxideret 25 form."Reducing conditions" refers to conditions which retain or bring, as appropriate in the present case, the CSF-1 protein in the monomeric reduced form. If CSF-1 is produced in an environment that initially brings it into a reduced form (i.e., the cysteines are in this form, not cystine), milder conditions may be sufficient than would be necessary if the protein were initially in oxidized form.

"Genfoldningsbetingelser" refererer til betingelse::, hvori et denatureret protein får lov til at antage en konformation, som er forbundet med fysiologisk aktivite:."Refolding conditions" refers to the condition in which a denatured protein is allowed to assume a conformation associated with physiological activity:.

30 Dette inkluderer specifik dannelse af disulfider og/eller associering i dimere eller multimere strukturer, som funktionelt er identiske med strukturerne for det native protein. Sådanne betingelser inkluderer langsom fjernelse af eller trinvis fortynding af chaotropiske midler i næ:-35 værelse eller fravær af midler, der tillader dannelsen af de disulfidbindinger, som normalt er til stede i den ac- DK 173693 B1 13 tive konformation. Hvis der til opløseliggørelsen anvendes høje koncentrationer af chaotropiske midler, eller hvis proteinet på anden måde denatureres af disse midler, kan de chaotropiske substanser, som er inkluderet i det 5 chaotropiske middel, fjernes ved simpel fortynding, ved dialyse, ved hulfiberdiafiltrering eller ved hjælp af en række andre kendte metoder inden for fagområdet, hvorved koncentrationen af små molekyler effektivt sænkes med eller uden en tilsvarende sænkning af koncentrationen af 10 proteinet.This includes specific formation of disulfides and / or association in dimeric or multimeric structures that are functionally identical to the native protein structures. Such conditions include slow removal or stepwise dilution of chaotropic agents in near-room or absence of agents which allow the formation of the disulfide bonds normally present in the active conformation. If high concentrations of chaotropic agents are used for solubilization, or if the protein is otherwise denatured by these agents, the chaotropic substances included in the chaotropic agent can be removed by simple dilution, by dialysis, by hollow fiber diafiltration or by a number of other known methods in the art, whereby the concentration of small molecules is effectively lowered with or without a corresponding decrease in the concentration of the protein.

Det er ønskeligt at fremme dannelsen af disulfidbindinger under denne proces. Dette kan opnås ved luftoxidation eller ved at inkludere reagenser, som er egnede til dette 15 formål, i genfoldningsmiljøet. Sådanne reagenser omfatter "redoxsystemer”, som muliggør den kontinuerlige oxidation og reduktion af thiol/disulfidparrene. Et af de mest almindeligt anvendte blandt disse systemer er glutathion, i både oxideret og reduceret form. Det er kendt, at oxide-20 ret glutathion og reduceret glutathion er naturligt forekommende bestanddele i pattedyrceller og kan faktisk ud over eller sammen med isomeraser katalysere denne reaktion, fremme thiol/ disulfidbindingsudveksling in vivo (Tietze, F., Anal. Biochem. (1969) 27:502). Der kan også 25 anvendes andre par af oxiderede (disulfid) og reducerede (thiol) reagenser; det er bestemt ikke nødvendigt, at disulfidet og thiolen stammer fra det samme molekyle. Nye disulfidbindinger kan endvidere dannes ved sulfitolyse efterfulgt af oxidation af de sulfonerede thiolgrupper.It is desirable to promote the formation of disulfide bonds during this process. This can be achieved by air oxidation or by including reagents suitable for this purpose in the refolding environment. Such reagents include "redox systems" which enable the continuous oxidation and reduction of the thiol / disulfide pairs. One of the most commonly used among these systems is glutathione, in both oxidized and reduced form. It is known that oxidized glutathione and reduced glutathione are naturally occurring components of mammalian cells and can, in fact, catalyze this reaction in addition to or together with isomerases, promote thiol / disulfide bond exchange in vivo (Tietze, F., Anal. Biochem. (1969) 27: 502). pairs of oxidized (disulfide) and reduced (thiol) reagents, it is certainly not necessary that the disulfide and thiol originate from the same molecule, and new disulfide bonds can also be formed by sulfitolysis followed by oxidation of the sulfonated thiol groups.

30 Denne fremgangsmåde er beskrevet i U.S. patent nr. 4.620.948 til Builder et al., se ovenfor.This process is described in U.S. Pat. Patent No. 4,620,948 to Builder et al., supra.

De heri omtalte rensningsmetoder omfatter en række metoder. Blandt adskillige typer, der kan anvendes, er stør-35 relsesfraktionering under anvendelse af molekylesigtekro-matografi, ionbytningskromatografi under egnede betingel- DK 173693 B1 14 ser, affinitetskromatografi under anvendelse af f.ek>. monoklonale antistoffer rettet mod den biologisk aktive form af proteinet, adsorptionskromatografi under anvendelse af ikke-specifikke baerematerialer, såsom hydroxyϊ-5 patit, siliciumoxid, aluminiumoxid osv., samt også elec-troforese i geler. Med CSF-1 har hydrofob vekselvirc-ningskromatografi, såsom anvendelse af phenyl-Sepharose eller phenyl-TSK, vist sig at være specielt nyttig. 5n initiel rensning af monomer CSF-1 under anvendelse af 10 ionbytningskromatografi {såsom DEAE-Sepharosekromatd- grafi) har endvidere vist sig at være en specielt effektiv fremgangsmåde til at forøge renheden af det dimere CSF-l-protein. Disse rensningsmetoder er i en generel betydning velkendte inden for fagområdet, og en detaljeret 15 beskrivelse af det særegne ved deres anvendelse over far CSF-l-proteiner er beskrevet i eksemplerne nedenfor.The purification methods mentioned herein include a variety of methods. Among several types that can be used are size fractionation using molecular sieve chromatography, ion exchange chromatography under suitable conditions, affinity chromatography using e.g. monoclonal antibodies directed against the biologically active form of the protein, adsorption chromatography using non-specific support materials such as hydroxyapatite, silica, alumina, etc., and also electrophoresis in gels. With CSF-1, hydrophobic interaction chromatography, such as the use of phenyl-Sepharose or phenyl-TSK, has been found to be particularly useful. Furthermore, 5n initial purification of monomer CSF-1 using 10 ion exchange chromatography (such as DEAE-Sepharose chromatography) has proved to be a particularly effective method of increasing the purity of the dimeric CSF-1 protein. These purification methods are generally well known in the art, and a detailed description of the peculiarities of their use over far CSF-1 proteins is described in the Examples below.

Som anvendt heri betyder "biologisk aktiv" en præparatian af human- CSF-1 frembragt rekombinant i bakterier, hvil-20 ken CSF-1 stort set har den samme specifikke aktivitet i human- og musebenmarvskolonidannende analyser som nativ human-CSF-1 frembragt af pattedyrceller.As used herein, "biologically active" means a preparation of human CSF-1 produced recombinantly in bacteria, which CSF-1 has substantially the same specific activity in human and mouse bone marrow colony forming assays as native human CSF-1 produced by mammalian cells.

"Klinisk ren" CSF-1 betyder en præparation af biologisk 25 aktiv, human- CSF-1 frembragt rekombinant i bakterier, hvilken præparation er mindst 95% CSF-1 vurderet ved enten HPLC med omvendt fase eller reducerende eller ikka-reducerende SDS-PAGE, og som har et endotoxinindhold oå mindre end ca. 1,0 ng/mg CSF-1 analyseret ved La(l-30 standardanalyse."Clinically pure" CSF-1 means a preparation of biologically active human CSF-1 produced recombinantly in bacteria, which preparation is at least 95% CSF-1 assessed by either reverse phase HPLC or reducing or non-reducing SDS-PAGE , and having an endotoxin content of less than approx. 1.0 ng / mg CSF-1 analyzed by La (l-30 standard analysis).

B. CSF-l-proteiner:B. CSF-1 proteins:

Som anført i baggrundsafsnittet er CSF-1 biologisk aktiv 35 i dens dimere form. Det har været muligt at opnå rekombinant DNA, som koder for CSF-l-monomerer, der består af £n DK 173693 B1 15 række aminosyresekvenser og -længder. Figur 5 henholdsvis 6 viser DNA- og aminosyresekvenserne for den korte form og den lange form, som begge har en foranstillet 32-aminosyre-lang signalsekvens, Sekvenserne af det monomere 5 CSF-l-protein betragtes heri af bekvemmelighedsgrunde til at være den 224-aminosyre-lange korte form (SCSF) og den 522-aminosyre-lange lange form (LCSF), som er vist i disse figurer.As stated in the background section, CSF-1 is biologically active in its dimeric form. It has been possible to obtain recombinant DNA encoding CSF-1 monomers consisting of a variety of amino acid sequences and lengths. Figures 5 and 6, respectively, show the DNA and amino acid sequences of the short form and the long form, both of which have a precursor 32 amino acid long signal sequence. The sequences of the monomeric 5 CSF-1 protein are considered herein to be the 224- amino acid long form (SCSF) and the 522 amino acid long form (LCSF) shown in these figures.

10 Der kan for tiden opnås plasmider, som koder for en række CSF-l-former, og de kan udtrykkes i bakterielle systemer.10 Currently, plasmids encoding a variety of CSF-1 forms can be obtained and can be expressed in bacterial systems.

Som umiddelbart ovenfor beskrevet kan det gen, som koder for den lange form af CSF-1, udtrykkes i sin helhed, eller genet kan trunkeres til ekspression af former, hvor 15 den C-terminale ende er fjernet. Endvidere kan de to eller tre første N-terminale codoner fjernes, således at det fremkomne protein er mere homogent. Specielt har det vist sig lettere at fjerne det N-terminale methionin, som kodes opstrøms den N-terminale ende af den modne, native 20 sekvens (hvilket N-terminale methionin bevares i proteinet, med mindre det fjernes ved posttranslationel proces-sering), fra disse konstruktioner med deletioner i den N-terminale ende. Endvidere findes der en væsentlig heterogenitet (som kan opløses ved RP-HPLC-analyse af den redu-25 cerede monomer), når det gen, som koder for den "native" N-terminale sekvens (f.eks. af den korte form, muteinet SCSF/CV150) udtrykkes. Denne heterogenitet elimineres, når det tilsvarende CSF-l-gel, som mangler de to N-terminale glutaminsyrecodoner, udtrykkes. Tilsvarende kan 30 der også anvendes N-terminalt trunkerede former af andre korte og lange CSF-l-genkonstruktioner.As immediately described above, the gene encoding the long form of CSF-1 may be expressed in its entirety or the gene may be truncated for expression of forms where the C-terminal end is removed. Furthermore, the two or three first N-terminal codons can be removed so that the resulting protein is more homogeneous. In particular, it has been found easier to remove the N-terminal methionine encoded upstream of the N-terminal end of the mature native sequence (which N-terminal methionine is retained in the protein unless removed by post-translational processing). from these constructions with deletions at the N-terminal end. Furthermore, there is a substantial heterogeneity (which can be resolved by RP-HPLC analysis of the reduced monomer) when the gene encoding the "native" N-terminal sequence (e.g., of the short form, the mutein SCSF / CV150) is expressed. This heterogeneity is eliminated when the corresponding CSF-1 gel, which lacks the two N-terminal glutamic acid codons, is expressed. Similarly, N-terminally truncated forms of other short and long CSF-1 gene constructs can also be used.

Af bekvemmelighedsgrunde vil den primære struktur af monomere proteiner, som kodes af de forskellige, beskrevne 35 cDNA-konstruktioner, heri få følgende korte betegnelse: LCSF betegner den 522-aminosyre-lange sekvens, der er om- ........... .... _ ---------- DK 173693 B1 16 talt i forbindelse med klonen pcCSF-4, anført i den ovenfor refererede artikel af Ladner et al., The EMBO. J.For convenience, the primary structure of monomeric proteins encoded by the various 35 cDNA constructs described herein will be briefly referred to herein: LCSF represents the 522-amino acid long sequence which is about ........ ... .... _ ---------- DK 173693 B1 16 referred to in the clone pcCSF-4, cited in the above referenced article by Ladner et al., The EMBO. J.

(1987) 6(9):2693-2698, og som er vist i figur 6. SCSF betegner den 224-aminosyre-lange sekvens, som er omtalt i 5 forbindelse med klonen pcCSF-17, der er vist i figur 5, og som er beskrevet i den ovenfor omtalte artikel af Kawasaki, Science (1985) 230:292-296, som også er medtaget her ved henvisning. Det skal bemærkes, at den bestemte pcCSF-17-klon har en tyrosinrest i position 59, hvorimod(1987) 6 (9): 2693-2698 and shown in Figure 6. SCSF represents the 224-amino acid long sequence referred to in connection with the clone pcCSF-17 shown in Figure 5, and which is described in the above article by Kawasaki, Science (1985) 230: 292-296, which is also incorporated herein by reference. It should be noted that the particular pcCSF-17 clone has a tyrosine residue at position 59, whereas

10 det gen, som er defineret af den genomiske klon, har vist sig at kode for asparginsyre i denne position. ASP59SCSFThe gene defined by the genomic clone has been found to encode aspartic acid in this position. ASP59SCSF

betegner derfor en mutein af den omtalte korte form med denne modifikation. (Den omtalte LCSF-klon koder for Asp i position 59). Muteiner, som svarer til aminosyresubsti-15 tutioner i de afbillede "native'' sekvenser betegnes tilsvarende med substitutionen efterfulgt af positionen. Mr-teinformer af CSF-1 er omtalt i europæisk patentansøgning nr. 87309409.8, indleveret 23. oktober 1987, og medtaget her ved denne henvisning. Når konstruktioner, som forme -20 des at kode for disse proteiner, udtrykkes som modne proteiner i bakterier, kan de også bevare et N-terminalt methionin. Eftersom tilstedeværelsen eller fraværet s.i det N-terminale methionin ikke kan forudsiges, er denr)e mulighed ikke inkluderet i notationen.therefore denotes a mutein of the said short form with this modification. (The LCSF clone referred to encodes Asp at position 59). Muteins corresponding to amino acid substitutions in the "native" sequences depicted are correspondingly designated by the substitution followed by the position. MRF informants of CSF-1 are disclosed in European Patent Application No. 87309409.8, filed October 23, 1987, and incorporated herein. When constructs that are -20 to encode these proteins are expressed as mature proteins in bacteria, they may also retain an N-terminal methionine. Since the presence or absence of the N-terminal methionine cannot be predicted, the option is not included in the notation.

25 C-terminale og N-terminale trunkeringer af disse basale SCSF- og LCSF-sekvenser vil blive betegnet som CV henholdsvis NV. De C-terminale deletioner vil blive efterfulgt af det tal, der repræsenterer det resterende antal 30 aminosyrer i den native struktur; for de N-terminale deletioner vil NV blive efterfulgt af det antal aminosyrer, som er fjernet fra den N-terminale ende. Således betegner f.eks. LCSF/CV150 en konstruktion, der koder for et protein, som indeholder de første 150 aminosyrer af 35 den lange CSF-sekvens; SCSF/CV158 betegner en konstrul:-tion, der koder for et protein, som indeholder de første j DK 173693 B1 17 158 aminosyrerester af den korte form; SCSF/NV2 betegner en konstruktion, som koder for den korte form, hvor to N-terminale aminosyrer er fjernet. (Som ovenfor anført divergerer LCSF og SCSF begyndende ved position 150 og fø-5 res tilbage nær den C-terminale ende). LCSF/NV2CV150 betegner en form, der er den samme som LCSF/CV150 med undtagelse af, at de to N-terminale glutaminsyrerester er fjernet.25 C-terminal and N-terminal truncations of these basal SCSF and LCSF sequences will be designated as CV and NV, respectively. The C-terminal deletions will be followed by the number representing the remaining 30 amino acids in the native structure; for the N-terminal deletions, NV will be followed by the number of amino acids removed from the N-terminal end. Thus, e.g. LCSF / CV150 a construct encoding a protein containing the first 150 amino acids of the long CSF sequence; SCSF / CV158 denotes a construct: -tion encoding a protein containing the first short form amino acid residues; SCSF / NV2 denotes a structure which encodes the short form in which two N-terminal amino acids are removed. (As stated above, LCSF and SCSF diverge starting at position 150 and being fed back near the C-terminal end). The LCSF / NV2CV150 represents a form that is the same as the LCSF / CV150 except that the two N-terminal glutamic acid residues are removed.

10 Specielt foretrukne konstruktioner, der resulterede i CSF-1-proteiner, som udsættes for fremgangsmåden ifølge den foreliggende opfindelse, inkluderer gener, der koder for LCSF/CV150, LCSF/CV190, LCSF/CV221, LCSF/CV223, LCSF og de tilsvarende NV2-, NV3-, tyr59-, ser157-, ser^g- 15 og ser^57ser25g-forroer. Ligeledes foretrukne er SCSF/CV158, SCSF/CV150, SCSF og de tilsvarende NV2-, NV3- og asp59~former.Particularly preferred constructs that resulted in CSF-1 proteins subjected to the method of the present invention include genes encoding LCSF / CV150, LCSF / CV190, LCSF / CV221, LCSF / CV223, LCSF and the corresponding NV2 -, NV3-, tyr59-, ser157-, ser ^ g- 15 and ser ^ 57ser25g windshields. Also preferred are SCSF / CV158, SCSF / CV150, SCSF and the corresponding NV2, NV3 and asp59 forms.

Specielt foretrukne udgangsmaterialer omfatter produkter-20 ne fra de gener, som koder for SCSF/NV3CV150, LCSF/NV3CV221, ser157LCSF/NV3CV221, ser159LCSF/NV3CV221 og ser157ser^59LCSF/CV221.Particularly preferred starting materials include the products of the genes encoding SCSF / NV3CV150, LCSF / NV3CV221, ser157LCSF / NV3CV221, ser159LCSF / NV3CV221 and ser157ser559LCSF / CV221.

På grund af de til ekspressionen anvendte forskellige 25 værtssystemers processering kan de fremkomne proteiner bevare eller ikke bevare den længde, som er foreskrevet af genet. Selv om udgangsmaterialeproteinerne til genfoldning omtales med den samme betegnelse, skal det derfor forstås, at disse betegnelser i realiteten refererer 30 til genkonstruktionen, og den faktiske længde af udgangsmaterialet for den foreliggende fremgangsmåde kan være kortere eller længere (hvis det har N-terminal Met) end det, som er specificeret af det C-terminale aminosyretal.Due to the processing of various host systems used for expression, the resulting proteins may or may not retain the length prescribed by the gene. Therefore, although the starting material proteins for refolding are referred to by the same designation, it should be understood that these designations in fact refer to the gene structure and the actual length of the starting material for the present process may be shorter or longer (if it has N-terminal Met) than that specified by the C-terminal amino acid number.

DK 173693 B1 18 C. Generel procedure;DK 173693 B1 18 C. General procedure;

Udgangsmaterialet for fremgangsmåden ifølge den forelig-5 gende opfindelse er CSF-l-proteinf der er frembragt f::a det CSF-l-kodende DNA, som er transformeret ind i en ba'c-terievært. CSF-l-genet kan udtrykkes som et modent protein ved at anvende det hensigtsmæssige CSF-l-kodende DNA, som har en umiddelbart foranstående Met-kodende AT<3-10 codon, eller som er et fusionsprotein, hvor CSF--L-sekvensen er anbragt i læseramme med en proteinkodende sekvens eller i en secerneret form ved at anvende en sig-nalsekvens, der kan fungere i den udvalgte vært. Hvis konstruktionen koder for den "modne" form af proteine:, 15 kan det N-terminale methionin være fuldstændigt process ϊ-ret, slet ikke eller delvist. Methionin er selvfølgelig ikke til stede i den N-terminale ende af secernerede former udtrykt fra gener med funktionsdygtigt koblede signalsekvenser. Signalsekvenser hidrører almindeligvis fra 20 bakteriesystemer, såsom penicillinase eller phosphatase A. Hvis den secernerede form anvendes, hvad enten secernering er lykkedes eller ej, produceres proteinet almindeligvis i en form, som er mere opløselig end den, der opnås, når det produceres som et modent protein eller et 25 fusionsprotein. Denne generalisering er ikke uden undtagelser.The starting material for the method of the present invention is the CSF-1 protein produced from the CSF-1 coding DNA transformed into a bacterial host. The CSF-1 gene can be expressed as a mature protein by using the appropriate CSF-1 coding DNA, which has an immediately preceding Met-coding AT <3-10 codon, or which is a fusion protein wherein CSF - L the sequence is arranged in reading frame with a protein coding sequence or in a secreted form using a signal sequence that can function in the selected host. If the construct encodes the "mature" form of protein: 15, the N-terminal methionine may be completely process-free, not at all or in part. Of course, methionine is not present at the N-terminal end of secreted forms expressed from genes with functionally coupled signal sequences. Signal sequences are generally derived from 20 bacterial systems, such as penicillinase or phosphatase A. If the secreted form is used, whether or not secretion is successful, the protein is usually produced in a form that is more soluble than that obtained when produced as a mature one. protein or a fusion protein. This generalization is not without exception.

Hvis det secernerede protein allerede er opløseligt, kan det chaotropiske miljø ikke desto mindre være nødvendigt 30 til udførelse af genfoldningsproceduren. Hvis proteinet dannes i en uopløselig form, er initiel opløseliggørelse nødvendig.Nevertheless, if the secreted protein is soluble, the chaotropic environment may be necessary to perform the refolding procedure. If the protein is formed in an insoluble form, initial solubilization is necessary.

Fremgangsmåden begynder derfor almindeligvis med den op-35 løseliggjorte monomer i et chaotropisk miljø, som bevares under reducerende betingelser. En sådan bevaring kan ih- DK 173693 B1 19 volvere anvendelsen af et egnet reducerende middel, såsom β-mercaptoethanol eller dithiothreitol (DTT), men CSF-l'en kan allerede være reduceret, og udelukkelsen af oxiderende midler kan være tilstrækkeligt. Det opløselig-5 gjorte protein holdes f.eks. typisk i 8 M urinstof eller 7 M guanidinhydrochlorid ved en pH-værdi på ca. 7-8,6 i nærværelse af ca. 2-100 mM thioforbindelse. Med udgangspunkt i denne opløseliggjorte form kan monomeren enten genfoldes direkte eller delvis renset fra de resterende 10 proteiner ved en egnet rensningsprocedure, såsom kromatografi på en adsorberende gel, kromatografi under anvendelse af en ionbytningssøjle eller gelgennemtrængningskromatografi forud for genfoldning. Anvendelse af et rensningstrin forud for genfoldning har den fordel, at 15 der fjernes kontaminerende værtsproteiner og materialer, som kan nedbryde eller ændre CSF-1. Gelgennemtrængningskromatografi er nyttig, eftersom den muliggør en let størrelsesseparation af den ønskede monomerlængde, som almindeligvis kendes i forvejen, fra urenheder med for-20 skellige molekylevægte. Kapaciteten af gelgennemtrængningssøjlerne bliver begrænsende, når materialevoluminet stiger. For større voluminer foretrækkes ionbytningskromatografi, f.eks. DEAE-kromatografi. Det er nødvendigt, at rensningen udføres under reducerende betingelser 25 for at forhindre dannelsen af disulfidforbundne aggregater. Uanset den anvendte kromatografiske procedure inkluderes derfor et egnet reducerende middel i de opløsninger, som anvendes til at lade de kromatografiske søjler eller til portioner, og i elueringsopløsningerne. I nogle 30 tilfælde kan det reducerende middel erstattes med lav pH, såsom pH 6, eftersom lav pH stort set vil forhindre dannelse af disulfidbindinger i nogle kromatografiske systemer selv i fravær af reducerende middel.Therefore, the process generally begins with the solubilized monomer in a chaotropic environment which is preserved under reducing conditions. Such preservation may permit the use of a suitable reducing agent, such as β-mercaptoethanol or dithiothreitol (DTT), but the CSF-1 may already be reduced and the exclusion of oxidants may be sufficient. The solubilized protein is maintained e.g. typically in 8 M urea or 7 M guanidine hydrochloride at a pH of approx. 7-8.6 in the presence of approx. 2-100 mM thio compound. Starting from this solubilized form, the monomer can be either directly refolded or partially purified from the remaining 10 proteins by a suitable purification procedure such as chromatography on an adsorbent gel, chromatography using an ion exchange column or gel permeation chromatography prior to refolding. Applying a purification step prior to refolding has the advantage of removing contaminating host proteins and materials that can degrade or alter CSF-1. Gel permeation chromatography is useful as it enables easy size separation of the desired monomer length, which is generally known in advance, from impurities of different molecular weights. The capacity of the gel penetration columns becomes limiting as the volume of material increases. For larger volumes, ion exchange chromatography is preferred, e.g. DEAE chromatography. It is necessary that the purification be performed under reducing conditions 25 to prevent the formation of disulfide bonded aggregates. Therefore, regardless of the chromatographic procedure used, a suitable reducing agent is included in the solutions used to charge the chromatographic columns or for portions and in the elution solutions. In some 30 cases, the reducing agent can be replaced by low pH, such as pH 6, since low pH will largely prevent formation of disulfide bonds in some chromatographic systems even in the absence of reducing agent.

35 Den delvis rensede monomer udsættes derefter for genfoldningsbetingelser til dannelsen af dimeren. Proteinkoncen- DK 173693 B1 20 trationen under dette trin er væsentlig. De endelige ui-bytteprocenter af diiner pr. volumen genfoldningsreaktiυη stiger, hvis proteinkoncentrationen er mindre end ca. 2 mg/ml CSF-l-protein; der foretrækkes et koncentrationson-5 råde på 0,03-0,5 mg/ml. Anvendelsen af proteinkoncentrationer, som er for høje, kan resultere i dannelsen af uønskede oligomerer af højere orden. Genfoldningsbetii-gelserne kan inkludere gradvis fjernelse af det chaotro-piske miljø i løbet af en passende tidsperiode {sædvan-10 ligvis adskillige timer) eller fortynding af prøven til den ønskede koncentration af protein og chaotropisk middel. Der kan ligeledes anvendes metoder, som giver en konstant proteinkoncentration, såsom dialyse eller hulfi-berdiafiltrering, medens det chaotropiske middel langsomt 15 fjernes. Ved slutningen af processen, når det chaotropiske miljø er udtømt, opnås et ikke-denaturerende niveau. Hvis f.eks. guanidinhydrochlorid anvendes som det chaotropiske middel, opnås en slutkoncentration på mindre end ca. 2 M, og hvis urinstof anvendes som det chaotro-20 piske middel, opnås en slutkoncentration på mindre epd ca. 1 M og fortrinsvis 0,1-0,5 M.The partially purified monomer is then subjected to refolding conditions to form the dimer. The protein concentration during this step is significant. The final onion exchange rates of diines per the volume of refolding reaction increases if the protein concentration is less than ca. 2 mg / ml CSF-1 protein; a concentration range of 0.03-0.5 mg / ml is preferred. The use of protein concentrations which are too high may result in the formation of higher order unwanted oligomers. The refolding conditions may include gradual removal of the chaotropic environment over an appropriate period of time (usually several hours) or dilution of the sample to the desired concentration of protein and chaotropic agent. Methods can also be used which provide a constant protein concentration such as dialysis or hollow fiber filtration while slowly removing the chaotropic agent. At the end of the process, when the chaotropic environment is depleted, a non-denaturing level is achieved. For example, guanidine hydrochloride is used as the chaotropic agent, a final concentration of less than ca. 2 M, and if urea is used as the chaotropic agent, a final concentration of less epd is obtained approx. 1 M and preferably 0.1-0.5 M.

Genfoldningen under fjernelse af det chaotropiske miljø udføres på en sådan måde, at oxidation af sulfhydrylgruo-25 perne til disulfider er mulig til tilvejebringelse af den fremkomne, biologisk aktive, dimere konfiguration, som i tilfældet med CSF-1 stabiliseres ved dannelsen af disulfider, hvoraf en eller flere kan sammenkoble de to kæder.The refolding during removal of the chaotropic environment is carried out in such a way that oxidation of the sulfhydryl groups to disulfides is possible to provide the resulting biologically active dimeric configuration, which in the case of CSF-1 is stabilized by the formation of disulfides, of which one or more can link the two chains.

Der kan også dannes intrakædedisulfider. Egnede redox-30 betingelser, som fremmer denne dimerdannelse, inkluderer sulfhydryl/disulfidreagenskombinationerne, såsom oxideret og reduceret glutathion. Forholdet mellem reduceret og oxideret glutathion eller en anden sulf-hydryl/disulfidkombination ligger typisk på fra ca. 2 35 mM/0,1 mM til 0,5 mM/1,0 mM. Der kan også accepteres al ternative metoder til tilvejebringelse af denne oxidati- DK 173693 B1 21 on. For eksempel virker simpel fjernelse eller fortynding af det reducerende middel uden forholdsregler til udelukkelse af luft og metalioner på dannelsen af ønskede disul fidbindinger. I alle tilfælde bør opløsningens pH-5 værdi under genfoldningsprocessen holdes ved ca. pH 7,5- 9,0. Det er klart, at i genfoldningsprocessen anvendes der ikke længere de meget reducerende betingelser, under hvilke den initielle rensning blev udført. En minimering af koncentrationen af salte, såsom natriumchlorid, under 10 genfoldningsprocessen muliggør anvendelse af ionbytnings-kromatografi som et efterfølgende koncentrerings-og/eller rensningstrin.Intra-chain disulfides can also be formed. Suitable redox conditions which promote this dimer formation include the sulfhydryl / disulfide reagent combinations, such as oxidized and reduced glutathione. The ratio of reduced to oxidized glutathione or another sulfhydryl / disulfide combination typically ranges from approx. 2 35 mM / 0.1 mM to 0.5 mM / 1.0 mM. Alternative methods for providing this oxidation may also be accepted. For example, simple removal or dilution of the reducing agent without precautions to exclude air and metal ions acts on the formation of desired disul fid bonds. In all cases, the pH of the solution during the refolding process should be maintained at approx. pH 7.5 - 9.0. It is clear that in the refolding process, the very reducing conditions under which the initial purification was carried out are no longer used. Minimizing the concentration of salts, such as sodium chloride, during the refolding process allows the use of ion exchange chromatography as a subsequent concentration and / or purification step.

Under genfoldningsprocessen kan der dannes adskillige di-15 mere og højere oligomere former af CSF-1, herunder former, som har en nedsat opløselighed i høj salt, og oligo-merer af højere orden, som kan adskillles ved størrelses-udelukkelseskroraatografi. Denne aggregeringsproces gøres mindst mulig ved temperaturstyring, hvor lave tempera-20 turer på ca. 0-4°C foretrækkes fremfor højere temperaturer på 25-37°C.During the refolding process, several di-15 and higher oligomeric forms of CSF-1 can be formed, including forms having a low salt solubility, and higher-order oligomers which can be separated by size exclusion chromatography. This aggregation process is made least possible by temperature control, where low temperatures of approx. 0-4 ° C is preferred over higher temperatures of 25-37 ° C.

Under genfoldningsprocessen kan der også dannes mindre stabile, dimere former af CSF-1, som under visse betin-25 gelser kan opløses som en tidlig elueringstop ved HPLC med omvendt fase. Disse mindre stabile former kan hidrøre fra dannelsen af uønskede disulfidbindinger. Cystein-resterne i positionerne 157 og 159, der er til stede i den lange form af CSF-1, er ikke nødvendige for biologisk 30 aktivitet. DNA-konstruktioner, der koder for CSF-1, som indeholder serinsubstitutioner for en eller begge disse cysteiner, giver højere udbytter ved den foreliggende rensningsmetode og kan også ændre op-løselighedsegenskaberne på en ønsket måde.During the refolding process, less stable, dimeric forms of CSF-1 can also be formed, which under certain conditions can be resolved as an early elution peak by reverse phase HPLC. These less stable forms may result from the formation of undesirable disulfide bonds. The cysteine residues at positions 157 and 159 present in the long form of CSF-1 are not required for biological activity. DNA constructs encoding CSF-1, which contain serine substitutions for one or both of these cysteines, provide higher yields by the present purification method and may also alter the solubility properties in a desired manner.

35 DK 173693 B1 2235 DK 173693 B1 22

Hvis rester af redoxreagenser er til stede i genfoldet CSF-1, kan det skabe problemer under efterfølgende rensningstrin. Der er mange måde, hvorpå man kan blokere e.L-ler forhindre de disulfidudskiftninger, som kunne ske i 5 nærværelse af sådanne resterende redoxreagenser (f.eks. glutathion), herunder fjernelse ved f.eks. diafiltrering eller dialyse, fortynding og en passende sænkning af opløsningens pH-værdi. Blandt ovennævnte fremgangsmåder sr de to mere foretrukne fremgangsmåder sænkning af pi-10 værdien til under 7,0 og diafiltrering.If residues of redox reagents are present in refolded CSF-1, it can create problems during subsequent purification steps. There are many ways in which e-clays can be blocked to prevent the disulfide replacements that could occur in the presence of such residual redox reagents (e.g., glutathione), including removal by e.g. diafiltration or dialysis, dilution and an appropriate lowering of the pH of the solution. Among the above methods, the two more preferred methods lower the pi-10 to below 7.0 and diafiltrate.

Efter at genfoldnings-, koncentrerings- og/eller de ini.-tielle rensningstrin er fuldført, renses diraeren yderligere fra resterende redoxmateriale og fra andre proteiner 15 under anvendelse af procedurer, som ligner de procedurer, der er anført ovenfor for monomeren. Det er selvfølgelig ikke nødvendigt at* vælge den samme rensningsprocedure; det kan faktisk være foretrukket at anvende en anden fremgangsmåde end den, der blev anvendt til rensning af 20 opløseliggjort monomer. Specielt egnede måder omfatter gelfiltrering, kromatografi med hydrofob vekselvirknng, ionbytningskromatografi og HPLC med omvendt fase.After the refolding, concentration, and / or initial purification steps are completed, the conductor is further purified from residual redox material and from other proteins using procedures similar to the procedures set forth above for the monomer. Of course, it is not necessary to * choose the same cleaning procedure; in fact, it may be preferable to use a method other than that used to purify 20 solubilized monomer. Particularly suitable methods include gel filtration, hydrophobic interaction chromatography, ion exchange chromatography and reverse phase HPLC.

Før yderligere rensning af den genfoldede, dimere CSF-1 25 kan fjernelse af redoxmaterialet, hvis det er til stede, og koncentrering af de genfoldede proteiner f.eks. udføres ved at sætte det genfoldede materiale direkte på en ionbytningskromatografisøj le under anvendelse af f.eks. DEAE-Sepharose. Sådanne procedurer udføres ofte ved pH-30 værdier på omkring 8, men det viste sig imidlertid, at sænkning af pH-værdien til området fra 5,5 til 7,0 nedsatte oligomerdannelse og forøgede udbyttet af dimer CSF-1.Prior to further purification of the refolded dimeric CSF-1, removal of the redox material, if present, and concentration of the refolded proteins, e.g. performed by placing the refolded material directly on an ion exchange chromatography column using e.g. DEAE-Sepharose. Such procedures are often performed at pH-30 values of about 8, but it was found that lowering the pH to the range of 5.5 to 7.0 decreased oligomer formation and increased the yield of dimer CSF-1.

35 Rensningen af dimeren er nødvendig for at fjerne urenheder, navnlig pyrogener og andre endotoxiner, som hidrører DK 173693 B1 23 fra den bakterielle frembringelse af proteinet. Ved en specielt vellykket protokol til fjernelse af disse uønskede urenheder anvendes kromatografi på en phenyl-TSK-eller phenyl-Sepharose-søjle. Kromatografien udføres un-5 der betingelser og med reagenser, som er endotoxinfrie.The purification of the dimer is necessary to remove impurities, in particular pyrogens and other endotoxins resulting from the bacterial production of the protein. In a particularly successful protocol for removing these unwanted impurities, chromatography is applied to a phenyl-TSK or phenyl-Sepharose column. The chromatography is performed under conditions and with reagents which are endotoxin free.

Den ønskede, dimere CSF-1 er opløselig og stabil i ca.The desired dimer CSF-1 is soluble and stable for approx.

1,5 M ammoniumsulfat ved neutral pH og sættes på søjlerne under disse betingelser ved lave temperaturer på ca. 2-10°C og fortrinsvis ca. 4°C. Aggregater og ustabile for-10 mer af genfoldet CSF-1 fjernes tilsyneladende fra stabile, dimere former af genfoldet CSF-1 ved fjernelse af et præcipitat, som dannes ved tilsætningen af ammoniumsulfat. Det ønskede, dimere protein kan elueres under anvendelse af en gradient med faldende ammoniumsulfat og 15 med stigende ethylenglycol i neutral puffer. CSF-1-dimeren eluerer ved ca. 0,6 M ammoniumsulfat, 35% ethylenglycol fra phenyl-TSK-søjlen. Der kan også anvendes alternative bærematerialer, og phenyl-Sepharose kan være foretrukket til fremstilling af det rensede, dimere CSF-20 1-protein i større skala.1.5 M ammonium sulfate at neutral pH and put on the columns under these conditions at low temperatures of approx. 2-10 ° C and preferably approx. 4 ° C. Apparatus and unstable forms of refolded CSF-1 are apparently removed from stable, dimeric forms of refolded CSF-1 by removal of a precipitate formed by the addition of ammonium sulfate. The desired dimeric protein can be eluted using a gradient with decreasing ammonium sulfate and 15 with increasing ethylene glycol in neutral buffer. The CSF-1 dimer elutes at ca. 0.6 M ammonium sulfate, 35% ethylene glycol from the phenyl-TSK column. Alternative carriers may also be used, and phenyl-Sepharose may be preferred for the preparation of the purified, dimeric CSF-20 1 protein on a larger scale.

Den fremkomne dimer er af klinisk renhed. Den specifikke aktivitet af sådanne præparationer er ca. ækvivalent med den specifikke aktivitet af nativ human-CSF-1, som er 25 frembragt af pattedyrceller. I situationer, hvor CSF-1-udgangsmaterialet er af lavere renhed, eller hvor man ønsker en højere slutrenhed, kan der anvendes et yderligere rensningstrin {såsom DEAE-kromatografi efter genfoldning) .The resulting dimer is of clinical purity. The specific activity of such preparations is approx. equivalent to the specific activity of native human CSF-1 produced by mammalian cells. In situations where the CSF-1 starting material is of lower purity or where a higher final purity is desired, an additional purification step (such as DEAE chromatography after refolding) can be used.

30 I de udførelsesformer, som inkluderer det yderligere, indledende trin med opløseliggørelse af den monomere form af proteinet, opnås udgangsmaterialerne som et uopløseligt, intracellulært protein, der kan adskilles fra oplø-35 selige bakterieproteiner ved lysering af cellerne under egnede betingelser og genfinding af det uopløselige pro- DK 173693 B1 24 tein ved centrifugering. Det genfundne, uopløselige protein anbringes derefter direkte i et chaotropisk miljø til adskillelse af aggregater og fremkaldelse af oplø£-ning/denaturering.In the embodiments which include the further initial step of solubilizing the monomeric form of the protein, the starting materials are obtained as an insoluble, intracellular protein which can be separated from soluble bacterial proteins by lysing the cells under suitable conditions and recovering the cells. insoluble products by centrifugation. The recovered insoluble protein is then placed directly in a chaotropic environment to separate aggregates and induce dissolution / denaturation.

55

De genfundne, rensede, dimere former vises at være biologisk aktive ved at anvende en hvilken som helst af adskillige prolifereringsanalyser. En standardanalyse, s Dm opfylder de påkrævede kriterier, er in vitro kolonistima-10 leringsanalysen af Metcalf, D., J. Cell Physiol. (1970) 76:89. Tilstedeværelsen af CSF-1 i dette system resultsrer i dannelsen af hovedsagelig makrofagkolonier. En ariden analyse er stigningen i celleproliferering målt ved ^H-thymidininkorporering i en CSF-l-afhængig cellelinie, 15 såsom musemakrofaglinien BAC. I en anden form af denne analyse kan der anvendes et kolorimetrisk påvisningssystem baseret på reduktionen af tetrazoliumsaltet MTT. De ved fremgangsmåden ifølge opfindelsen opnåede CSF-1-dimerer er aktive i sådanne analyser og er stort set frie 20 for andre proteiner, som produceres af bakterierne.The recovered, purified, dimeric forms are shown to be biologically active using any of several proliferation assays. A standard assay, if Dm meets the required criteria, is the in vitro colonization assay of Metcalf, D., J. Cell Physiol. (1970) 76:89. The presence of CSF-1 in this system results in the formation of mainly macrophage colonies. An arid analysis is the increase in cell proliferation measured by 3 H-thymidine incorporation into a CSF-1 dependent cell line, such as the mouse macrophage line BAC. In another form of this analysis, a colorimetric detection system based on the reduction of the tetrazolium salt MTT can be used. The CSF-1 dimers obtained by the process of the invention are active in such assays and are substantially free of other proteins produced by the bacteria.

Det er vigtigt, at CSF-l-præparationerne er klinisk rene.It is important that the CSF-1 preparations are clinically pure.

De er stort set frie for endotoxin, idet de indeholder mindre end ca. 1,0 ng endotoxin/mg CSF-1 målt ved limulu-25 samebocytlysatstandardanalysen (LAL-standardanalysen), Associates of Cape Cod, Inc., Woods Hole, MA. Yderligere rensning kan være ønsket, men ved fremgangsmåden ifølge opfindelsen opnås præparationer med en CSF-1- proteinrenhed på ca. 95% eller mere, vurderet ved SES-30 PAGE. Endvidere er den specifikke aktivitet omtrent ækvivalent med eller højere end den specifikke aktivitet af det native protein.They are virtually free of endotoxin in that they contain less than approx. 1.0 ng of endotoxin / mg CSF-1 as measured by the limulu-25 co-lysate lysate standard assay (LAL standard assay), Associates of Cape Cod, Inc., Woods Hole, MA. Further purification may be desired, but in the process of the invention, preparations with a CSF-1 protein purity of approx. 95% or more, assessed by SES-30 PAGE. Furthermore, the specific activity is approximately equivalent to or higher than the specific activity of the native protein.

DK 173693 B1 25 D. Farmaceutiske sammensætninger:DK 173693 B1 25 D. Pharmaceutical compositions:

De genfoldede og klinisk rene CSF-l-præparationer kan 5 derefter ved traditionelle protokoller og måder formuleres til indgivelse, fortrinsvis systemisk, herunder intravenøs indgivelse. Sammensætningerne kan omfatte traditionelle excipienser, såsom vand til injektion, puffere, opløsningsmidler og stabiliseringsmidler, hvilket er 10 kendt inden for fagområdet. En oversigt over formuleringsmetoder til farmaceutiske sammensætninger, som inkluderer protein, findes f.eks. i Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, sidste udgave.The refolded and clinically pure CSF-1 preparations can then be formulated by conventional protocols and methods for administration, preferably systemically, including intravenous administration. The compositions may comprise conventional excipients such as water for injection, buffers, solvents and stabilizers, which are known in the art. An overview of formulation methods for pharmaceutical compositions which include protein is found, e.g. in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, last edition.

15 E. HeterodimerdannelseE. Heterodimer Formation

Det skal bemærkes, at fremgangsmåden ifølge opfindelsen muliggør dannelsen af heterodimerer fra forskellige mono-20 mere enheder af CSF-1. Det store antal CSF-l-proteiner, som dannes ved variationer i C-terminal processering, tilvejebringer f.eks. en række udgangsmaterialer, som kan anvendes ved dimerdannelsen. Der kan således let dannes nye, heterodimere materialer. For eksempel kan den mono-25 mere form af SCSF/CV150 blandes med den monomere form af LCSF/C0190 og behandles ved fremgangsmåden ifølge opfindelsen; heterodimeren kan derefter adskilles fra homodi-merbiprodukterne ved forskellige kromatografiske metoder. Lignende blandinger, der behandles ved fremgangsmådenIt should be noted that the process of the invention enables the formation of heterodimers from different monomer units of CSF-1. For example, the large number of CSF-1 proteins produced by variations in C-terminal processing provides e.g. a variety of starting materials which can be used in the dimer formation. Thus, new heterodimeric materials can easily be formed. For example, the monomeric form of SCSF / CV150 can be mixed with the monomeric form of LCSF / C0190 and treated by the process of the invention; the heterodimer can then be separated from the homodimer by-products by various chromatographic methods. Similar mixtures treated by the process

30 ifølge opfindelsen, fører til heterodimerer af komponenter med aminosyresubstitutioner, f.eks. glU52 LCSF30 of the invention leads to heterodimers of components with amino acid substitutions, e.g. glU52 LCSF

og LCSF/CV190.and LCSF / CV190.

De forskellige monomerer kan blandes in vitro eller frem-35 bringes i den samme celle. Hvis de frembringes i den samme celle, indføres en konstruktion til ekspression af DK 173693 B1 26 hver monomer i den samme vært/ ved sådanne udførelsesfor-mer foretrækkes det, at hver konstruktion bærer en forskellig markør (såsom TcR og AmpR), således at cotrans-formerede værter selekteres. De cotransformerede celler 5 dyrkes derefter og induceres til opnåelse af blandinger af de to former.The various monomers can be mixed in vitro or produced in the same cell. If produced in the same cell, a construct for expression of each monomer is introduced into the same host / in such embodiments it is preferred that each construct carries a different marker (such as TcR and AmpR) so that cotrans -formed hosts are selected. The cotransformed cells are then grown and induced to obtain mixtures of the two forms.

Eksempler 10 De efterfølgende eksempler har til hensigt at illustrere opfindelsen uden at begrænse den.Examples 10 The following examples are intended to illustrate the invention without limiting it.

Eksempel 1 15 Dette eksempel beskriver udvindingen af et renset, biologisk aktivt protein, der udtrykkes fra en konstruktion, som koder for asp5gSCSF/CVl50 i E. coli under styring af PL-promotoren i en vektor, der er konstrueret som beskrevet i europæisk patentansøgning nr. 87309409.8, indlevo-20 ret 23. oktober 1987, overdraget til samme assignator og medtaget her ved denne henvisning. Proteinet frembringes intracellulært i en uopløselig, monomer form.Example 1 15 This example describes the recovery of a purified biologically active protein expressed from a construct encoding Asp5SSCSF / CV150 in E. coli under the control of the PL promoter in a vector constructed as described in European Patent Application no. 87309409.8, filed October 23, 1987, assigned to the same assignor and incorporated herein by reference. The protein is produced intracellularly in an insoluble monomeric form.

En E. coli λ lysogen, DG116, transformeret med det over-25 udtrykte (O/E) plasmid pPLSCSFasp59/CVl50, CMCC- cellelinie deponerings nr. 2948, blev dyrket i en 10 1:.- ter fermentor i basalt medium indeholdende 72 mM (NH4)2S04, 20 mM KH2P04, 2,0 ml/1 TK9 med sterile til sætninger af 10 g/1 glucose, 3,0 mM MgS04 · 7H20, 72 μΜ 30 FeS04, 20 mg/1 thiamin · HCl og 50 mg/1 ampicillin. !An E. coli λ lysogen, DG116, transformed with the expressed (O / E) plasmid pPLSCSFasp59 / CV150, CMCC cell line deposition # 2948, was grown in a 10 1: 5 fermentor in basal medium containing 72 mM (NH4) 2SO4, 20mM KH2PO4, 2.0 ml / 1 TK9 with sterile for 10 g / l glucose, 3.0 mM MgSO4 · 7H20, 72 μΜ 30 FeSO4, 20 mg / l thiamine · HCl mg / l ampicillin. !

Cellerne blev dyrket ved 30°C til OD680nin = 12; der blev tilsat casaminosyrer til 2%, og derefter blev CSF-·:.- ekspression induceret ved skift til 42°C. Cellerne blev 35 dyrket i endnu 3 timer til en OD680nin-slutværdi på 16,5, DK 173693 B1 27The cells were grown at 30 ° C to OD 680nin = 12; casamino acids were added to 2%, and then CSF expression was induced by changing to 42 ° C. The cells were cultured for another 3 hours to an OD680nin final value of 16.5, DK 173693 B1 27

Cellerne blev høstet ved centrifugering og homogeniseret under anvendelse af 30 min. lydbehandling ved 4°C. Homo-genatet blev derefter centrifugeret, og celleresterne 5 blev bevaret. Resterne indeholdt det uopløselige protein, som blev resuspenderet i 30% saccharose og centrifugeret ved 15.000 x g i 10 minutter ved 4°C til berigelse for det uopløselige protein.The cells were harvested by centrifugation and homogenized using 30 min. sound treatment at 4 ° C. The homogenate was then centrifuged and cell debris 5 was preserved. The residues contained the insoluble protein, which was resuspended in 30% sucrose and centrifuged at 15,000 x g for 10 minutes at 4 ° C to enrich the insoluble protein.

10 Pelletten fra centrifugeringen blev opløst i 7 m guanidin • HC1 i 0,1 M natriumphosphat, pH 7, indeholdende 50 mM ’ DTT og 5 mM EDTA i 30 minutter. Suspensionen blev deref ter opvarmet til 40°C i 5 minutter, og supernatanten blev indvundet efter centrifugering. Den indvundne supernatant 15 blev sat på en 90 x 2,6 cm Sephacryl-søjle (S-200) ækvi-libreret i den samme puffer, men indeholdende 2 mM DTT i stedet for 50 mM. Søjlen blev kørt med den samme puffer, og proteinkoncentrationen blev overvåget ved absorption ved 280 nm med de i figur 1 viste resultater. Hovedparten 20 af bakterieproteinerne var separeret fra CSF-1, der blev indvundet som en 17 kd top, som ca. repræsenterede 80% ren CSF-1-monomer.The pellet from the centrifugation was dissolved in 7 m guanidine • HCl in 0.1 M sodium phosphate, pH 7 containing 50 mM 'DTT and 5 mM EDTA for 30 minutes. The suspension was then heated to 40 ° C for 5 minutes and the supernatant was recovered after centrifugation. The recovered supernatant 15 was placed on a 90 x 2.6 cm Sephacryl column (S-200) equilibrated in the same buffer but containing 2 mM DTT instead of 50 mM. The column was run with the same buffer and the protein concentration was monitored by absorption at 280 nm with the results shown in Figure 1. The majority of 20 bacterial proteins were separated from CSF-1, which was recovered as a 17 kd peak, which was ca. represented 80% pure CSF-1 monomer.

CSF-l-puljen blev derefter fortyndet til 0,25 mg/ml pro-25 tein i en tilsvarende puffer indeholdende 7 M guanidin-hydrochlorid, 50 mM Tris, pH 8,5 og 5 mM EDTA, som indeholdt et redoxsystem, der bestod af 2 mM reduceret glu-tathion (GSH) og 1 mM oxideret glutathion (GSSG). Til genfoldning af det delvis rensede CSF-1 blev puljen fra 30 S-200-søjlen dialyseret mod denne puffer (indeholdende 7 M guanidinhydrochlorid og GSH/GSSG), fik derefter lov til at folde ved langsom tilsætning af en opløsning af 50 mM Tris, pH 8,5, 5 mM EDTA og GSH/GSSG i 0,1 M NaCl til dialysebeholderen. Tilsætningen blev udført ved 4°C i løbet 35 af 48 timer, indtil guanidinslutkoncentrationen var ca.The CSF-1 pool was then diluted to 0.25 mg / ml protein in a corresponding buffer containing 7 M guanidine hydrochloride, 50 mM Tris, pH 8.5, and 5 mM EDTA containing a redox system comprising of 2 mM reduced glutathione (GSH) and 1 mM oxidized glutathione (GSSG). To refold the partially purified CSF-1, the pool from the 30 S-200 column was dialyzed against this buffer (containing 7 M guanidine hydrochloride and GSH / GSSG), then allowed to fold by slowly adding a solution of 50 mM Tris. pH 8.5, 5 mM EDTA and GSH / GSSG in 0.1 M NaCl to the dialysis vessel. The addition was carried out at 4 ° C over 35 of 48 hours until the guanidine concentration was ca.

0,2 M. Dialysatet indeholdt på dette tidspunkt dimer CSF- DK 173693 B1 28 1, som blev sat direkte på en Sepharose 12 molekylesorteringssøjle ækvilibreret i phosphatpufferet saltvand til yderligere rensning. Elueringen blev igen fulgt ved absorption ved 280 nm, Elueringsmønstret er vist i figur 2.0.2 M. The dialysate at this time contained dimer CSF-DK 173693 B1 28 1 which was placed directly on a Sepharose 12 molecule sorting column equilibrated in phosphate buffered saline for further purification. The elution was again followed by absorption at 280 nm. The elution pattern is shown in Figure 2.

5 Før udsættelse for genfoldningsbetingelser eluerede CSF-l'en som forventet for monomeren (figur 2a), men når proteinet blev udsat for genfoldningsbetingelser ved 0,3 mg/ml, som oven beskrevet (eller alternativt ved 0,1 mg/ml), viste resultaterne dannelsen af det dimerstore 10 materiale, hvilket er indiceret i figur 2b henholdsvis 2c.Prior to exposure to refolding conditions, the CSF-1 eluted as expected for the monomer (Figure 2a), but when the protein was subjected to refolding conditions at 0.3 mg / ml, as described above (or alternatively at 0.1 mg / ml), the results showed the formation of the dimer-sized material, which is indicated in Figure 2b and 2c, respectively.

Det dimere produkt kromatograferede som en enkelt top ved HPLC med omvendt fase, hvilket er vist i figur 3b. Mere 15 end 90% af det dimere produkt er en enkelt form ved HPIC med omvendt fase (se figur 3b) og viser tilfredsstillende stabilitet og fuld biologisk aktivitet. Med hensyn til andre proteiner er det ved SDS-PAGE-analyse under reducerende og ikke-reducerende betingelser vist, at CSF-1 er 20 over 95% ren (figur 7) . Resultatet for udgangsmaterialet i S-200-puljen før genfoldning, hvilket er vist i figur 3a, indicerer en monomerdominans (der eluerer som to hc-vedtoppe af CSF-1). Den enkelte dimertop, som er illustreret i figur 3b, viste sig imidlertid at bestå af to 25 hovedkomponenter efter genreducering til monomeren (figur 3c) og adskilt ved HPLC med omvendt fase.The dimeric product was chromatographed as a single peak by reverse phase HPLC, as shown in Figure 3b. More than 90% of the dimeric product is a single reverse phase HPIC form (see Figure 3b) and shows satisfactory stability and full biological activity. With respect to other proteins, SDS-PAGE analysis showed, under reducing and non-reducing conditions, that CSF-1 is 20 above 95% pure (Figure 7). The result of the starting material in the S-200 pool prior to refolding, as shown in Figure 3a, indicates a monomer dominance (eluting as two hc tops of CSF-1). However, the single dimer peak illustrated in Figure 3b was found to consist of two major components after gene reduction to the monomer (Figure 3c) and separated by reverse phase HPLC.

Proteinproduktet blev karakteriseret for opløselighed ved UV-synlig spektroskop!. Der blev optegnet spektre med 10 30 minutters intervaller efter fortynding af den oprensere dimerpulje i phosphatpufferet saltvand, hvilket er vist i figur 4. Som vist i panel A forblev spektret af slutproduktet konstant over en to-timers periode, hvilket indicerer, at det genfoldede protein var stabilt og opløse-35 ligt under fysiologiske betingelser. I modsætning hertil viste en lignende spektralanalyse af det monomere uc- DK 173693 B1 29 gangsmateriale, som vist i panel B i figur 4, med 90-sekunders intervaller, at proteinet var ustabilt og let dannede uopløselige, lysspredende aggregater.The protein product was characterized for solubility by UV-visible spectroscope. Spectra were recorded at 10 30 minute intervals after dilution of the purifier dimer pool in phosphate buffered saline, as shown in Figure 4. As shown in panel A, the spectrum of the final product remained constant over a two-hour period, indicating that the refolded protein was stable and soluble under physiological conditions. In contrast, a similar spectral analysis of the monomeric uc-material as shown in panel B of Figure 4, at 90-second intervals, showed that the protein was unstable and readily formed insoluble, light-scattering aggregates.

5 Det ovenfor fremstillede, rensede, dimere materiale blev analyseret i musebenmarvskolonianalysen i dobbeltbestemmelse sammen med en "kontrol", som bestod af renset, re-kombinant CSF opnået fra et gen med en lignende sekvens (SCSF) udtrykt som et aktivt secerneret molekyle med ca.The above purified dimeric material was assayed in the mouse bone marrow colony assay in duplicate, together with a "control" consisting of purified, recombinant CSF obtained from a gene of similar sequence (SCSF) expressed as an active secreted molecule of ca. .

10 158 aminosyrer i pattedyrcellelinien CV-1. Det genfoldede E. coli CSF-1 havde en specifik aktivitet i musebenmarvs-analysen (i enheder/mg) på 2-4 x 107 sammenlignet med ca.10 158 amino acids in the mammalian cell line CV-1. The refolded E. coli CSF-1 had a specific activity in the mouse bone marrow assay (in units / mg) of 2-4 x 10 7 compared to ca.

3 x 107 enheder/mg for CSF-1 opnået fra CV-l-celler. Det rensede, ikke-genfoldede udgangsmateriale havde en speci-15 fik aktivitet, som var ca. 1000 gange lavere. (Museben-marvsanalysen blev beskrevet af Moore, R. et al., J. Immunol. (1983) 131:2397 og af Prystowsky, M. et al., Am.3 x 107 units / mg for CSF-1 obtained from CV-1 cells. The purified non-refolded starting material had a specific activity which was approx. 1000 times lower. (The mouse bone marrow assay was described by Moore, R. et al., J. Immunol. (1983) 131: 2397, and by Prystowsky, M. et al., Am.

J. Pathol. (1984) 114:149. Human-CSF-1 viser ca. 10 gange højere aktivitet i en musebenmarvsanalyse sammenlignet 20 med aktiviteten i en human-benmarvsanalyse).J. Pathol. (1984) 114: 149. Human CSF-1 shows approx. 10 times higher activity in a mouse bone marrow assay compared to 20 activity in a human bone marrow assay).

Nativ CSF-1, som var oprenset fra MIA PaCa-celler, havde en specifik aktivitet på 4-8 x 107 enheder/mg i en musebenmarvsanalyse.Native CSF-1 purified from MIA PaCa cells had a specific activity of 4-8 x 10 7 units / mg in a mouse bone marrow assay.

2525

Det genfoldede E. coli-proteins spektrum ved cirkulær dichroisme (CD) var inden for forsøgsfejl stort set identisk med spektret for "naturligt foldet" CSF-1 fra CV-l-celler.The spectrum of the E. coli protein by circular dichroism (CD) protein was within experimental error substantially identical to the spectrum of "naturally folded" CSF-1 from CV-1 cells.

3030

Eksempel 2Example 2

Tyve g frosset E. coli DG116-pasta fra celler, som udtrykker en konstruktion, der koder for asp5gSCSF/CVl50 35 under styring af PL-promotoren, blev resuspenderet i 200 j---------- DK 173693 B1 30 ml 50 mM Tris, 10 mM EDTA {pH 8,5) og blev lydbehandlet ji 30 minutter i et isbad, 60% impuls, intensitet 9.Twenty g of frozen E. coli DG116 paste from cells expressing a construct encoding asp5gSCSF / CV150 under the control of the PL promoter was resuspended in 200 µl ---------- DK 173693 B1 30 ml of 50 mM Tris, 10 mM EDTA (pH 8.5) and was sonicated for 30 minutes in an ice bath, 60% pulse, intensity 9.

Celleresterne blev bevaret efter 10 minutters centrifuge-5 ring ved 15.000 x g. Celleresterne blev resuspenderet i 200 ml 30% saccharose (i 10 mM EDTA, pH 8,0) og blev lyd-behandlet i 3 minutter til opbrydning af klumper og frigørelse af uopløseligt protein. Suspensionen blev derefter centrifugeret i 15 minutter ved 15.000 x g, og pej-10 letten blev bevaret.The cell residues were retained after 10 minutes centrifugation at 15,000 x g. The cell residues were resuspended in 200 ml of 30% sucrose (in 10 mM EDTA, pH 8.0) and sonicated for 3 minutes to break up clumps and release insoluble protein. The suspension was then centrifuged for 15 minutes at 15,000 x g and the pellet light was preserved.

Det saccharoserensede, uopløselige protein blev derefter opløst i 15 ml gennem 0,45 μ filtreret 7 M guanidin · HOlThe sucrose-purified, insoluble protein was then dissolved in 15 ml through 0.45 µ filtered 7 M guanidine · HOl

(GuHCl), 0,1 M natriumphosphat, 5 mM EDTA, 50 mM DTT {pH(GuHCl), 0.1 M sodium phosphate, 5 mM EDTA, 50 mM DTT {pH

15 7,5-8,0) i ca. 15 minutter og blev derefter opvarmet t:.l ca. 37-40°C i 10 minutter for at sikre, at disulfidbindinger blev reduceret. Det opløseliggjorte materiale blev derefter centrifugeret i 10 minutter ved 15.000 x g.7.5-8.0) for approx. 15 minutes and then heated for about 1 hour. 37-40 ° C for 10 minutes to ensure that disulfide bonds were reduced. The solubilized material was then centrifuged for 10 minutes at 15,000 x g.

20 Seks til ti ml af den klarede, opløste CSF-1 blev sat på en 2,6 x 95 cm S-200-søjle ækvilibreret i filtreringsste riliseret S-200-puffer {7 M GuHCl, 0,1 M natriumphospha:, 5 mM EDTA, 2 mM DTT, pH 6,8) og adskilt efter størrelse natten over ved stuetemperatur med 1 ml/ minut. Proteinet 25 elueredes som en veladskilt top og indeholdt i sammenhældt tilstand 40-70 mg protein med ca. 1,2-1,5 mg/ml (40-60 ml) .20 Six to 10 ml of the clarified dissolved CSF-1 was loaded onto a 2.6 x 95 cm S-200 column equilibrated in filtration-controlled S-200 buffer {7 M GuHCl, 0.1 M sodium phospha: 5 mM EDTA, 2 mM DTT, pH 6.8) and separated by size overnight at room temperature at 1 ml / minute. Protein 25 was eluted as a well-separated peak and contained in a condensed state 40-70 mg protein with approx. 1.2-1.5 mg / ml (40-60 ml).

Proteinindholdet blev bestemt ved absorbans ved 280 nm 30 under antagelse af, at 1 A28O er lig med i mg/ml. Opløsningen blev derefter fortyndet til 0,1-0,15 mg/ml proti-in, 0,5-0,7 M GuHCl i pufferholdig 50 mM Tris (pH 8,5i, 100 mM NaCl, 5 mM EDTA, 2 mM reduceret glutathion (GSHi, lmM oxideret glutathion (GSSG) ved tilsætning af den hei-35 sigtsmæssige puffer til proteinopløsningen og henstand i 24 timer ved 4°C.The protein content was determined by absorbance at 280 nm 30 assuming 1A28O is equal to mg / ml. The solution was then diluted to 0.1-0.15 mg / ml protiin, 0.5-0.7 M GuHCl in buffer containing 50 mM Tris (pH 8.5i, 100 mM NaCl, 5 mM EDTA, 2 mM reduced glutathione (GSHi, 1mM oxidized glutathione (GSSG)) by adding the appropriate buffer to the protein solution and standing for 24 hours at 4 ° C.

DK 173693 B1 31DK 173693 B1 31

Der blev tilsat fast ammoniumsulfat til en slutkoncentra-tion på 1,2 M, og pH-vaerdien blev derefter indstillet til 7,0. På dette tidspunkt dannedes et præcipitat, som inde-5 holdt ukorrekt foldede former af CSF-1. Disse kan i det mindste delvis indvindes og recirkuleres (se nedenfor). CSF-l-præparationen blev derefter forberedt til yderligere fjernelse af pyrogener/endotoxiner og resterende kontaminerende forbindelser på en phenyl-TSK-søjle. Alle 10 puffere og reagenser blev fremstillet pyrogenfrie. CSF-1-præparationen blev centrifugeret 10 minutter ved 15.000 x g og blev filtreret gennem et 0,45 μ filter i en en-gangsenhed (500 ml), før den blev pumpet på en phenyl-TSK-HPLC-søjle aekvilibreret i 1,5 M ammoniumsulfat, 0,1 M 15 natriumphosphat (pH 7,0) kørt ved 4°C.Solid ammonium sulphate was added to a final concentration of 1.2 M, and the pH was then adjusted to 7.0. At this time, a precipitate was formed containing incorrectly folded forms of CSF-1. These can be recovered and recycled at least in part (see below). The CSF-1 preparation was then prepared for further removal of pyrogens / endotoxins and residual contaminants on a phenyl-TSK column. All 10 buffers and reagents were prepared pyrogen-free. The CSF-1 preparation was centrifuged for 10 minutes at 15,000 xg and filtered through a 0.45 µ filter in a single unit (500 ml) before being pumped on a phenyl-TSK-HPLC column equilibrated for 1.5 hours. M ammonium sulfate, 0.1 M sodium phosphate (pH 7.0) run at 4 ° C.

Efter påsætningen af CSF-1 blev søjlen vasket i 30 minutter. Proteinet blev derefter elueret med en 45-minutters gradient af faldende ammoniumsulfat, stigende ethylengly-20 col B puffer (B puffer = 60% e thyl engly col, 0,01 M natriumphosphat (pH 7,0)). CSF-l-proteinet eluerede ved ca.After the application of CSF-1, the column was washed for 30 minutes. The protein was then eluted with a 45-minute gradient of decreasing ammonium sulfate, increasing ethylene glycol B buffer (B buffer = 60% ethyl thly col, 0.01 M sodium phosphate (pH 7.0)). The CSF-1 protein eluted at ca.

0,6 M ammoniumsulfat, 35% ethylenglycol.0.6 M ammonium sulfate, 35% ethylene glycol.

Den første hovedtop, som eluerede, var biologisk aktiv, 25 dimer CSF-1. CSF-l-toppen blev hældt sammen og derefter grundigt dialyseret mod 5% mannitol, 25 mM natriumphosphat (pH 7,4), blev filtersteriliseret og opbevaret ved 4°C. Endotoxinindholdet varierede fra 0,1 til 1 ng/ mg.The first major peak that eluted was biologically active, 25 dimer CSF-1. The CSF-1 peak was pooled and then thoroughly dialyzed against 5% mannitol, 25 mM sodium phosphate (pH 7.4), filter sterilized and stored at 4 ° C. The endotoxin content ranged from 0.1 to 1 ng / mg.

30 På lignende måde blev E. coli-protein fremstillet under styring af PL-promotoren fra DNA, som koder for asp59SCSF/NV2CVl50, asp59SCSF/NV3CVl50, NV3CV158, LCSF/CV190 og LCSF/CV221, genfoldet og renset. Slutpræ-35 parationerene indeholdt 6-15 mg renset CSF-1 med et omtrentligt samlet udbytte på 15-30% og en specifik aktivi- DK 173693 B1 32 n tet på 5-10 x 10 enheder/mg i musebenmarvsanalysen (under anvendelse af A2qq og under antagelse af, at en værdi på 1,0 svarer til 1 mg CSF-1 pr. ml). Præparationerne havde også omtrent den samme specifikke aktivitet i hr-5 man-benmarvsanalysen som renset, nativ MIA PaCa-CSF-1.Similarly, E. coli protein prepared under the control of the PL promoter from DNA encoding asp59SCSF / NV2CV150, asp59SCSF / NV3CV150, NV3CV158, LCSF / CV190 and LCSF / CV221 was refolded and purified. The final preparations contained 6-15 mg of purified CSF-1 with an approximate overall yield of 15-30% and a specific activity of 5-10 x 10 units / mg in the mouse bone marrow assay (using A2qq and assuming that a value of 1.0 corresponds to 1 mg CSF-1 per ml). The preparations also had approximately the same specific activity in the hr-5 human bone marrow assay as purified native MIA PaCa-CSF-1.

Eksempel 3Example 3

Direkte genfoldning af opløseliggjorte, lysbrydende lege|-10 mer:Direct refolding of solubilized, refractive games | -10 more:

Saccharoserensede, opløseliggjorte, lysbrydende legemer med asp5gSCSF/CV150 blev fremstillet som i eksempel 2, og de havde en proteinkoncentration på 20 mg/ml. Til gen-15 foldning blev proteinkoncentrationen nedsat ved fortynding til 1,5 mg/ml asp59SCSF/CVl50 (total CSF-1 var 38 mg) i 7 M GuHCl, 0,1 M natriumphosphat (pH 7,0), 5 nM EDTA, ImM DTT. Genfoldning blev påbegyndt ved en tigange-fortynding til 0,15 mg/ml i 50 mM Tris (pH 8,5), 100 nM 20 NaCl, 5 mM EDTA, 2 mM GSH og 1 mM GSSG (samme genfoldningspuffer som ovenfor) ved 4°C og genfoldningen fik løv til at forløbe i 24 timer.Sucrose-purified, solubilized, light refractive bodies with asp5gSCSF / CV150 were prepared as in Example 2 and had a protein concentration of 20 mg / ml. For refolding, the protein concentration was reduced by dilution to 1.5 mg / ml asp59SCSF / CV150 (total CSF-1 was 38 mg) in 7 M GuHCl, 0.1 M sodium phosphate (pH 7.0), 5 nM EDTA, ImM DTT. Refolding was started by a titanium dilution to 0.15 mg / ml in 50 mM Tris (pH 8.5), 100 nM 20 NaCl, 5 mM EDTA, 2 mM GSH and 1 mM GSSG (same refolding buffer as above) at 4 ° C and the refolding allowed leaves to run for 24 hours.

Ca. 35% af CSF-1-monomeren genfoldede til den dimere fojrm 25 (baseret på den kendte tilbageholdelsestid for dimer CSF- 1), hvilket blev påvist ved HPLC med omvendt fase. Renheden af de genfoldede dimerer blev ved HPLC med omvendt fase vurderet til at være ca. 63%.Ca. 35% of the CSF-1 monomer refolded to the dimeric foil 25 (based on the known retention time of dimer CSF-1), which was detected by reverse phase HPLC. The purity of the refolded dimers was estimated by reverse phase HPLC to be approx. 63%.

30 Eksempel 4Example 4

Recirkulering af aggregater:Recycling of aggregates:

Det i eksempel 2 beskrevne præcipitat indeholdt antagelLg 35 ukorrekt foldede former af CSF-1. Når det blev dannet v.»d genfoldning af ca. 38 mg protein, udgjorde det ca. 10 tøg DK 173693 B1 33 præcipitat, som kunne bundfældes. Dette præcipitat blev opløst i S-200-pufferen, som indeholdt 7 M GuHCl og 2 mM DTT (beskrevet i eksempel 2). Suspensionen blev opvarmet ved 37°C i 15 minutter til reduktion af enhver disulfid-5 binding, og den fremkomne, klare opløsning blev afkølet til 4°C. Opløsningen blev derefter fortyndet til 0,7 M GuHCl i genfoldningspuffer og fik lov til at genfolde som ovenfor beskrevet. Der blev derefter tilsat ammoniumsul-fat, og den genfoldede CSF-l-dimer blev oprenset fra den 10 fremkomne opløsning til fjernelse af pyroge-ner/endotoxiner ved phenyl-TSK-HPLC, som er beskrevet ovenfor. Dette gav over 3 mg opløselig, dimer CSF-1.The precipitate described in Example 2 contained assumption 35 incorrectly folded forms of CSF-1. When it was formed, the refolding of ca. 38 mg of protein, it amounted to approx. 10 cloth DK 173693 B1 33 precipitate which could be precipitated. This precipitate was dissolved in the S-200 buffer containing 7 M GuHCl and 2 mM DTT (described in Example 2). The suspension was heated at 37 ° C for 15 minutes to reduce any disulfide bond, and the resulting clear solution was cooled to 4 ° C. The solution was then diluted to 0.7 M GuHCl in refolding buffer and allowed to refold as described above. Ammonium sulphate was then added and the refolded CSF-1 dimer was purified from the resulting solution to remove pyrogens / endotoxins by phenyl-TSK-HPLC described above. This yielded over 3 mg of soluble dimer CSF-1.

Denne recirkuleringsproces forventes, når den udføres i 15 større skala, at forbedre det samlede udbytte ved fremgangsmåden til fremstilling af genfoldet CSF-1.This recycling process, when carried out on a larger scale, is expected to improve the overall yield of the refold CSF-1 preparation process.

Eksempel 5 20 E. coli stamme DG116 blev transformeret med plasmidvektor pLCSF221A, et plasmid, der indeholdt genet, som koder for asp59LCSF/NV3CV221. Den transformerede E. coli stamme DG116 blev deponeret hos American Type Culture Collection under deponerings nr. ATCC 67390 den 14. april 1987. Den 25 transformerede vært blev dyrket i en 100 1 luftgen-Example 5 20 E. coli strain DG116 was transformed with plasmid vector pLCSF221A, a plasmid containing the gene encoding asp59LCSF / NV3CV221. The transformed E. coli strain DG116 was deposited with the American Type Culture Collection under Deposit No. ATCC 67390 on April 14, 1987. The transformed host was grown in a 100 L air gene.

nemstrømmet, Rushton standardturbinefermentor i basalt medium, som indeholdt 96 mM (ΝΗ,^^δΟή, 28 mM KH2PO4, 4 mMeasy flow, Rushton standard turbine fermentor in basal medium containing 96 mM (ΝΗ, ^^ δΟή, 28 mM KH 2 PO 4, 4 mM

Na3-citrat ♦ 2 H20, 1,7 ml/1 TK9 (30 mM ZnS04, 30 mMNa 3 Citrate ♦ 2 H2 O, 1.7 ml / 1 TK9 (30 mM ZnSO 4, 30 mM

MgS04, 1 mM CUSO4), med sterile tilsætninger af 6,5 g/1 30 glucose, 2,2 mM MgS04 · 7 H20, 95 μΜ FeSo^ · 7 H20 og 26 mg/1 thiamin · HC1 ved 30°C, indtil der blev nået en OD680nm 1·®· Kulturen blev derefter induceret ved temperaturskift til 37°c med samtidige sterile tilsætninger af casaminosyrer til en s lut koncentrat ion på 2,3% 35 (vægt/volumen) og MgS04 · 7 H20 til en slutkoncentration på 1,7 mM.MgSO 4, 1 mM CUSO 4), with sterile additions of 6.5 g / l 30 glucose, 2.2 mM MgSO 4 · 7 H 2 O, 95 μΜ FeSo 2 · 7 H 2 O and 26 mg / l thiamine · HCl at 30 ° C an OD680nm 1 · ® was reached. The culture was then induced by temperature change to 37 ° C with concurrent sterile additions of casamino acids to a final concentration of 2.3% 35 (w / v) and MgSO4 · 7 H2 O to a final concentration. of 1.7 mM.

DK 173693 B1 34DK 173693 B1 34

Fire timer efter induktionen blev cellerne høstet ved fem-gange koncentrering og diafiltrering mod 10 voluminer af 5 mM EDTA, pH 8,5, under anvendelse af Dorr-Oliver 5 tangential tværstrømsmikroporefiltrering. Cellerne sprængtes ved tre passager ved 51.710 kPa i en mekanisk Manton-Gaulin højtrykscellehomogenisator. Der blev tilsat 1-Octanol til 0,1% (v/v), og homogenatet blev holdt vejd 4°C natten over.Four hours after induction, the cells were harvested by five-fold concentration and diafiltration against 10 volumes of 5 mM EDTA, pH 8.5, using Dorr-Oliver 5 tangential cross-stream micropore filtration. The cells were blasted at three passages at 51,710 kPa in a mechanical Manton-Gaulin high-pressure cell homogenizer. 1-Octanol was added to 0.1% (v / v) and the homogenate kept weighed 4 ° C overnight.

1010

Homogenatet blev ført til 25% saccharose ved tilsætning af en 63% vægt/volumen saccharoseopløsning. Den uopløselige proteinfraktion (lysbrydende legemer) blev adskilt fra celleresterne ved skivestackcentrifugering med konti.-15 nuerligt flow (Westphalia SB7) ved 9000 x g, 1 li-ter/minut og 4-6°C. Den våde pellet blev blandet med deioniseret vand i et forhold på 50:50 (vægt/volumen) cg blev opbevaret ved -20°c i delmængder på 45 g.The homogenate was brought to 25% sucrose by the addition of a 63% w / v sucrose solution. The insoluble protein fraction (light refractive bodies) was separated from the cellular residues by disc stack centrifugation with continuous flow (Westphalia SB7) at 9000 x g, 1 liter / minute and 4-6 ° C. The wet pellet was mixed with deionized water at a ratio of 50:50 (w / v) cg stored at -20 ° C in 45 g aliquots.

20 Halvfems gram suspension af lysbrydende legemer blev optøet ved stuetemperatur og blev homogeniseret i 200 ml 0,1 M Tris, pH 8,5, som indeholdt 25 mM EDTA og 10 eM DTT, under anvendelse af en Tekmar-vævshomogenisator i 1 minut ved 50% hastighed. Suspensionen blev indstillet til 25 1 liter 8 M urinstof, 2 mM DTT, 5 mM EDTA og 20 mM Tria, pH 8,5, og blev omrørt i ca. 30 minutter ved stuetemperatur. Uopløselige rester blev fjernet med en 929 cm2 stor 0,8-0,2 μπι Sartorius-membranfilterpatron til engangsbruc .Twenty-nine grams of suspension of light refracting was thawed at room temperature and homogenized in 200 ml of 0.1 M Tris, pH 8.5 containing 25 mM EDTA and 10 µM DTT, using a Tekmar tissue homogenizer for 1 minute at 50 % velocity. The suspension was adjusted to 25 L of 8 M urea, 2 mM DTT, 5 mM EDTA and 20 mM Tria, pH 8.5, and stirred for approx. 30 minutes at room temperature. Insoluble residues were removed with a 929 cm2 0.8-0.2 μπι Sartorius disposable membrane filter cartridge.

30 Efter filtrering blev suspensionen, som indeholdt reduceret CSF-l-monomer, delvis renset ved DEAE-kromatograf:..After filtration, the suspension containing reduced CSF-1 monomer was partially purified by DEAE chromatograph:

Prøver med en A280 På 10 (500 blev påsat hver af to 5 x 45 cm DEAE-Sepharosesøjler med hurtigt gennemløb og æk-bilibreret i 0,1 M Tris, pH 8,5. Hver søjle blev elueret 35 med en 3.600 ml 0-0,4 M NaCl-gradient i 4 M urinstof, 0,1 M Tris, pH 8,5, 5 mM EDTA og 2 mM DTT. Baseret på anta- DK 173693 B1 35 gelsen af, at 1 A280 «r li?f 1 ™g/ml blev der indvun det 4,5 g protein.Samples with an A280 of 10 (500 were loaded on each of two 5 x 45 cm rapid-pass DEAE-Sepharose columns and equilibrated in 0.1 M Tris, pH 8.5. Each column was eluted with a 3,600 ml of 0- 0.4 M NaCl gradient in 4 M urea, 0.1 M Tris, pH 8.5, 5 mM EDTA and 2 mM DTT Based on the assumption that 1 A280 1 g / ml was obtained in the 4.5 g protein.

DEAE-renset CSF-l-monomer blev afkølet til 4°C og fortyn-5 det 1:10 i forafkølet 50 mM Tris, pH 8,5, som indeholdt 5 mM EDTA, 2 mM reduceret glutathion og 1 mM oxideret glu-tathion til en beregnet slutproteinabsorbans ved A2go P^ 0,2. Skønt den initielle dimerdannelse stort set var afsluttet i løbet af 24 timer vurderet ved SDS-PAGE, blev 10 genfoldningsblandingen (22,5 liter) holdt ved 4°C i 5 dage for at få størst muligt udbytte af CSF-l-dimer med den korrekte konformation. Konformationen af dimer CSF-1 i genfoldningsblandingen blev vurderet ved HPLC med omvendt fase. Med en C^j-søjle og en 35-55% acetonitrilgradient 15 eluerede dimer CSF-1 som to adskilte former; stabil, aktiv CSF-1 var den mere hydrofobe. Denne stabile, aktive CSF-l-form repræsenterede 65% af proteinet efter 5 dages inkubering.DEAE-purified CSF-1 monomer was cooled to 4 ° C and diluted 1:10 in pre-cooled 50 mM Tris, pH 8.5 containing 5 mM EDTA, 2 mM reduced glutathione and 1 mM oxidized glutathione to a calculated final protein absorbance at A2go P ^ 0.2. Although the initial dimer formation was virtually complete within 24 hours assessed by SDS-PAGE, the 10 refolding mixture (22.5 liters) was kept at 4 ° C for 5 days to maximize the CSF-1 dimer yield with the correct conformation. The conformation of dimer CSF-1 in the refolding mixture was assessed by reverse phase HPLC. With a C ^ j column and a 35-55% acetonitrile gradient, dimer CSF-1 eluted as two distinct forms; stable, active CSF-1 was the more hydrophobic. This stable, active CSF-1 form represented 65% of the protein after 5 days of incubation.

20 Reduceret og oxideret glutathion blev fjernet ved diafiltrering mod 20 mM natriumphosphat, pH 7, og proteinet blev koncentreret til en A28CTabsorbans på 1,2 under anvendelse af en 0,929 m2 stor Amicon PM10-hulfiberpatron.20 Reduced and oxidized glutathione was removed by diafiltration against 20 mM sodium phosphate, pH 7, and the protein was concentrated to an A28CT absorbance of 1.2 using a 0.929 m2 Amicon PM10 hollow fiber cartridge.

Der blev tilsat ammoniumsulfat til det diafiltrerede ma-25 teriale til en koncentration op 1,2 M. Præcipiterede, ustabile konformationer (den mindre hydrofobe form påvist ved HPLC med omvendt fase) blev fjernet ved filtrering. Filtratet (2 g stabil, dimer CSF-1) blev sat på et 5 x 20 cm leje af phenyl-Sepharose med hurtigt gennemløb og 30 ækvilibreret i 1,2 M ammoniumsulfat, som indeholdt 0,0025 M natriumphosphat, pH 7,0, og blev elueret på 6 timer i en samtidigt faldende (0,72 M til 0 M ammoniumsulfat) og stigende (24% til 60% v/v ethylenglycol) gradient på 1500 ml i 0,01 M natriumphosphatpuffer, pH 7,0. Dimer CSF-1 35 blev elueret ved ca. 30-35% ethylenglycol og var vel adskilt fra tetramer CSF-1 og endotoxin, som begge blev DK 17 3693 B1 36 elueret senere. Dimer CSF-1 blev diafiltreret mod 20 i\M natriumphosphat, pH "7,5, og blev koncentreret til en A28 0-værcli på 10 under anvendelse af en 929 cm2 stor Am:.- con spiralpatron (YM10). Genfindingen var 1,3 g stabil, 5 dimer CSF-1 baseret på A2qø. Det fremstillede CSF-1 havde en biologisk aktivitet på ca. 6 x 107 enheder/mg i an CSF-l-afhængig celleprolifereringsanalyse til bestemmelse af aktivitet. Slutproduktet var 98,6% dimer og 93% reducerbar dimer, hvilket blev bestemt ved ikke-reducerenieAmmonium sulfate was added to the diafiltered material to a concentration up to 1.2 M. Precipitated, unstable conformations (the less hydrophobic form detected by reverse phase HPLC) were removed by filtration. The filtrate (2 g of stable dimer CSF-1) was placed on a 5 x 20 cm rapid-pass phenyl Sepharose bed and equilibrated in 1.2 M ammonium sulfate containing 0.0025 M sodium phosphate, pH 7.0, and was eluted at 6 hours in a simultaneously decreasing (0.72 M to 0 M ammonium sulfate) and increasing (24% to 60% v / v ethylene glycol) gradient of 1500 ml in 0.01 M sodium phosphate buffer, pH 7.0. Dimer CSF-1 35 was eluted at ca. 30-35% ethylene glycol and was well separated from tetramer CSF-1 and endotoxin, both of which were eluted later. Dimer CSF-1 was diafiltered against 20 µM sodium phosphate, pH 7.5, and was concentrated to an A28 0 reaction of 10 using a 929 cm 2 Am: .con spiral cartridge (YM10). The recovery was 1 , 3 g of stable, 5 dimer CSF-1 based on A2qo. The produced CSF-1 had a biological activity of about 6 x 10 7 units / mg in an CSF-1-dependent cell proliferation assay to determine activity. % dimer and 93% reducible dimer as determined by non-reducer

10 og reducerende SDS-PAGE-analyse. Endotoxinindholdet var 0,01 ng/mg CSF-1 bestemt ved LAL-analysen og A28O10 and reducing SDS-PAGE analysis. The endotoxin content was 0.01 ng / mg CSF-1 determined by the LAL assay and A28O

Eksempel 6 15 DEAE-kromatografi efter genfoldning:Example 6 DEAE chromatography after refolding:

En E. coli stamme HW22, der var transformeret med plasmi-det pJN653, som indeholdt asp59SCSF/NV3CVl58-genet, blev dyrket i en 10-liter fermentor i det samme medium som fce-20 skrevet i eksempel 5. Cellerne blev dyrket ved 30°C til en absorbans på 10 ved 680 nm, og der blev derefter tilsat casaminosyrer til 2%. CSF-l-ekspression blev induceret ved at skifte kulturens temperatur til 37°C. Efter 4 timer nåede absorbansen ved 680 nm 79; cellerne blev 25 høstet, homogeniseret, og der blev fremstillet lysbrydende legemer som beskrevet i eksempel 5.An E. coli strain HW22 transformed with the plasmid pJN653 containing the asp59SCSF / NV3CV158 gene was grown in a 10-liter fermentor in the same medium as fce-20 written in Example 5. The cells were grown at 30 ° C to an absorbance of 10 at 680 nm and then casamino acids were added to 2%. CSF-1 expression was induced by changing the temperature of the culture to 37 ° C. After 4 hours, the absorbance reached 680 nm 79; the cells were harvested, homogenized and light refractory bodies prepared as described in Example 5.

Femogtyve gram suspension med lysbrydende legemer (ca.Twenty-five grams of suspension with light refractory bodies (approx.

390 g protein) blev opløst i 250 ml 8 M urinstof, som in-30 deholdt 25 mM Tris, 10 mM natriumphosphatpuffer (pH 8,4),390 g of protein) was dissolved in 250 ml of 8 M urea containing 25 mM Tris, 10 mM sodium phosphate buffer (pH 8.4),

1 mM EDTA og 4 mM DTT. Efter 2 timer ved stuetemperatur blev opløsningen klaret ved centrifugering ved 15.000 j g i 15 minutter. En prøve på 150 ml af det opløseliggjoite CSF-1 blev derefter sat på en 5 x 8 cm DEJE-35 Sepharosesøjle (Pharmacia), der var ækvilibreret i 6 M1 mM EDTA and 4 mM DTT. After 2 hours at room temperature, the solution was clarified by centrifugation at 15,000 µg for 15 minutes. A sample of 150 ml of the soluble CSF-1 was then placed on a 5 x 8 cm DEJE-35 Sepharose column (Pharmacia) equilibrated in 6 M

DK 173693 B1 37 urinstof, som indeholdt 25 mM Tris og 10 mM na-triumphosphatpuffer (pH 7,0). Søjlen blev vasket med 1 lejevolumen af ovennævnte opløsnng, som var modificeret til at indeholde 1 mM DTT og 1 mM EDTA, og CSF-1 blev 5 derefter elueret med en 1,4 1 saltgradient af 0-0,6 M na-triumchlorid i vaskepufferen. CSF-l-toppen eluerede ved ca. 0,06 M natriumchlorid. De resterende 90 ml opløselig-gjorte, lysbrydende legemer blev derefter renset over DEAE-Sepharosesøjlen på lignende måde. De kombinerede 10 CSF-l-puljer (165 ml) indeholdt ca. 250 mg protein med en renhed på ca. 50%.DK 173693 B1 37 urea containing 25 mM Tris and 10 mM sodium phosphate buffer (pH 7.0). The column was washed with 1 bed volume of the above solution, modified to contain 1 mM DTT and 1 mM EDTA, and CSF-1 was then eluted with a 1.4 l salt gradient of 0-0.6 M sodium chloride in wash buffer. The CSF-1 peak eluted at ca. 0.06 M sodium chloride. The remaining 90 ml of soluble, light refractive bodies were then purified over the DEAE-Sepharose column in a similar manner. The combined 10 CSF-1 pools (165 ml) contained approx. 250 mg protein with a purity of approx. 50%.

CSF-1 blev derefter genfoldet ved at fortynde DEAE-puljen 10 gange i genfoldningspuffer, som indeholdt 50 mM Tris 15 (pH 8,5), 5 mM EDTA, 2 mM reduceret glutathion, 1 mM oxideret glutathion, som var forafkølet til 4°C. CSF-1 fik lov til at genfolde i 30 timer ved 4°C. pH-værdien af den genfoldede CSF-1 blev indstillet til 6,8 med en 8,5% phosphorsyreopløsning. Opløsningen blev klaret ved cen-20 trifugering i 10 minutter ved 15.000 x g og blev sat på en 5 x 4 cm DEAE-Sepharosesøjle, som var forækvilibreret i 10 mM natriumphosphat, 25 mM Tris (pH 6,8). Søjlen blev vasket med 300 ml af denne puffer og blev elueret med en 700 ml 0-0,6 M natriumchloridgradient i det samme puffer-25 system. CSF-1 eluerede ved ca. 120 mM natriumchlorid. Der blev tilsat ammoniumsulfat (4M stamopløsning, pH 7,0} til den 95 ml store DEAE-pulje til en slutkoncentration på 1 M. CSF-1 blev filtreret gennem et Nalgene 0,45 μ filter og blev (ved 4°C) sat på en 21,5 x 150 mm Bio-Rad TSK-30 Phenyl-5-PW-søjle ækvilibreret i depyrogeneret 1,5 M ammoniumsulfat og 0,1 M natriumphosphat (pH 7,0). Søjlen blev vasket med 2 lejevoluminer af denne påsætningspuffer og blev elueret i 0,1 M natriumphosphat (pH 7,0) under anvendelse af en 45 minutters gradient, hvori ammonium-35 sulfatkoncentrationen faldt fra 1,5 M til 0 M, og ethy-lenglycolkoncentrationen steg fra 0 til 60%. Alle opera- DK 17^693 B1 38 tioner blev udført ved 4°C under stort set pyrogenfria betingelser. CSF-1 eluerede ved ca. 0, 6 M ammoniumsulfat i 30% ethylenglycol. CSF-1 blev dialyseret grundigt i 10 mM HEPES-puffer (pH 7,5), som indeholdt 150 mM natriumch-5 lorid, og blev filtersteriliseret gennem et 0,45 μ Mil>-lex-filter.CSF-1 was then refolded by diluting the DEAE pool 10 times in refolding buffer containing 50 mM Tris 15 (pH 8.5), 5 mM EDTA, 2 mM reduced glutathione, 1 mM oxidized glutathione, which was pre-cooled to 4 ° C. CSF-1 was allowed to refold for 30 hours at 4 ° C. The pH of the refolded CSF-1 was adjusted to 6.8 with an 8.5% phosphoric acid solution. The solution was clarified by cen-20 trifugation for 10 minutes at 15,000 x g and placed on a 5 x 4 cm DEAE-Sepharose column pre-equilibrated in 10 mM sodium phosphate, 25 mM Tris (pH 6.8). The column was washed with 300 ml of this buffer and eluted with a 700 ml of 0-0.6 M sodium chloride gradient in the same buffer-25 system. CSF-1 eluted at ca. 120 mM sodium chloride. Ammonium sulfate (4M stock solution, pH 7.0} was added to the 95 ml large DEAE pool to a final concentration of 1 M. CSF-1 was filtered through a Nalgene 0.45 µ filter and (at 4 ° C) added on a 21.5 x 150 mm Bio-Rad TSK-30 Phenyl-5-PW column equilibrated in depyrogenated 1.5 M ammonium sulfate and 0.1 M sodium phosphate (pH 7.0). The column was washed with 2 bed volumes of this loading buffer and was eluted in 0.1 M sodium phosphate (pH 7.0) using a 45 minute gradient in which the ammonium sulfate concentration decreased from 1.5 M to 0 M and the ethylene glycol concentration increased from 0 to 60%. All operations were performed at 4 ° C under substantially pyrogen-free conditions CSF-1 eluted at approximately 0. 6 M ammonium sulfate in 30% ethylene glycol CSF-1 was dialyzed thoroughly in 10 mM HEPES. buffer (pH 7.5), containing 150 mM sodium chloride, and was filter sterilized through a 0.45 μ Mil> -lex filter.

Der blev opnået ca. 50 mg renset asp5gSCSF/NV3CVl58-CSF- 1. Mere end 90% af det endelige CSF-l-produkt var ved 10 SDS-PAGE-analyse en enkelt form og ca. 96% ren ved HPIC med omvendt fase i acetonitril/TFA. Den specifikke aktivitet var 1,7 x io8 enheder/mg (enheder bestemt som kolc-nidannende enhedsækvivalenter under anvendelse af en CSE- 1-afhængig cellelinie og proteinkoncentration bestemt ved 15 A280nm °9 en staget ekstinktionkoefficient på 1,0). Der- ne specifikke aktivitet er mindst ækvivalent med, hvis ikke større end, den specifikke aktivitet af nativ MIA PaCa-CSF-1. Endotoxinindholdet bestemt ved LAL-analysen var 0,5-1 ng/mg CSF-1.Approx. 50 mg of purified asp5gSCSF / NV3CV158-CSF- 1. By 10 SDS-PAGE analysis, more than 90% of the final CSF-1 product was a single form and ca. 96% pure by reverse phase HPIC in acetonitrile / TFA. The specific activity was 1.7 x 10 8 units / mg (units determined as carbon-forming unit equivalents using a CSE-1 dependent cell line and protein concentration determined at 15 A280nm ° 9 a sustained extinction coefficient of 1.0). This specific activity is at least equivalent to, if not greater than, the specific activity of native MIA PaCa-CSF-1. The endotoxin content determined by the LAL assay was 0.5-1 ng / mg CSF-1.

2020

Eksempel 7Example 7

Der blev anvendt en alternativ rensningsmetode til at b€-handle en genfoldningsreaktion med LCSF/NV3CV221, sera 25 var fremstillet ifølge fremgangsmåden i eksempel 5, til og med genfoldningstrinnet. Ved denne modificerede fremgangsmåde blev den genfoldede CSF-1 sat direkte på en anionbyttersøjle. Ved pH 6,8 strømmer redoxsysten-reagenserne direkte gennem anionbyttersøj len, medens CS!‘-30 1 forbliver bundet og koncentreret på søjlen. På denr e måde blev CSF-1 separeret fra redoxsystemet ved en pi -værdi, hvor thio-disulfidudvekslingsreaktioner var mind«t mulige, hvorved den væsentlige oligomerdannelse, som fandtes at finde sted, hvis dette trin blev udført ved ^n 35 højere pH-værdi (8,5), blev forhindret.An alternative purification method was used to treat a refolding reaction with LCSF / NV3CV221, sera 25 prepared according to the procedure of Example 5, up to and including the refolding step. In this modified method, the refolded CSF-1 was directly applied to an anion exchange column. At pH 6.8, the redox cyst reagents flow directly through the anion exchange column, while CS1 - 30 l remains bound and concentrated on the column. In this way, CSF-1 was separated from the redox system at a pi value, where thio-disulfide exchange reactions were least possible, thereby enabling the significant oligomer formation to occur if this step was carried out at a higher pH. value (8.5), was prevented.

i DK 173693 B1 39in DK 173693 B1 39

Fem ml genfoldet CSF-1 (1 mg totalt protein fra den i eksempel 5 beskrevne genfoldningsreaktion) blev sat direkte på en 7,5 x 75 mm Bio-Rad TSK-DEAE-5-PW-søjle, efter at pH-værdien af den genfoldede CSF-1 var blevet nedsat til 5 6,8 under anvendelse af en 1M phosphorsyreopløsning.Five ml of refolded CSF-1 (1 mg total protein from the refolding reaction described in Example 5) was loaded directly onto a 7.5 x 75 mm Bio-Rad TSK-DEAE-5-PW column after the pH of the refolded CSF-1 had been reduced to 5 6.8 using a 1M phosphoric acid solution.

DEAE-søjlen var blevet ækvilibreret i 10 mM na-triumphosphat, 25 mM Tris (pH 6,8). Efter påsætningen af CSF-1 blev søjlen vasket med 2 lejevoluminer af denne puffer og blev derefter elueret med en 45 minutters 0-0,6 10 M natriumchloridgradient i den samme puffer. Søjlen adskilte dimer CSF-1 fra monomere og oligomere former af CSF-1 (bestemt ved ikke-reducerende SDS-PAGE og Westernn-analyse af DEAE-fraktionerne). Udbyttet af dimer CSF-1 var ca. 70%. Dette er et 5 gange højere udbytte end det 15 udbytte, som blev opnået, når den samme rensning blev udført ved pH 8,5. Efter dette DEAE-rensningstrin vil CSF-1 som beskrevet i eksempel 6 blive renset fri for kontami-nerende endotoxiner og den ustabile form af CSF-1-dimeren begyndende med den ammoniumsulfattilsætning, som går for-20 ud for phenyl-Sepharose-trinnet.The DEAE column had been equilibrated in 10 mM sodium phosphate, 25 mM Tris (pH 6.8). After the CSF-1 was applied, the column was washed with 2 bed volumes of this buffer and then eluted with a 45 minute 0-0.6 10 M sodium chloride gradient in the same buffer. The column separated dimer CSF-1 from monomeric and oligomeric forms of CSF-1 (determined by non-reducing SDS-PAGE and Westernn analysis of the DEAE fractions). The yield of dimer CSF-1 was approx. 70%. This is a 5 times higher yield than the 15 obtained when the same purification was carried out at pH 8.5. Following this DEAE purification step, as described in Example 6, CSF-1 will be purified free of contaminating endotoxins and the unstable form of the CSF-1 dimer beginning with the ammonium sulfate addition preceded by the phenyl-Sepharose step.

Eksempel 8Example 8

Der blev anvendt en alternativ metode til genfoldning af 25 CSF-1. Plasmidet pLCSF221A blev induceret i E. coli, og det udtrykte protein blev behandlet stort set som beskrevet i eksempel 5 med nogle modifikationer. For eksempel blev de høstede celler diafiltreret mod 5 mM EDTA uden pH-indstilling. Efter den anden gennemføring i homogeni-30 satoren blev pH-værdien indstillet til 6 med eddikesyre. Desuden blev der anvendt luftoxidation til dannelse af disulfidbindinger under genfoldning af CSF-l-molekylet.An alternative method of refolding CSF-1 was used. The plasmid pLCSF221A was induced in E. coli and the expressed protein was treated largely as described in Example 5 with some modifications. For example, the harvested cells were diafiltered against 5 mM EDTA without pH adjustment. After the second run in the homogenizer, the pH was adjusted to 6 with acetic acid. In addition, air oxidation was used to form disulfide bonds during refolding of the CSF-1 molecule.

DEAE-renset CSF-l-monomer blev fortyndet til et slutkon-35 centration på 0,2 mg/ml i 50 mM Tris, pH 8,5, 5 mM EDTA og blev genfoldet i 4 dage ved 4°C i nærværelse eller ..... ... I------------ DK 173693 B1 40 fravær af glutathionredoxsystemet. De genfoldede proteiner blev yderligere renset ved stort set at følge de i eksempel 5 beskrevne procedurer igen med nogle modifikc-tioner. Den genfoldede, dimere blanding blev diafiltreret 5 og koncentreret til en OD-værdi på 1. Efter ammoniumsu]-fatpræcipiteringen blev prøven sat på en phenyl-Sepharose-søjle med hurtig gennemstrømning og blev derefter elueret i en faldende (0,78 til 0,18 M ammoniumsu] -fat) gradient på 1.800 ml i 0,01 M natriumphosphatpuffer 10 (pH 7). Dimeren eluerede ved ca. 0,6 M ammoniumsulfat;.DEAE-purified CSF-1 monomer was diluted to a final concentration of 0.2 mg / ml in 50 mM Tris, pH 8.5, 5 mM EDTA and refolded for 4 days at 4 ° C in the presence or. .... ... I ------------ DK 173693 B1 40 absence of the glutathione redox system. The refolded proteins were further purified by roughly following the procedures described in Example 5 again with some modifications. The refolded dimeric mixture was diafiltered and concentrated to an OD value of 1. After the ammonium sulphate precipitation, the sample was put on a phenyl-Sepharose column with rapid flow and then eluted in a descending (0.78 to 0). 18 M ammonium sulfate gradient of 1,800 ml in 0.01 M sodium phosphate buffer 10 (pH 7). The dimer eluted at ca. 0.6 M ammonium sulfate;

Til sidst blev den dimere CSF-1 diafiltreret mod 0,581% natriumcitrat og 0,645% NaCl ved pH 7. Hvis glutathionredoxsystemet ikke er til stede, kan det diafiltre-ringstrin, som er nødvendigt for fjernelse af glutathiorL 15 undlades.Finally, the dimeric CSF-1 was diafiltered against 0.581% sodium citrate and 0.645% NaCl at pH 7. If the glutathione redox system is not present, the diafiltration step necessary for glutathiorL removal can be omitted.

Slutprodukter fra genfoldninger udført i nærværelse eller fravær af et redoxsystem blev sammenlignet ved SDS-PAGI1, HPLC med omvendt fase, isoelektrisk fokusering og bioane-20 lyse. For begge genfoldede prøver observeres lignende molekylevægte og renheder (95% ved densitometriscanning) under både reducerende og ikke-reducerende betingelser på en 12% SDS-PAGE synliggjort ved Coomassiefarvning. Der blev også anvendt HPLC-analyse med omvendt fase til et 25 sammenligne genfoldningskinetikken efter 5 eller 12 dages CSF-l-genfoldning i nærværelse eller fravær af glutathionredoxsystemet. Disse prøver blev straks kørt på en C^-Vydac-søjle med en 35-55% acetonitril, 0,1% TFA gre- dienteluering i løbet af 30 minutter. I begge systemer 30 opnåedes to dimere hovedformer med lignende retentione-tider, og som syntes at være i en forholdsvis stabil ligevægt i løbet af den analyserede tidsperiode. Geler fra Phast (Pharmacia) isoelektrisk fokusering (IEF) af 1,0 pg af hver af de genfoldede CSF-l-præparationer viste lig-35 nende ioniske mønstre, som indeholdt en ionisk hovedform med en pi-værdi på ca. 4,7 og en lidt mere sur mindre DK 173693 B1 41 form. Både den spontant genfoldede CSF-1 og CSF-1 genfoldet under anvendelse af redoxsystemet havde specifikke aktiviteter på 1,2 x 10® enheder/mg i NSF-60-celleprolifereringsanalysen. Den CSF-1, som blev frem-5 stillet i disse to genfoldningssystemer, synes således stort set at være identisk med hensyn til produktrenhed og biologisk aktivitet, når de blev anlyseret ved de ovenfor beskrevne kriterier. De samlede udbytter var også sammenlignelige for de to fremgangsmåder.End products of refolding performed in the presence or absence of a redox system were compared by SDS-PAGI1, reverse phase HPLC, isoelectric focusing and bioanalysis. For both refolded samples, similar molecular weights and purities (95% by densitometry scanning) are observed under both reducing and non-reducing conditions on a 12% SDS-PAGE visualized by Coomassie staining. Reverse phase HPLC analysis was also used to compare the refolding kinetics after 5 or 12 days of CSF-1 refolding in the presence or absence of the glutathione redox system. These samples were immediately run on a C ^-Vydac column with a 35-55% acetonitrile, 0.1% TFA gradient elution over 30 minutes. In both systems 30, two dimer main forms with similar retention times were obtained and which appeared to be in a relatively stable equilibrium over the analyzed time period. Phast (Pharmacia) isoelectric focusing (IEF) gels of 1.0 µg of each of the refolded CSF-1 preparations showed similar ionic patterns which contained a major ionic form having a pi value of ca. 4.7 and a slightly more acidic minor form. Both the spontaneously refolded CSF-1 and CSF-1 refold using the redox system had specific activities of 1.2 x 10 6 units / mg in the NSF-60 cell proliferation assay. Thus, the CSF-1 produced in these two refolding systems appears to be largely identical in product purity and biological activity when analyzed by the criteria described above. The overall yields were also comparable for the two approaches.

1010

Udover at undlade diafiltreringstrinnet til fjernelse af glutathion kan koncentreringstrinnet erstattes med et alternativt rensningstrin, hvori det store volumen genfoldet dimer CSF-1 sættes direkte på en anden anionbytter-15 søjle til koncentrering forud for ammonium-sul fatpræcipitering og efter rensning ved kromatografi med hydrofob vekselvirkning.In addition to failing the diafiltration step to remove glutathione, the concentration step can be replaced by an alternative purification step in which the large volume of refolded dimer CSF-1 is applied directly to another anion exchange column for concentration prior to ammonium sulphate precipitation and after purification by chromatography by hydrofluorocarbon wetting .

Eksempel 9 20 CSF-l-konstruktioner, i hvilke visse cysteiner er blevet udskiftet til seriner, er også med held blevet genfoldet.Example 9 CSF-1 constructs in which certain cysteines have been replaced by serines have also been successfully refolded.

Disse genfoldede proteiner er helt aktive in vitro, men har lidt forskellige tilbageholdelsestider ved HPLC med 25 omvendt fase. For eksempel blev dobbeltserinkonstruktio-nen, ser157ser159LCSF/NV3CV221, genfoldet under anvendelse af den i eksempel 5 beskrevne fremgangsmåde, og dette resulterede i en CSF-l-præparation, som udviste en enkelt top ved HPLC med omvendt fase. Når den ene eller 30 den anden af enkeltserinkonstruktionerne, ser157LCSF/NV3CV221 eller ser159LCSF/ NV3CV221, blev genfoldet, var en modificeret genfoldningsprotokol nødvendig for at opnå et produkt, som var homogent, når det blev analyseret ved HPLC raed omvendt fase. Disse to pro-35 dukter eluerede begge to med en længere tilbageholdelses- DK 173693 B1 42 tid end det genfoldede ser157Ser159LCSF/NV3CV221-produkt, men var igen helt aktive in vitro.These refolded proteins are fully active in vitro, but have slightly different retention times by 25-phase HPLC. For example, the double serine construct, ser157ser159LCSF / NV3CV221, was refolded using the procedure described in Example 5, resulting in a CSF-1 preparation which exhibited a single peak by reverse phase HPLC. When one or the other of the single-serine constructs, ser157LCSF / NV3CV221 or ser159LCSF / NV3CV221, were refolded, a modified refolding protocol was necessary to obtain a product which was homogeneous when analyzed by reverse phase HPLC. These two products both eluted with a longer retention time than the refolded ser157Ser159LCSF / NV3CV221 product, but were again fully active in vitro.

E. coli stamme DG116 blev transformeret med enten 5 plasmidvektoren pLCSF221B eller pLCSF221C, plasmider, scm indeholdt det gen, der koder for seri57LCSF/NV3CV221 henholdsvis ser159LCSF/NV3CV221. Disse to E. coli stammer blev dyrket i rysteflasker ved 30°C i 500 ml af det samme medium, som er beskrevet i eksempel 5 (slut Agg0rm 10 på 0,2). CSF-l-ekspression blev induceret ved at skifte kulturens temperatur til 42°C. Efter 4 timer blev kulturen høstet ved centrifugering, og cellerne blev resusper-deret i 30 ml 50 mM Tris-puffer (pH 8,5), 5 mM EDTA. Cellerne blev lyseret ved lydbehandling, og celleresterre 15 blev bevaret efter centrifugering. Lysbrydende legem«r blev derefter isoleret ved at resuspendere celleresterre i 30% saccharose og pellettere de lysbrydende legemer v«d centrifugering. De lysbrydende legemer blev opløst i 10 M urinstof, 10 mM Tris (pH 8,5), 1 mM EDTA og 5 mM DTT.E. coli strain DG116 was transformed with either the plasmid vector pLCSF221B or pLCSF221C, plasmids, scm contained the gene encoding seri57LCSF / NV3CV221 and ser159LCSF / NV3CV221, respectively. These two E. coli strains were grown in shake flasks at 30 ° C in 500 ml of the same medium as described in Example 5 (end Aggorm 10 of 0.2). CSF-1 expression was induced by changing the temperature of the culture to 42 ° C. After 4 hours, the culture was harvested by centrifugation and the cells were resuspended in 30 ml of 50 mM Tris buffer (pH 8.5), 5 mM EDTA. The cells were lysed by sonication and cell residues 15 were retained after centrifugation. Light refracting bodies were then isolated by resuspending cell esters in 30% sucrose and pelleting the light refracting bodies by centrifugation. The light refractory bodies were dissolved in 10 M urea, 10 mM Tris (pH 8.5), 1 mM EDTA and 5 mM DTT.

20 Uopløseligt materiale blev fjernet ved centrifugerinc, efterfulgt af filtrering gennem et 0,2 μ Millex-filter.20 Insoluble material was removed by centrifugerinc followed by filtration through a 0.2 μ Millex filter.

CSF-1-monomererne blev derefter renset fra filtratet ur-der anvendelse af ionbytningskromatografi på en Bio-Rid TSK-DEAE-5-PW-søjle (7,5 x 75 mm) ækvilibreret i 6 M 25 urinstof, 10 mM Tris (pH 8,5), som indeholdt 1 mM EDTA cg 1 mM DTT. CSF-1 blev elueret med en 45 minutters 0-0,4 M natriumchloridgradient. CSF-1 blev elueret tidligt i gradienten som en enkelt proteinhovedtop. Proteinet blev sammenhældt, og absorbansen ved 280 nm blev bestemt. CSF-30 1 blev genfoldet ved fortynding i en opløsning, som inde holdt 30 mM Tris (pH 8,5), 5 mM EDTA, 2mM reduceret gli-tathion og 1 mM oxideret glutathion til en A280nn-slutværdi på 0,2 beregnet ud fra A23ønm-absorbansen fer den ikke-fortyndede DEAE-pulje. CSF-1 fik lov til at ger-35 folde i 48 timer ved 4°C.The CSF-1 monomers were then purified from the filtrate using ion exchange chromatography on a Bio-Rid TSK-DEAE-5-PW column (7.5 x 75 mm) equilibrated in 6 M urea, 10 mM Tris (pH 8.5) which contained 1 mM EDTA and 1 mM DTT. CSF-1 was eluted with a 45 minute 0-0.4 M sodium chloride gradient. CSF-1 was eluted early in the gradient as a single protein head. The protein was pooled and the absorbance at 280 nm was determined. CSF-30 1 was refolded by dilution in a solution containing 30 mM Tris (pH 8.5), 5 mM EDTA, 2 mM reduced glutathione and 1 mM oxidized glutathione to an A280nn final value of 0.2 calculated from the A23 nm absorbance leaves the undiluted DEAE pool. CSF-1 was allowed to fold for 35 hours at 4 ° C.

DK 173693 B1 43 På dette punkt blev der tilføjet et yderligere oxidationstrin til genfoldningsprotokollen for at opnå et produkt, som stort set var homogent ved HPIC-analyse med omvendt fase. Det genfoldede CSF-l-protein blev dialyseret 5 ved 4°C i 24 timer i 0,4 M urinstof, 50 mM Tris (pH 8,5), 5 mM EDTA, som kun indeholdt reduceret glutathion (2 mM).At this point, an additional oxidation step was added to the refolding protocol to obtain a product which was substantially homogeneous by reverse phase HPIC analysis. The refolded CSF-1 protein was dialyzed 5 at 4 ° C for 24 hours in 0.4 M urea, 50 mM Tris (pH 8.5), 5 mM EDTA containing only reduced glutathione (2 mM).

Dette trin kan fjerne glutathion, som er bundet til proteinet via et blandet disulfid. Derefter blev der anvendt 1 M phosphorsyre for at indstille pH-værdien til 6,5, 10 hvorved thio-disulfidudskiftningshastigheden blev nedsat.This step can remove glutathione, which is bound to the protein via a mixed disulfide. Then, 1M phosphoric acid was used to adjust the pH to 6.5, thereby reducing the thio-disulfide exchange rate.

CSF-1 blev renset ved ionbytningskromatografi på en Bio-Rad TSK-DEAE-5-PW-søjle, som var ækvilibreret i 10 mM na-triumphosphat, 25 mM Tris-puffer (pH 6,5). Ved dette trin fjernes resterende glutathion, og proteinet oprenses 15 yderligere. Proteinet blev elueret med en 45 minutters 0-, 0, 6 M natriumchloridgradient. Puljen med den genfoldede CSF-1-dimer blev derefter udsat for kuprichloridoxidation under anvendelse af en modifikation af den fremgangsmåde, som er beskrevet i U.S. patent nr. 4.572.798, hvilket pa- 20 tent er medtaget her ved denne henvisning. CSF-1 blev fortyndet til 0,2 absorbansenheder (^sonm* i 1° mM na- triumphosphat, 25 mM Tris-puffer (pH 6,5) og blev behandlet med 50 micromolær kuprichlorid i 2 timer ved stuetemperatur.CSF-1 was purified by ion exchange chromatography on a Bio-Rad TSK-DEAE-5-PW column equilibrated in 10 mM sodium phosphate, 25 mM Tris buffer (pH 6.5). At this step, residual glutathione is removed and the protein is further purified. The protein was eluted with a 45 minute 0-, 0, 6M sodium chloride gradient. The pool of the refolded CSF-1 dimer was then subjected to cupric chloride oxidation using a modification of the process described in U.S. Pat. Patent No. 4,572,798, which is hereby incorporated by reference. CSF-1 was diluted to 0.2 absorbance units (3 sonm * in 1 ° mM sodium phosphate, 25 mM Tris buffer (pH 6.5) and treated with 50 micromolar cuprichloride for 2 hours at room temperature.

2525

Den oxiderede CSF-l-dimer fandtes at være opløselig i 1,2 M ammoniumsulfat. Der kan udføres en yderligere rensning ved kromatografi med hydrofob vekselvirkning på en phe-nyl-Sepharose-søjle, som beskrevet i eksempel 5.The oxidized CSF-1 dimer was found to be soluble in 1.2 M ammonium sulfate. Further purification can be performed by hydrophobic interaction chromatography on a phenyl-Sepharose column as described in Example 5.

3030

Det er hensigten, at modifikationer af de ovenfor beskrevne udførelsesformer til udøvelse af opfindelsen, hvilke modifikationer er indlysende for en fagmand inden for biokemiområdet, navnlig inden for proteinrensning og 35 beslægtede områder, ligger inden for de tilhørende kravs rækkevidde.It is intended that modifications of the above-described embodiments of the practice of the invention which are obvious to those skilled in the art of biochemistry, particularly in the field of protein purification and related fields, are within the scope of the appended claims.

Claims (14)

1. Fremgangsmåde til opnåelse af renset, biologisk aktiv CSF-l-dimer ud fra produktet fra et CSF-l-gen, som ud-5 trykkes rekombinant i bakterier, kendetegnet ved: (a) at en reduceret, monomer, opløseliggjort form af CSE- 1-produktet udvindes fra bakterieceller i et chaotropisk 10 miljø og under reducerende betingelser, (b) at monomeren fra (a) genfoldes ved 4°C over mindst 2 4 timer ved at reducere koncentrationen af det chaotrope middel med mindst en faktor 10 under tilstedeværelse éf 15 redoxsystemet GSH/GSSG, herunder at CSF-l-dimeren dannes, og (c) at den genfoldede, dimeriserede CSF-1 fra (b) renses.A method for obtaining purified, biologically active CSF-1 dimer from the product of a CSF-1 gene which is recombinantly expressed in bacteria, characterized by: (a) a reduced, monomeric, solubilized form of The CSE-1 product is recovered from bacterial cells in a chaotropic environment and under reducing conditions, (b) refolding the monomer from (a) at 4 ° C for at least 2 4 hours by reducing the concentration of the chaotropic agent by at least a factor of 10 in the presence of the GSH / GSSG redox system, including the formation of the CSF-1 dimer and (c) purifying the refolded dimerized CSF-1 from (b). 2. Fremgangsmåde ifølge krav 1, kendetegnet ved, at den yderligere omfatter et rensningstrin for den reducerede, monomere form fra (a) ved en fremgangsmåce udvalgt fra gruppen bestående af: 25 (i) størrelsessorteringsgelkromatografi, (ii) ionbytningskromatografi og (iii) kromatografi med hydrofob vekselvirkning.Process according to claim 1, characterized in that it further comprises a purification step for the reduced, monomeric form from (a) by a process selected from the group consisting of: (i) sizing gel chromatography, (ii) ion exchange chromatography and (iii) chromatography. with hydrophobic interaction. 3. Fremgangsmåde ifølge krav 1, kendetegnet 30 ved, at genfoldningsbetingelserne i (b) omfatter fortyn ding af det chaotropiske miljø, hvor disulfidbindinger dannes ved luftoxidation. 1 Fremgangsmåde ifølge krav 1, kendetegnet 35 ved, at genfoldningsbetingelserne i (b) omfatter fortyrb DK 173693 B1 45 ding af det chaotropiske miljø i nærværelse af et redox-system.Process according to claim 1, characterized in that the refolding conditions in (b) comprise dilution of the chaotropic environment where disulfide bonds are formed by air oxidation. A method according to claim 1, characterized in that the refolding conditions in (b) comprise destroying the chaotropic environment in the presence of a redox system. 5. Fremgangsmåde ifølge krav 4, kendetegnet 5 ved, at fremgangsmåden yderligere omfatter diafiltrering til fjernelse af redoxsystemet forud for rensningstrinnet (c) .Process according to claim 4, characterized in that the method further comprises diafiltration to remove the redox system prior to the purification step (c). 6. Fremgangsmåde ifølge krav 1, kendetegnet 10 ved, at den dimere form fra (c) renses ved én eller flere fremgangsmåder udvalgt fra gruppen bestående af: (i) størrelsessorteringsgelkromatografi, (ii) ionbytningskromatografi, 15 (iii) affinitetskromatografi og (iv) kromatografi med hydrofob vekselvirkning.Process according to claim 1, characterized in that the dimeric form of (c) is purified by one or more methods selected from the group consisting of: (i) size sorting gel chromatography, (ii) ion exchange chromatography, (iii) affinity chromatography and (iv) chromatography with hydrophobic interaction. 7. Fremgangsmåde ifølge krav 6, kendetegnet ved, at der ved ionbytningskromatografien anvendes DEAE-Process according to claim 6, characterized in that the DEAE-ion exchange chromatography is used. 20 Sepharose-kromatografi ved fra ca. pH 5,5 til ca. 7,0.20 Sepharose chromatography at from ca. pH 5.5 to approx. 7.0. 8. Fremgangsmåde ifølge krav 7, kendetegnet ved, at proteinet renses yderligere ved kromatografi med hydrofob vekselvirkning under anvendelse af phenyl-Process according to claim 7, characterized in that the protein is further purified by chromatography with hydrophobic interaction using phenyl 25 Sepharose eller phenyl-TSK.Sepharose or phenyl-TSK. 9. Fremgangsmåde ifølge krav 1, kendetegnet ved, at den yderligere omfatter, at alle uopløselige aggregater, som dannes ud fra CSF-l-proteinet under gen- 30 foldningsbetingelserne i (b), separeres fra, at aggregaterne opløseliggøres, og at de opløseliggjorte aggregater udsættes for genfoldningsbetingelser.Method according to claim 1, characterized in that it further comprises separating all the insoluble aggregates formed from the CSF-1 protein under the refolding conditions in (b) from the solubilizers and the solubilized ones. aggregates are subjected to refolding conditions. 10. Fremgangsmåde ifølge krav 1, kendetegnet 35 ved, at CSF-1 udgangsproduktet er ekspressionsproduktet fra konstruktioner udvalgt fra gruppen bestående af: DK 173693 B1 46 asp5gSCSF/CVl50 og de tilsvarende NV2- eller NV3* analoger deraf, asp5gSCSF/CVl58 og den tilsvarende NV3- analog deraf, LCSF/CV190, LCSF/CV221 og den tilsvarende NV3-analog deraf, ser157LCSF/cV221, ser15gLCSF/CV221 oj 5 ser257ser259LCSF/CV221 og NV2- eller NV3-analogerne der af.Method according to claim 1, characterized in that the CSF-1 starting product is the expression product of constructs selected from the group consisting of: DK 173693 B1 46 asp5gSCSF / CV150 and the corresponding NV2 or NV3 * analogs thereof, asp5gSCSF / CV135 and the corresponding NV3 analog thereof, LCSF / CV190, LCSF / CV221 and the corresponding NV3 analog thereof, ser157LCSF / cV221, ser15gLCSF / CV221 and ser257ser259LCSF / CV221 and the NV2 or NV3 analogs thereof. 11. Fremgangsmåde ifølge krav 1, kendetegnet ved, at CSF-1 er ekspressionsproduktet fra konstruktioner 10 udvalgt fra gruppen bestående af: aspjgSCSF og NV2- eller NV3-analogerne deraf, asp5gSCSF/cVl58 og den tilsvarende NV2-analog deraf, LCSF, LCSF/CV150, LCSF/CV190, og ser257ser^5gLCSF/CV223 og de tilsvarende NV2- eller NV3- analoger deraf, LCSF/CV221 og den tilsvarende NV2-analog 15 deraf.Method according to claim 1, characterized in that CSF-1 is the expression product of constructs 10 selected from the group consisting of: aspjg SCSF and the NV2 or NV3 analogs thereof, asp5gSCSF / cVl58 and the corresponding NV2 analog thereof, LCSF, LCSF / CV150, LCSF / CV190, and ser257ser ^ 5gLCSF / CV223 and the corresponding NV2 or NV3 analogs thereof, LCSF / CV221 and the corresponding NV2 analog thereof. 12. Fremgangsmåde ifølge krav 1 til opnåelse af en CSF-1-heterodimer, kendetegnet ved, at en blanding af mindst to forskellige, opløseliggjorte, monomere, pri- 20 mære CSF-l-sekvenser udsættes for gen foldningsbetingelser, og at heterodimeren renses fra de genfoldede CSF-l-homodimerer.A method according to claim 1 for obtaining a CSF-1 heterodimer, characterized in that a mixture of at least two different solubilized monomeric primary CSF-1 sequences is subjected to gene folding conditions and the heterodimer is purified from they refolded CSF-1 homodimers. 13. Fremgangsmåde ifølge krav 1, kendetegnet 25 ved, at CSF-l-genet koder for LCSF/NV3CV221, asp59SCSF/NV3CVl50, asp5gSCSF/ NV3CV158, ser157LCSF/NV3CV221, serx59LCSF/NV3CV221 eller ser157-ser15gLCSF/NV3CV221.The method of claim 1, characterized in that the CSF-1 gene encodes LCSF / NV3CV221, asp59SCSF / NV3CV150, asp5gSCSF / NV3CV158, ser157LCSF / NV3CV221, serx59LCSF / NV3CV221 or ser157-ser157