HRP980604A2 - Purification process - Google Patents

Description

Cilj izuma je postupak za kromatografsko pročišćavanje ciklosporina A iz sirovog produkta koji sadrži kompleks ciklosporina upotrebom stupca napunjenog sa silikagelom i uz primjenu kromatografije više stupnjeva sa stupcem napunjenim s normalnom fazom silikagela i sa smjesom otapala koja sadrži toluen kao glavnu komponentu. The aim of the invention is a process for the chromatographic purification of cyclosporine A from a crude product containing a cyclosporine complex using a column filled with silica gel and with the application of multistage chromatography with a column filled with a normal phase of silica gel and with a solvent mixture containing toluene as the main component.

Ciklosporini su ciklički undekapeptidi (peptidi s 11 amino kiselina) koji su N-metilirani na nekoliko mjesta i većina njih ima dokazane farmakološke učinke. 25 članova ove skupine spojeva koji su već odavno poznati su označeni sa slovima A do Z. Kao prvi između njih je odijeljen ciklosporin A, koji je prirodna tvar i izoliranje iz kulture Tolypocladium inflatum Gams strain (Helv. Chim. Acta 59. 1075/19767). Najprije je taj spoj postao poznat kao slabi antifugalni antibiotik, kasnije je privukao pozornost njegov imunosupresivan učinak (J.F. Borel i ostali: Immunology 32 1017/19777). Radi toga je uglavnom upotrebljavan kod presađivanja organa od drugih osoba (pluća, srce, bubreg, koštana srž, koža). Farmakološka ispitivanja su dokazala da on inhibira i humoralni i celularni imuni odgovor spriječavanjem proliferacije T-stanica i prekidanjem sinteze interleukina-2. Kasnije je rasčišćeno da je djelotvoran kod različitih vrsta autoimunih i upalnih oboljenja, kao štp su autoimuna hematolška oboljenja, ulceroznog kolitisa, Gravesove bolesti, multiple skleroze, psorijaze, kao i reumatskog artritisa. Daljnji pokusi su učinjeni radi liječenja zaraza uzrokovanih protozoom i također tumora. Važnost ove skupine je ukazana činjenicom da se brojni odgovarajući sintetski spojevi mogu pripraviti ugradnjom različitih aminokiselina i supstituenata (na primjer patenti EP 56782, CH 630062 i EP 29122). Cyclosporins are cyclic undecapeptides (peptides with 11 amino acids) that are N-methylated in several places and most of them have proven pharmacological effects. 25 members of this group of compounds that have been known for a long time are marked with the letters A to Z. Cyclosporin A was the first to be isolated, which is a natural substance isolated from the culture of Tolypocladium inflatum Gams strain (Helv. Chim. Acta 59. 1075/19767 ). First, this compound became known as a weak antifungal antibiotic, later its immunosuppressive effect attracted attention (J.F. Borel et al.: Immunology 32 1017/19777). For this reason, it is mainly used when transplanting organs from other people (lungs, heart, kidney, bone marrow, skin). Pharmacological tests have proven that it inhibits both humoral and cellular immune responses by preventing the proliferation of T-cells and interrupting the synthesis of interleukin-2. It was later clarified that it is effective in various types of autoimmune and inflammatory diseases, such as autoimmune hematological diseases, ulcerative colitis, Graves' disease, multiple sclerosis, psoriasis, as well as rheumatic arthritis. Further experiments were done to treat infections caused by protozoa and also tumors. The importance of this group is indicated by the fact that numerous corresponding synthetic compounds can be prepared by incorporating different amino acids and substituents (for example patents EP 56782, CH 630062 and EP 29122).

Najveći dio ciklosporina je pripravljen fermentacijom. Kao primjeri kultura upotrebljenih mikroorganizama su Cylindrocarpon Lucidum Booth (patentna specifikacija br. CH 589,716); Trichoderma polysporum Rifai (patentna specifikacija br. CH 603,790); Tolypocladium varium (patentna specifikacija br. HU 201,577). Na kraju fermentacije nastaje kompleks ciklosporina koji može sadržavati također druge nečistoće (sastojke kulturne sredine, sredstva protiv pjenjenja, metabolite, itd.) ovisno o karakteru postupka. Most cyclosporine is prepared by fermentation. Examples of cultures of microorganisms used are Cylindrocarpon Lucidum Booth (patent specification no. CH 589,716); Trichoderma polysporum Rifai (Patent Specification No. CH 603,790); Tolypocladium varium (Patent Specification No. HU 201,577). At the end of fermentation, a cyclosporine complex is formed, which may also contain other impurities (culture medium ingredients, anti-foaming agents, metabolites, etc.) depending on the nature of the procedure.

Općenito je produkt izoliran iz juhe postupcima ekstrakcije. To se može načiniti odijeljivanjem micelija od juhe centrifiigiranjem ili filtriranjem, nakon toga otapanjem aktivnog sastojka iz micelija s metanolom ili acetonom i ekstrahirajući filtrat s otapalima koja se ne miješaju s vodom. Jedan drugi poznati način provedbe je postupak bez filtriranja, upotrebom ekstrakcije cijele juhe s organskim otapalima koja se ne miješaju s vodom. Sadržaj otapala u organskom dijelu se uparava vakuumskom destilacijom. Generally, the product is isolated from the broth by extraction procedures. This can be done by separating the mycelium from the broth by centrifugation or filtration, then dissolving the active ingredient from the mycelium with methanol or acetone and extracting the filtrate with solvents immiscible with water. Another known method of implementation is the process without filtration, using the extraction of the whole broth with organic solvents that are immiscible with water. The solvent content in the organic part is evaporated by vacuum distillation.

Međutim, tijekom ekstrakcije s organskim otapalima se spojevi koji imaju lipidni karakter također prenose u organski dio, što uzrokuje poteškoće kod daljnjeg pročišćavanja. Radi odijeljivanja ovih spojeva postoje poznati postupci (na primjer švicarski patent br. 589,716 ili publicirana njemačka patentna prijava br. 2455859), gdje se nakon uklanjanja otapala s kojim se ekstrahiralo ostatak otopi u smjesi metanola i vode i tada se ekstrahira nekoliko puta s istim volumenom petrol etera. However, during extraction with organic solvents, compounds that have a lipid character are also transferred to the organic part, which causes difficulties in further purification. In order to separate these compounds there are known procedures (for example Swiss Patent No. 589,716 or published German Patent Application No. 2455859), where after removal of the extraction solvent, the residue is dissolved in a mixture of methanol and water and then extracted several times with the same volume petroleum ether.

Dijelovi petrol etera se skupe i ciklosporini se izoliraju sa smjesom metanola i vode. Aktivna tvar se prenese s višestrukom ekstrakcijom iz skupljenih dijelova metanola i vode u etilen klorid, koji se tada ispere s vodom i upari do suhog. Sirovi produkt pripravljen gornjim načinom i koji sadržava ciklosporine može se čistiti učinkovitije s jednom od kromatografskih metoda. The petroleum ether portions are collected and the cyclosporins are isolated with a mixture of methanol and water. The active substance is transferred with multiple extractions from the collected portions of methanol and water into ethylene chloride, which is then washed with water and evaporated to dryness. The crude product prepared by the above method and containing cyclosporins can be purified more efficiently with one of the chromatographic methods.

Prema načinu opisanom u US patentu br. 4,117,118 je smjesa ciklosporina prebačena prvo na stupac sa Sephadexom LH-20 i ispirana s metanolom, tada je ispirana redom na stupcu aluminij e vog oksida sa smjesom toluena i etilnog acetata (15%) i na stupcu silikagela sa smjesom kloroforma i etanola (2%). Uprkos ponovljenoj kromatografiji nastali produkt nije čist, nego je smjesa ciklosporina A i B. According to the method described in US patent no. 4,117,118, the cyclosporin mixture was first transferred to a column with Sephadex LH-20 and washed with methanol, then it was washed successively on an aluminum oxide column with a mixture of toluene and ethyl acetate (15%) and on a silica gel column with a mixture of chloroform and ethanol (2% ). Despite repeated chromatography, the resulting product is not pure, but a mixture of cyclosporine A and B.

Sličan kromatografski postupak je otkriven između ostalih u US patentu br. 4,215,199, gdje je grubo čišćenje sa smjesom kloroforma i metanola (98:2% volumski) na stupcu silikagela. Eluat je zatim uparen do suhog. Ostatak je otopljen u metanolu i podvrgnut kromatografiji na stupcu Sephadex LH-20 upotrebom metanola kao eluensa. Frakcije eluata su uparene do suhog, ostatak je otopljen u 98:2% volumskoj smjesi kloroforma i metanola. Podvrgnut je ponovno kromatografiji na silikagelu. Ciklosporin A se pojavio u prvom eluatu. Ova i slijedeće frakcije su odijeljene i čiste komponente su dobivene uparavanjam eluata. A similar chromatographic procedure is disclosed in, among others, US patent no. 4,215,199, where rough cleaning is with a mixture of chloroform and methanol (98:2% by volume) on a silica gel column. The eluate was then evaporated to dryness. The residue was dissolved in methanol and subjected to chromatography on a Sephadex LH-20 column using methanol as eluent. The eluate fractions were evaporated to dryness, the residue was dissolved in a 98:2 volume mixture of chloroform and methanol. It was re-chromatographed on silica gel. Cyclosporin A appeared in the first eluate. This and the following fractions were separated and the pure components were obtained by evaporating the eluates.

Prema njemačkom patentu br, DD 298276 uljasti sirovi produkt je otopljen u maloj količini kloroforma, zatim podvrgnut kromatografiji na stupcu aluminijevog oksida s kloroformom. Frakcije koje sadže ciklosporin A su uparene u vakuumu, otopljene u kloroformu, nanesene na sličan stupac i tada eluirane s kloroformom. Frakcije koje su sadržavala aktivnu tvar su ponovno uparene u vakuumu. Ostatku je dodan heksan i cikosporin A je kristalizirao. Produkt je ispran sa heksanom, zatim sušen i konačno prekristaliziran iz smjese etera i heksana ili acetona. According to German patent no., DD 298276, the oily crude product was dissolved in a small amount of chloroform, then subjected to chromatography on an aluminum oxide column with chloroform. Fractions containing cyclosporin A were evaporated in vacuo, dissolved in chloroform, applied to a similar column, and then eluted with chloroform. The fractions containing the active substance were re-evaporated in vacuo. Hexane was added to the residue and cycosporin A crystallized. The product was washed with hexane, then dried and finally recrystallized from a mixture of ether and hexane or acetone.

Prema mađarskom patentu br. 201577 sirovi produkt dobiven nakon uparavanja se može čistiti na stupcu silikagela eluiranjem sa smjesom kloroforma i metanola, postepeno povećavajući koncentraciju metanola. According to Hungarian patent no. 201577 crude product obtained after evaporation can be purified on a silica gel column by elution with a mixture of chloroform and methanol, gradually increasing the concentration of methanol.

Postupak počinje s čistim kloroformom i nastavlja se povećavajući metanol u eluensu u koracima od 0,5% volumski. Ciklosporin A je eluiran s 2% volumskih, ciklosporin B s 2,5% volumskih, ciklosporin C s 3% volumskim metanolom koji sadrži kloroform sa stupca. Komponente su dobivene uparavanjem frakcija. The procedure starts with pure chloroform and continues by increasing the methanol in the eluent in steps of 0.5% by volume. Cyclosporine A was eluted with 2% by volume, cyclosporine B with 2.5% by volume, cyclosporine C with 3% by volume methanol containing chloroform from the column. The components were obtained by matching the fractions.

Postupci koji su gore spomenuti opisuju uglavnom fermentacijske postupke ciklosporina, gdje je prvi cilj stupnjeva čišćenja utvrđivanje dobivenog produkta. Stoga je produkt izoliran samo u malim količinama i dane su samo fizikalne i kemijske osobine bez publiciranja podataka koji se odnose na čistoću produkta i količine nečistoća. The processes mentioned above describe mainly the fermentation processes of ciclosporin, where the first objective of the purification steps is to determine the product obtained. Therefore, the product is isolated only in small quantities and only physical and chemical properties are given without publishing data related to the purity of the product and the amount of impurities.

Rileger i ostali /Helv. Chim. Acta 59(4), str. 1075-92 (l976)7 su izolirali male količine čistog ciklosporina A i V ponavljajućim kromatografijama i s ostalim koracima čišćenja za identifikaciju i strukturnu analizu. Prema ovom članku sirovi produkt dobiven fermentacijom Trichoderma polysporum Rifai koji uglavnom sadrži ciklosporine A i C je odmašćen s metanolom i petrol eterom. Nakon uparavanja ostatak je otopljen u kloroformu i podvrgnut kromatografiji uz eluiranje s 98,5:1,5% volumskim gradijentom kloroforma i metanola kao eluensa. Čisti kristaliničan ciklospoprin A je dobiven daljnjom kromatografijom. Frakcija koja sadrži ciklosporin A je otopljena u metanolu i podvrgnuta kromatografiji na Sephadex LH-20 stupcu upotrebom metanola kao eluensa. Frakcije s produktom su uparene, otopljene u toluenu i podvrgnute kromatografiji na stupcu napunjenim s aluminij oksidom, upotrebom toluena kao eluensa, u nazočnosti povećavajuće koncentracije octene kiseline. Kristalinični produkt je dobiven nakon uparavanja frakcija i tretiranja s aktivnim ugljenom u alkoholnoj otopini. Rileger and others /Helv. Chem. Acta 59(4), p. 1075-92 (1976)7 isolated small amounts of pure cyclosporine A and V by repeated chromatography and with other purification steps for identification and structural analysis. According to this article, the crude product obtained from the fermentation of Trichoderma polysporum Rifai, which mainly contains cyclosporins A and C, was defatted with methanol and petroleum ether. After evaporation, the residue was dissolved in chloroform and subjected to chromatography eluting with a 98.5:1.5% volume gradient of chloroform and methanol as eluent. Pure crystalline cyclosporine A was obtained by further chromatography. The fraction containing cyclosporin A was dissolved in methanol and subjected to chromatography on a Sephadex LH-20 column using methanol as eluent. Fractions with the product were evaporated, dissolved in toluene and subjected to chromatography on a column filled with aluminum oxide, using toluene as eluent, in the presence of increasing concentrations of acetic acid. The crystalline product was obtained after evaporation of the fractions and treatment with activated carbon in an alcoholic solution.

Postupak pročišćavanja pogodan na industrijskom mjerilu je opisan u US patentu br. 5,382,655. Prema postupku je sirovi produkt, koji sadrži različite komponente ciklosporina, podvrgnut tretiranju zagrijavanjem prije kromatografije na stupcu silikagela sa smjesom kloroform-diklorometan-etanol i kloroform-etilni acetat-etanol. Dobiveni produkt je podvrgnut daljnjoj kromatografiji i prekristalizaciji, što je rezultiralo u čistoj kvaliteti produkta, dobroj za proizvodnju injekcija. A purification process suitable on an industrial scale is described in US patent no. 5,382,655. According to the procedure, the crude product, which contains various cyclosporin components, was subjected to treatment by heating before chromatography on a silica gel column with a mixture of chloroform-dichloromethane-ethanol and chloroform-ethyl acetate-ethanol. The obtained product was subjected to further chromatography and recrystallization, which resulted in a pure product quality, good for the production of injections.

Pročišćavanje sirovog produkta koji sadrži smjesu ciklosporina je vrlo teško, jer nečistoće koje imaju sličnu kemijsku strukturu su i vrlo slične u kromatografskim osobinama ciklosporina A kao glavnog produkta. Kao što ranije opisani postupci dokazuju, da bez obzira na upotrebljenu smjesu otapala i radi preklapanja kromatografskih pikova daljni kromatografski ili ostali koraci pročišćavanja se moraju provesti radi dobivanja određenih komponenti u čistom obliku. Dosad poznate postupke pročišćavanja općenito karakterizira upotreba velike količine otapala, primjena 3-4 različite vrste otapala ili smjesa otapala i 2-3 vrste punjenja stupca. Kao posljedica ovih činjenica se traži nekoliko vrsti kromatografskih tehnika i načina obnove (regeneracije) u postupku, što čini poteškoće u razvoju jednostavno konstruirane i jednoliko izvedive gospodarske tehnologije na idustrijskom mjerilu. Purification of a crude product containing a cyclosporin mixture is very difficult, because impurities that have a similar chemical structure are also very similar in the chromatographic properties of cyclosporin A as the main product. As the previously described procedures prove, regardless of the solvent mixture used and due to the overlapping of chromatographic peaks, further chromatographic or other purification steps must be carried out in order to obtain certain components in pure form. The previously known purification processes are generally characterized by the use of a large amount of solvent, the use of 3-4 different types of solvents or solvent mixtures and 2-3 types of column loading. As a consequence of these facts, several types of chromatographic techniques and methods of restoration (regeneration) are required in the process, which makes it difficult to develop a simply constructed and uniformly feasible economic technology on an industrial scale.

S točke gledišta zaštite okoline, daljni problemi izrastaju upotrebom kloriranih ugljikovodika, jer sve više i više zemalja čini napore za ograničenje njihove upotrebe. From an environmental point of view, further problems arise with the use of chlorinated hydrocarbons, as more and more countries make efforts to limit their use.

S obzirom na tvari za punjenje stupaca, primjena aluminij oksida za industrijske svrhe je vrlo dvojbena, jer kao posljedica male specifične površine su kapacitet punjenja i mogućnosti odijeljivanja vrlo mali. Nadalje, nema prednosti za industrijske svrhe, jer je aluminij oksid krut i lako lomljiv, pa stoga treba posebnu opremu i tehnologiju. Ona se ne može upotrebiti u često mijenjajućim čeličnim stupcima koji se upotrebljavaju u kemijskoj industriji. With regard to substances for filling the columns, the use of aluminum oxide for industrial purposes is very doubtful, because as a result of the small specific surface, the filling capacity and the separation possibilities are very small. Furthermore, it has no advantages for industrial purposes, because aluminum oxide is rigid and easily brittle, and therefore needs special equipment and technology. It cannot be used in frequently changing steel columns used in the chemical industry.

Punila vrste Sephadex su vrlo skupa i u slučaju kompleksa ciklosporina je njihova djelotvornost vrlo ograničena, jer su veličine molekula vrlo blizu jedna drugoj. Sephadex type fillers are very expensive and in the case of cyclosporine complexes their effectiveness is very limited, because the sizes of the molecules are very close to each other.

Cilj sadašnjeg izuma je razvoj nekog lako primjenjivog načina kromatografskog pročišćavanja u industrijskom mjerilu koje je pogodno za proizvodnju ciklosporina A koji sadrži mnogo manje nečistoća tako da je njegova upotreba u medicinskoj praksi sigurna. The aim of the present invention is to develop an easily applicable method of chromatographic purification on an industrial scale which is suitable for the production of cyclosporin A which contains much less impurities so that its use in medical practice is safe.

Naš cilj je bio razviti takvu tehnologiju kromatografskog pročišćavanja, koja zahtjeva samo jednu vrstu smjese otapala i samo jednu vrstu punjenja stupca. Our goal was to develop such a chromatographic purification technology, which requires only one type of solvent mixture and only one type of column loading.

Zbog svojih prednosnih osobina - visoke specifične površine, visoke veličine pora, dobre mogućnosti sorpcije, lakog rukovanja i relativno niske cijene - silikagel je upotrebljen kao punilo stupca. Trebala se odabrati idealna smjesa otapala i način za ovo punilo, što je pogodno za odijeljivanje ciklosporinskih komponenata uz visoku selektivnost. Due to its advantageous properties - high specific surface area, high pore size, good sorption capacity, easy handling and relatively low price - silica gel is used as a column filler. An ideal mixture of solvents and a method for this filler had to be chosen, which is suitable for the separation of ciclosporin components with high selectivity.

Kao rezultat naših pokusa smo prepoznali da se naš cilj može ostvariti s kromatografijom u više stupnjeva na stupcu silikagela upotrebljavajući kao smjesu otapala eluens čiji je glavni sastojak toluen. Iznenađujuće je nađeno da se čak ciklosporin U i L komponentne koje su najbliže ciklosporinu A mogu odijeliti kromatografijom na stupcu silikagela u tri stupnja upotrebljavajući toluen koji sadrži aceton kao eluens. Ove komponente se razlikuju od ciklosporina A samo u metilnoj skupini. As a result of our experiments, we recognized that our goal can be achieved with multi-step chromatography on a silica gel column using an eluent whose main ingredient is toluene as a solvent mixture. Surprisingly, it was found that even cyclosporin U and L components, which are closest to cyclosporin A, can be separated by chromatography on a silica gel column in three stages using toluene containing acetone as eluent. These components differ from cyclosporine A only in the methyl group.

Prema prvom primjeru prijave WO 94/16091 je također upotrebljena smjesa otapala toluena i acetona u jednom stupnju. Dobiveni produkt je prekristaliziran iz smjese otapala etera i heksana. (Iskorištenje 66,6%). Optičko skretanje produkta je dovoljno, ali je njegova točka tališta značajno niža od one opisane u literaturi, što ukazuje da produkt nije čist. Stoga, čak i prekristalizacija iz otapala će dati produkt slabe čistoće i niskog iskorištenja. According to the first example of the application WO 94/16091, a solvent mixture of toluene and acetone was also used in one step. The obtained product was recrystallized from a solvent mixture of ether and hexane. (Utilization 66.6%). The optical deviation of the product is sufficient, but its melting point is significantly lower than that described in the literature, which indicates that the product is not pure. Therefore, even recrystallization from a solvent will give a product of low purity and low yield.

Postupak prema sadašnjem izumu je pogodan za odijeljivanje najučestalijih nečistoća, kao što su ciklosporini B i C koji su nazočni u najvećim količinama i još više ciklosporin D, U i L komponente koji su nazočni u tragovima. Sadržaj ciklosporina B i C u konačnom produktu ciklosporina A dobivenog na ovaj način je manji od 0,02% težinski, dok je sadržaj ciklosporina L, U i D ispod 0,05% težinski. The process according to the present invention is suitable for separating the most common impurities, such as cyclosporin B and C, which are present in the largest amounts, and even more cyclosporin D, U and L components, which are present in traces. The content of cyclosporine B and C in the final product cyclosporine A obtained in this way is less than 0.02% by weight, while the content of cyclosporine L, U and D is below 0.05% by weight.

Cilj našeg izuma je poboljšani postupak pročišćavanja ciklosporina A iz sirovog produkta koji sadrži ciklosporinski kompleks, koji također omogućava proizvodnju krajnje čistog ciklosporina A u velikom mjerilu, s kromatografskom metodom na stupcu silikagela upotrebom kromatografije u više stupnjeva sa smjesom otapala koja sadrži toluen kao glavni sastojak. Druga novost postupka je to što mi primjenjujemo krajnje visoko opterećenje stupca. U uobičajenoj kromatografskoj opterećenje stupca sa sirovim produktom nije veće od 5-10% punjenja stupca, a ta vrijednost je niža za ciklosporine. The aim of our invention is an improved process for the purification of cyclosporine A from a crude product containing a cyclosporine complex, which also enables the production of extremely pure cyclosporine A on a large scale, with a silica gel column chromatographic method using multistage chromatography with a solvent mixture containing toluene as the main ingredient. Another novelty of the procedure is that we apply an extremely high column load. In the usual chromatographic column loading with the crude product is not higher than 5-10% of the column loading, and this value is lower for ciclosporins.

Kromatografija u više stupnjeva, smjesa otapala koja sadrži toluen kao glavni sastojak i visoko opterećenje stupca sa sirovim produktom su međusobno povezani i željeni rezultat se može postići samo njihovom kombiniranom upotrebom. Stoga se sve tri gornje osobine trebaju upotrijebiti istovremeno tako da bi se dobio krajnje čisti produkt. Chromatography in multiple stages, solvent mixture containing toluene as the main ingredient and high loading of the column with the crude product are interconnected and the desired result can be achieved only by their combined use. Therefore, all three of the above properties should be used simultaneously in order to obtain an extremely pure product.

Prema sadašnjem izumu se poželjno primjenjuju 2-4 kromatografska stupnja jedan za drugim i najkorisnije tri stupnja. According to the present invention, 2-4 chromatographic stages are preferably applied one after the other, and three stages are most useful.

Preopterećenje stupca sa sirovim produktom je najviše u prvom stupnju kromatografije. Konačno odijeljivanje aktivne tvari i nečistoća dozvoljava upotrebu visokog opterećenja stupca u dva slijedeća kromatografska stupnja. Column overload with crude product is highest in the first stage of chromatography. The final separation of the active substance and impurities allows the use of high column loading in the two subsequent chromatographic stages.

Za pročišćavanje je poželjna upotreba smjese otapala toluen-aceton, koja sadržava uglavnom 30% volumnih acetona. For purification, it is preferable to use a toluene-acetone solvent mixture, which contains mostly 30% acetone by volume.

Prema jednom drugom poželjnom načinu je upotrebljena smjesa otapala toluen-etilni acetat u kojoj je koncentracija etilnog acetata ispod 35% volumnih. According to another preferred method, a toluene-ethyl acetate solvent mixture is used in which the concentration of ethyl acetate is below 35% by volume.

U postupku pročišćavanja najmanje jedanput je poželjna eluacija s gradijentom. In the purification process, elution with a gradient is preferred at least once.

Prema našim pokusima je nađeno da je za pročišćavanje kompleksa ciklosporina provedenog u našem slučaj1; poželjna upotreba 10-30% volumnih i poželjnije upotreba 13-18% volumnih acetona i 10-35% volumnih ili 15-20% volumnih etilnog acetata. According to our experiments, it was found that for the purification of the cyclosporine complex carried out in our case1; preferably use 10-30% by volume and more preferably use 13-18% by volume of acetone and 10-35% by volume or 15-20% by volume of ethyl acetate.

Prema mogućem načinu sadašnjeg izuma eluiranje je učinjeno u slučaju kromatografije u tri stupnja s toluenom koji je sadržavao 15% volumnih acetona ili 18% volumnih etilnog acetata. U prvom stupnju je odijeljen veći dio ciklosporina C, u drugom stupnju uklonjen je veći dio ciklosporinskih komponenti B, L i U, i konačno u trećem stupnju količine L i U komponenti i ostalih neutvrđenih nečistoća se mogu smanjiti ispod 0,05% težinskih. U prvom stupnju su gubici ciklosporina A minimalni, međutim u predfrakcijama drugog i trećeg stupnja određena količina ciklosporina A je uklonjena skupa s ciklosporin D komponentom koja je vrlo blizu ciklosporinu A. Ciklosporin A se može povratiti u vrlo čistom obliku u četvrtom stupnju. According to the possible method of the present invention, the elution was done in the case of chromatography in three stages with toluene containing 15% by volume of acetone or 18% by volume of ethyl acetate. In the first stage, most of cyclosporine C is separated, in the second stage, most of cyclosporin components B, L and U are removed, and finally in the third stage, the amounts of L and U components and other undetermined impurities can be reduced below 0.05% by weight. In the first stage, the losses of cyclosporin A are minimal, however, in the prefractions of the second and third stages, a certain amount of cyclosporin A is removed together with the cyclosporin D component, which is very close to cyclosporin A. Cyclosporin A can be recovered in a very pure form in the fourth stage.

Za usporedbu su u tablici ispod dani profili nečistoća ciklosporina A u USP standardu, ciklosporin A aktivni sastojak od SANDIMMUN® injekcija i također podaci ciklosporin A produkta pripravljenog u sadašnjem izumu. For comparison, the impurity profiles of cyclosporin A in the USP standard, the cyclosporin A active ingredient from SANDIMMUN® injections and also the data of the cyclosporin A product prepared in the present invention are given in the table below for comparison.

USP standard SANDIMMUN® injekcija. Produkt prema Primjeru 1 USP standard SANDIMMUN® injection. Product according to Example 1

nečistoće %tež. %tež. %tež. impurities %wt. %wt. %wt.

CiklosporinC - 0,17 0,05 CyclosporinC - 0.17 0.05

CiklosporinB - 0,21 0,05 CyclosporinB - 0.21 0.05

nepoznat 0,09 0,35 0,05 unknown 0.09 0.35 0.05

CiklosporinL 0,05 0,35 0,05 CyclosporinL 0.05 0.35 0.05

CiklosporinU - - 0,05 CyclosporinU - - 0.05

CiklosporinD 0,12 - 0,05 CyclosporinD 0.12 - 0.05

Iz podataka se može vidjeti, kvaliteta ciklosporina A dobivenog sadašnjim izumom značajno premašuje parametre SANDIMMUN® injekcije, štoviše premašuje također čak i zahtjeve USP. Postupak prema sadašnjem izumu je primjenjiv za pročišćavanje sirovog produkta također i u malom i u velikom mjerilu. It can be seen from the data, the quality of cyclosporin A obtained by the present invention significantly exceeds the parameters of SANDIMMUN® injection, moreover, it also exceeds even USP requirements. The process according to the present invention is applicable for the purification of the raw product also on a small and large scale.

Pored činjenice da je postupak prema sadašnjem izumu sposoban pripraviti čisti ciklosporin A, on posjeduje također i neke tehnološke prednosti.. Zbog toga što je upotrebljena samo jedna tehnološka metoda (kromatografija), postupak pročišćavanja se provodi jednoliko, štoviše on je ponovljiv i može se pretvoriti u kontinuirani postupak. Nadalje, postoji posebna prednost, da s obzirom da je upotrebljena samo jedna vrsta smjese otapala u tri kromatografska stupnja, regeneracija i stupaca i otapala je postala jednostavnija. In addition to the fact that the process according to the present invention is capable of preparing pure cyclosporine A, it also has some technological advantages. Due to the fact that only one technological method (chromatography) is used, the purification process is carried out uniformly, moreover it is reproducible and can be converted in a continuous process. Furthermore, there is a particular advantage, that since only one type of solvent mixture is used in three chromatographic stages, the regeneration of both columns and solvents has become simpler.

Daljnja posebna korist postupka je što u prvom stupnju kromatografije visoko vezajuće nečistoće ostaju na stupcu, što uvelike olakšava regeneraciju punila stupca u dva slijedeća stupnja. Izum je predstavljen s primjerima dolje bez ograničavanja zaštitnih zahtjeva samo na njih. A further special benefit of the process is that in the first step of chromatography, highly binding impurities remain on the column, which greatly facilitates the regeneration of the column filler in the two following steps. The invention is presented with the examples below without limiting the claims to them only.

U usporednom primjeru (Primjer 4) je prikazano da sa primjenom smjese otapala diklorometan-aceton u kromatografiji u 3 stupnja, dosad općenito upotrebljena u poznatim postupcima, ne bi mogla proizvesti konačni produkt u sličnoj čistoći. In a comparative example (Example 4) it is shown that with the use of a dichloromethane-acetone solvent mixture in 3-stage chromatography, generally used in known procedures, it would not be able to produce the final product in a similar purity.

Primjer 1 Example 1

Pročišćavanje ciklosporinskog sirovog produkta s tri kromatografska stupnja uz primjenu smjese otapala toluen-aceton Purification of cyclosporine crude product with three chromatographic stages using a toluene-acetone solvent mixture

Kvaliteta početnog sirovog produkta: Quality of the initial raw product:

Ciklosporin A sadržaj 60,9% težinski Cyclosporine A content 60.9% by weight

Ciklosporin B sadržaj 11,2% težinski Cyclosporine B content 11.2% by weight

Ciklosporin C sadržaj 8,3% težinski Cyclosporin C content 8.3% by weight

Ciklosporin L sadržaj 1,79% težinski Cyclosporine L content 1.79% by weight

Ciklosporin U sadržaj 1,58% težinski Cyclosporin U content 1.58% by weight

Ciklosporin D sadržaj 1,25% težinski Cyclosporin D content 1.25% by weight

1. stupanj 1st degree

Kromatografija je načinjena s dva kromatografska stupca povezana u seriju, svaki je 8 litarski s omotačem, promjera 10 cm, dužine 100 cm. Svaki od dva stupca sadrži 3,95-3,95 kg Merck vrste Kieselgel silikagela s veličinom zrna 0,04-0,063 mm. U prvoj kromatografiji oba stupca sadrže svježi silikagel. U slučaju slijedeće kromatografije je prvi stupac odijeljen i drugi stupac je povezan na njegovo mjesto sadržavajući svježi silikagel. Nadalje je za svaku kromatografiju upotrebljen samo jedan novi stupac. Chromatography was performed with two chromatographic columns connected in series, each 8-liter column with a jacket, diameter 10 cm, length 100 cm. Each of the two columns contains 3.95-3.95 kg of Merck type Kieselgel silica gel with a grain size of 0.04-0.063 mm. In the first chromatography, both columns contain fresh silica gel. In the case of the following chromatography, the first column is separated and the second column is connected in its place containing fresh silica gel. Furthermore, only one new column was used for each chromatography.

Priprava sirovog produkta Preparation of raw product

4,1 kg, čistoće 60,9% volumnih, sirovog produkta, prethodno otopljeno u 15 L toluena, je naneseno na par stupaca povezanih u seriju,. Dobiveno je 19 L otopine koja je nanesena na vrh prvog stupca kroz filter s brzinom od 2,4 L/h. Nakon nanašanja tvar je eluirana sa smjesom otapala od 13:87% volumnih aceton-toluena dok volumen eluata na dnu drugog stupca nije bio 39 litara. Sadržaj ciklosporina u eluatu je analiziran s TLC. Frakcije koje nisu sadržavale ciklosporin su skupljene kao otpad. 28 litara eluata nakon pojave ciklosporina je smatrano kao glavna frakcija. Sadržaj suhe tvari međuprodukta I dobivenog na taj načinje 3,23 kg. 4.1 kg, purity 60.9% by volume, crude product, previously dissolved in 15 L of toluene, was applied to a pair of columns connected in series. 19 L of solution was obtained, which was applied to the top of the first column through a filter at a rate of 2.4 L/h. After application, the substance was eluted with a solvent mixture of 13:87% acetone-toluene by volume until the volume of eluate at the bottom of the second column was 39 liters. The content of cyclosporine in the eluate was analyzed by TLC. Fractions that did not contain ciclosporin were collected as waste. 28 liters of eluate after the appearance of ciclosporin was considered as the main fraction. The dry matter content of intermediate product I obtained in this way is 3.23 kg.

Kvaliteta: ciklosporin A 75% težinski Quality: cyclosporin A 75% by weight

ciklosporin B 10,1% težinski cyclosporin B 10.1% by weight

ciklosporin C 1,6% težinski ciclosporin C 1.6% by weight

ciklosporin L 1,7% težinski ciclosporin L 1.7% by weight

ciklosporin U 1,5% težinski ciclosporin U 1.5% by weight

ciklosporin D 1,3% težinski ciclosporin D 1.3% by weight

Iskorištenje preračunato na ciklosporin A: 97%. Recovery converted to ciclosporin A: 97%.

2. stupanj 2nd degree

Odijeljivanje je načinjeno u l metar dugačkom stupcu od 8 litara s plastom. Stupac je sadržavao Merck Kieselgel 60 silikagel (0,015-0,040 mm). Masa punila 3,95 kg. Oko 3 litre volumena s 370 g suhe tvari koja je sadržavala otopinu međuprodukta I dobivenog u prvoj kromatografiji je naneseno na stupac s brzinom od 2,4 L/h i tada je isprana s l litrom toluena. Separation was done in a 1 meter long column of 8 liters with a layer. The column contained Merck Kieselgel 60 silica gel (0.015-0.040 mm). Weight of filler 3.95 kg. About 3 liters of volume with 370 g of dry matter containing a solution of intermediate I obtained in the first chromatography was applied to the column at a rate of 2.4 L/h and then washed with 1 liter of toluene.

Nakon nanašanja tvari stupac je eluiran sa smjesom 10 litara 15:85% volumnih aceton:toluen i zatim s 20 litara 25:75% volumnih otopina aceton:toluen. After applying the substance, the column was eluted with a mixture of 10 liters of 15:85% by volume acetone:toluene and then with 20 liters of 25:75% by volume acetone:toluene solutions.

Brzina protijeka otapala do 17 litara frakcija je 2,4 L/h, a zatim od 18 litarske frakcije 5 L/h. Frakcije se analizirane s TLC. Frakcije od 1-11 litara su otpad, frakcije 12-19 litara se smatraju kao kritične I frakcije i uzorci su uzimani iz njih. Profili nečistoća u njima su analizirani s HPLC i one su smatrane kao predfrakcije ili su skupljene s glavnim frakcijama. Na taj način se može dobiti 80 g suhe tvari koja sadrži predfrakcije, koje su tada uparene do suhog. Frakcije od 20-25 litara su skupljene kao glavne frakcije. Frakcije od 26-31 litre su se smatrale kritičnim i nakon analize su skupljene s glavnom frakcijom ili tretirane kao postfrakcije. The solvent flow rate up to 17 liter fractions is 2.4 L/h, and then from the 18 liter fraction 5 L/h. Fractions were analyzed by TLC. Fractions of 1-11 liters are waste, fractions of 12-19 liters are considered critical I fractions and samples were taken from them. Their impurity profiles were analyzed by HPLC and they were considered as pre-fractions or pooled with the main fractions. In this way, 80 g of dry matter containing pre-fractions can be obtained, which are then evaporated to dryness. Fractions of 20-25 liters were pooled as main fractions. Fractions of 26-31 liters were considered critical and after analysis were pooled with the main fraction or treated as post-fractions.

Glavna frakcija dobivena na gornji način je uparena do suhog u film uparivaču opremljenom s oscilirajućom mješalicom. Dobiveno je 234 g međuprodukta II s iskorištenjem od 80% i s kvalitetom kako slijedi: The main fraction obtained in the above manner was evaporated to dryness in a film evaporator equipped with an oscillating mixer. 234 g of intermediate II were obtained with a yield of 80% and the quality as follows:

ciklosporin A 95% težinski, cyclosporin A 95% by weight,

ciklosporin U 1,2% težinski ciclosporin U 1.2% by weight

ciklosporin L 0,7% težinski ciclosporin L 0.7% by weight

ciklosporin B <0,1 % težinski ciclosporin B <0.1% by weight

ciklosporin D 0,5% težinski ciclosporin D 0.5% by weight

ciklosporin C 0,1 % težinski ciclosporin C 0.1% by weight

3. stupanj 3rd degree

Kromatografija je načinjena na stupcima iste konstrukcije i geometrijske veličine kako je opisano u stupnjevima 1.12. Pripravljena je otopina koja sadrži 220 g suhe tvari u 1,7 litara toluena iz međuprodukta II dobivenog u drugom stupnju i nanesena je na vrh stupca s brzinom od 2,4 L/h, zatim je isprana s 1 litrom toluena. Chromatography was performed on columns of the same construction and geometric size as described in steps 1.12. A solution containing 220 g of dry matter in 1.7 liters of toluene from intermediate II obtained in the second step was prepared and applied to the top of the column at a rate of 2.4 L/h, then washed with 1 liter of toluene.

Stupac je eluiran sa smjesom 20 litara 15:85% volumnih aceton:toluena i zatin je ciklosporin eluiran sa smjesom 20 L 25:75% volumnih aceton:toluena. Brzina eluiranja je do 31 litre frakcija bila 2,4 L/h, zatim od 32 litre frakcija 5 L/h. Frakcije 1-18 litara su otpad, frakcije od 19-23 litre su predfrakcije i smatrane su kao kritične frakcije L Od njih su uzeti uzorci radi analize sadržaja suhe tvari i profila nečistoća s HPLC. Nakon analize su tretirane kao predfrakcije ili skupljene s glavnim frakcijama. Frakcije 29-38 litara su skupljene kao glavne frakcije. Frakcije 39-41 litara su skupljene u obroke od 1 litre i smatrane su kao kritične frakcije II. Nakon analize one su spojene s glavnom frakcijom ili tretirane kao postfrakcije. Kao rezultat skupljanja frakcija i nakon uparavanja do suhog može se dobiti 70 g predfrakcija. The column was eluted with a mixture of 20 liters of 15:85% by volume acetone:toluene and the cyclosporine was eluted with a mixture of 20 L 25:75% by volume acetone:toluene. The elution rate up to 31 liter fractions was 2.4 L/h, then from 32 liter fractions it was 5 L/h. Fractions of 1-18 liters are waste, fractions of 19-23 liters are pre-fractions and were considered as critical fractions L Samples were taken from them for analysis of dry matter content and impurity profile with HPLC. After analysis, they were treated as pre-fractions or pooled with the main fractions. Fractions 29-38 liters are pooled as main fractions. Fractions of 39-41 liters were collected in portions of 1 liter and were considered as critical fractions II. After analysis, they were combined with the main fraction or treated as post-fractions. As a result of collection of fractions and after evaporation to dryness, 70 g of pre-fractions can be obtained.

Glavne frakcije su skupljene i nakon uparavanja do suhog dobiveno je 157 g čistog ciklosporina s iskorištenjem od 75%. The main fractions were collected and, after evaporation to dryness, 157 g of pure ciclosporin were obtained with a yield of 75%.

Kvalitet produkta je slijedeći: The quality of the product is as follows:

ciklosporin A 99,6% težinski cyclosporin A 99.6% by weight

ciklosporin L <0,05% težinski ciclosporin L <0.05% by weight

ciklosporin U <0,05% težinski ciclosporin U <0.05% by weight

ciklosporin D <0,05% težinski ciclosporin D <0.05% by weight

ciklosporin B <0,02% težinski cyclosporine B <0.02% by weight

ciklosporin C <0,02% težinski ciclosporin C <0.02% by weight

Primjer 2 Example 2

Kromatografsko pročišćavanje sirovog ciklosporinskog produkta na stacionarnom stupcu silikagela u četiri stupnja s primjenom smjese otapala toluen-aceton ili toluen-etilni acetat. Chromatographic purification of the crude ciclosporin product on a stationary column of silica gel in four stages using a solvent mixture of toluene-acetone or toluene-ethyl acetate.

Sirovi ciklosporinski produkt je pročišćen s kromatografijom u tri stupnja opisanom u Primjeru 1. Dobivene predfrakcije u kromatografiji s tri stupnja su pročišćene u četvrtom stupnju na stacionarnoj fazi sa smjesom otapala toluen-etilni acetat. The crude ciclosporin product was purified with the three-stage chromatography described in Example 1. The obtained pre-fractions in the three-stage chromatography were purified in the fourth stage on the stationary phase with a solvent mixture of toluene-ethyl acetate.

4. stupanj 4th degree

Konstrukcija i geometrijska veličina kromatografskog stupca je ista kako je opisana u Primjeru 1. Stupac sadrži Merck Kieselgel 60 vrstu silikagela (0,015-0,040 mm) kako je napisano u Primjeru 1. The construction and geometric size of the chromatographic column is the same as described in Example 1. The column contains Merck Kieselgel 60 grade silica gel (0.015-0.040 mm) as described in Example 1.

Na stupac je nanesen koncentrat dobiven iz predfrakcija u količini od 260 g tvari otopljene u 2,5 litre toluena s brzinom proticanja od 2,4 L/h. The concentrate obtained from the pre-fractions was applied to the column in the amount of 260 g of substance dissolved in 2.5 liters of toluene with a flow rate of 2.4 L/h.

sadržaj ciklosporina A 80,6% težinski cyclosporin A content 80.6% by weight

sadržaj ciklosporina D 4,2% težinski ciclosporin D content 4.2% by weight

Naneseni uzorak je ispran s l litrom toluena, zatim je stupac eluiran sa smjesom 20 litara 17:83% volumnih etilni acetattoluen s brzinom proticanja od 2,4 L/h, zatim se eluiranje nastavilo sa smjesom 40 litara 28:72% volumnih etilni acetattoluen. The applied sample was washed with 1 liter of toluene, then the column was eluted with a mixture of 20 liters of 17:83% by volume ethyl acetate toluene with a flow rate of 2.4 L/h, then the elution continued with a mixture of 40 liters of 28:72% by volume ethyl acetate toluene.

U smislu skupljanja su frakcije od 1-19 litara otpad. Nakon HPLC analize su frakcije od 20-25 litara su otpad ili skupljene s glavnom frakcijom. Frakcije od 26-35 litara su skupljene kao glavne frakcije. Nakon uzorkovanja i HPLC analize frakcije od 36-42 litre su ili skupljene kao glavne ili tretirane kao otpad. Glavne frakcije skupljene od 23-42 litre, opisane gore, su uparene do suhog. Na taj načinje dobiveno 195 g čistog ciklosporina A koji je sadržavao 99,6% težinski aktivnog sastojka s iskorištenjem od 75% i sa slijedećom kvalitetom: In terms of collection, fractions of 1-19 liters are waste. After HPLC analysis, fractions of 20-25 liters are waste or collected with the main fraction. Fractions of 26-35 liters were pooled as main fractions. After sampling and HPLC analysis, fractions of 36-42 liters were either collected as main or treated as waste. The main fractions collected from 23-42 liters, described above, were evaporated to dryness. In this way, 195 g of pure cyclosporine A was obtained, which contained 99.6% by weight of the active ingredient with a utilization of 75% and with the following quality:

ciklosporin A 99,6% težinski cyclosporin A 99.6% by weight

ciklosporin D <0,05% težinski ciclosporin D <0.05% by weight

ciklosporin U - ciclosporin U -

ciklosporin L - ciclosporin L -

Primjer 3 Example 3

Kromatografsko pročišćavanje sirovog ciklosporinskog produkta na stacionarnom stupcu silikagela u dva stupnja s primjenom smjese otapala toluen-aceton Chromatographic purification of the crude ciclosporin product on a stationary silica gel column in two stages using a toluene-acetone solvent mixture

Ciklosporinski sirovi produkt je pročišćen prema istom postupku opisanom u 1. stupnju Primjera 1 dobijajući ciklosporinski međuprodukt I iste kvalitete. Daljnji postupak je slijedeći: The cyclosporine crude product was purified according to the same procedure described in the 1st step of Example 1, obtaining cyclosporine intermediate I of the same quality. The further procedure is as follows:

2. stupanj 2nd degree

Na stupac je naneseno 3 litre međuprodukta I koji je sadržavao 370 g suhe tvari s brzinom proticanja od 2,4 L/h, zatim je naneseni uzorak ispran s 1 litrom toluena. 3 liters of intermediate product I containing 370 g of dry matter was applied to the column at a flow rate of 2.4 L/h, then the applied sample was washed with 1 liter of toluene.

Nakon nanašanja je stupac eluiran sa smjesom 10 litara 15:85% vol, aceton:toluena, zatim je eluiranje nastavljeno sa smjesom 20 litara 25:75% vol. aceton:toluena. After application, the column was eluted with a mixture of 10 liters of 15:85% by volume, acetone:toluene, then the elution was continued with a mixture of 20 liters of 25:75% by volume of acetone:toluene.

Brzina proticanja otapala je 2,4 L/h do 17 litara i 5L/h od 18 litre. Prema TLC i HPLC određivanju su frakcije od 1-11 litre otpad, od 12-20 su frakcije predfrakcije, od 21-24 frakcije se smatraju glavnim frakcijama i frakcije od 25 do kraja su tretirane kao postfrakcije. Frakcije dobivene ispiranjem s acetonom su otpad. The solvent flow rate is 2.4 L/h up to 17 liters and 5 L/h from 18 liters. According to TLC and HPLC determination, fractions from 1-11 liters are waste, fractions from 12-20 are pre-fractions, fractions from 21-24 are considered main fractions and fractions from 25 to the end are treated as post-fractions. Fractions obtained by washing with acetone are waste.

Nakon frakcioniranja su skupljene glavne frakcije i uparene do suhog dajući 114 g ciklosporina A s iskorištenjem od 41% i s dobrom kvalitetom produkta kako je napisano u Primjeru 1. After fractionation, the main fractions were collected and evaporated to dryness to give 114 g of cyclosporin A in 41% yield and with good product quality as described in Example 1.

Primjer 4 Example 4

Usporedni primjer za pročišćavanje sirovog produkta s kromatografiiom u 3 stupnja s primjenom smjese otapala diklorometan-aceton Comparative example for the purification of the crude product with chromatography in 3 stages using the dichloromethane-acetone solvent mixture

Kvaliteta početnog ciklosporinskog sirovog produkta (isti kako je upotrebljen u Primjeru 1): Quality of starting cyclosporine crude product (same as used in Example 1):

ciklosporin A 60,9% težinski cyclosporine A 60.9% by weight

ciklosporin B 11,2% težinski cyclosporin B 11.2% by weight

ciklosporin C 8,3% težinski ciclosporin C 8.3% by weight

ciklosporin L 1,79% težinski ciclosporin L 1.79% by weight

ciklosporin U 1,58% težinski ciclosporin U 1.58% by weight

ciklosporin D 1,25% težinski ciclosporin D 1.25% by weight

1. stupanj 1st degree

Kromatografska oprema i nanašnje je isto kako je opisano u Primjeru 1. Na jedan par stupaca povezan u seriju je naneseno 4,1 kg sirovog produkta čistoće 60,9% u 15 litara diklorometanske otopine. Chromatographic equipment and application is the same as described in Example 1. 4.1 kg of crude product with a purity of 60.9% in 15 liters of dichloromethane solution was applied to one pair of columns connected in series.

Nakon nanašnja uzorka stupac je eluiran s diklorometanom s brzinom proticanja od 2,4 L/h dok se nije skupilo 35 litara eluata. After sample application, the column was eluted with dichloromethane at a flow rate of 2.4 L/h until 35 liters of eluate were collected.

Frakcije od 1-10 litara su otpad, dok su frakcije od 11-35 litara smatrane kao glavne. Sadržaj suhe tvari međuprodukta I dobivenog na ovaj načinje 2,9 kg, sadržaj aktivnog sastojka je 75% težinski. Fractions of 1-10 liters are waste, while fractions of 11-35 liters are considered major. The content of dry matter of the intermediate product I obtained in this way is 2.9 kg, the content of the active ingredient is 75% by weight.

2. stupanj 2nd degree

Kromatografska oprema i punjenje je isto kako je opisano u Primjeru l. Chromatographic equipment and charge is the same as described in Example 1.

Na stupac je na vrhu naneseno 3 litre međuprodukta I koji je sadržavao 350 g suhe tvari s brzinom proticanja od 2,4 L/h. Eluiranje je načinjeno sa smjesom 10 litara aceton:diklorometana 1:9 vol. omjer, zatim nastavljeno sa smjesom 25 litara aceton:diklorometana 2:8 vol. omjer i okončano s acetonom 2,4 L/h. Prema TLC analizi je volumen predfrakcija 13 litara, volumen glavne frakcije je bio 22 litre i volumen postfrakcija je bio 11 litara. 22 litre glavne frakcije je upareno do suhog i kao posljedica toga je dobiveno 220 g međuprodukta II s čistoćom od 91%. The column was top loaded with 3 liters of intermediate I containing 350 g of dry matter at a flow rate of 2.4 L/h. Elution was done with a mixture of 10 liters of acetone:dichloromethane 1:9 volume ratio, then continued with a mixture of 25 liters of acetone:dichloromethane 2:8 volume ratio and finished with acetone 2.4 L/h. According to TLC analysis, the volume of the prefractions was 13 liters, the volume of the main fraction was 22 liters and the volume of the postfractions was 11 liters. 22 liters of the main fraction were evaporated to dryness and as a result 220 g of intermediate II with a purity of 91% were obtained.

3. stupanj 3rd degree

Kromatografska oprema i nanošenje je isto kako je opisano u Primjeru 1. Chromatographic equipment and application is the same as described in Example 1.

Na punilo je naneseno 220 g međuprodukta II propuštajući diklorometanski koncentrat s brzinom od 2,4 L/h na stupac. Eluiranje je načinjeno sa smjesom 20 litara aceton:diklorometana 1:9 vol. omjer, zatim nastavljeno sa smjesom 30 litara aceton:diklorometan 2:8 vol. omjer i okončano s acetonom 2,4 L/h. Prvih 26 litara frakcija je smatrano kao predfrakcije, zatim je skupljeno26 litara glavne frakcije i konačno 11 litara postfrakcija. 220 g of intermediate II was applied to the packing by passing dichloromethane concentrate at a rate of 2.4 L/h per column. Elution was done with a mixture of 20 liters of acetone:dichloromethane 1:9 volume ratio, then continued with a mixture of 30 liters of acetone:dichloromethane 2:8 volume ratio and finished with acetone 2.4 L/h. The first 26 liters of fractions were considered as pre-fractions, then 26 liters of the main fraction and finally 11 liters of post-fractions were collected.

26 litara glavne frakcije je upareno do suhog i kao posljedica je dobiveno 140 g međuprodukta III sa slijedećom kvalitetom: 26 liters of the main fraction were evaporated to dryness and as a result 140 g of intermediate product III with the following quality were obtained:

ciklosporin A 98,6% težinski cyclosporine A 98.6% by weight

ciklosporin U 0,6% težinski ciclosporin U 0.6% by weight

ciklosporin D 0,3% težinski ciclosporin D 0.3% by weight

ciklosporin L 0,2% težinski ciclosporin L 0.2% by weight

ciklosporin B 0,1% težinski cyclosporin B 0.1% by weight

ciklosporin C 0,1% težinski ciclosporin C 0.1% by weight

Claims (13)

1. Postupak pročišćavanja za pripravu ciklosporina A visoke čistoće iz ciklosporinskog kompleksa koji sadržava sirovi produkt s kromatografskom metodom na stupcu silikagela, naznačen time, što se upotrebljava kromatografija u više stupnjeva sa smjesom otapala koja sadržava toluen kao glavni sastojak.1. Purification procedure for the preparation of high-purity cyclosporine A from the cyclosporine complex containing the crude product with the chromatography method on a silica gel column, indicated by the fact that multistage chromatography is used with a mixture of solvents containing toluene as the main ingredient. 2. Postupak prema zahtjevu 1, naznačen time, što se 2-4 kromatografska stupnja provode jedan za drugim.2. The method according to claim 1, characterized in that 2-4 chromatographic stages are carried out one after the other. 3. Postupak prema zahtjevu 1, naznačen time, što se 3 kromatografska stupnja provode jedan za drugim.3. The method according to claim 1, characterized in that 3 chromatographic stages are carried out one after the other. 4. Postupak prema zahtjevu 1, naznačen time, što je kao smjesa otapala upotrebljen toluen-aceton.4. The method according to claim 1, characterized in that toluene-acetone is used as a solvent mixture. 5. Postupak prema zahtjevu 4, naznačen time, što je upotrebljeni toluena sadrži najviše 30% acetona.5. The method according to claim 4, characterized in that the toluene used contains a maximum of 30% acetone. 6. Postupak prema zahtjevu 1, naznačen time, što je upotrebljena smjesa otapala toluen-etilni acetat6. The method according to claim 1, characterized in that the solvent mixture used is toluene-ethyl acetate 7. Postupak prema zahtjevu 6, naznačen time, što je upotrebljeni toluena sadrži najviše 35% vol. etilnog acetata.7. The method according to claim 6, characterized in that the toluene used contains at most 35% by volume of ethyl acetate. 8. Postupak prema zahtjevu 1, naznačen time, što je upotrebljeno eluiranje s gradijentom u slučaju od barem jednog kromatografskog stupnja.8. The method according to claim 1, characterized in that gradient elution is used in the case of at least one chromatographic stage. 9. Visoko čisti ciklosporin A, naznačen time, što je njegov sadržaj ciklosporina L, ciklosporina U i ciklosporina D manji od 0,05% težinski i njegov sadržaj ciklosporina B i C manji od 0,02% težinski.9. Highly pure cyclosporine A, characterized in that its content of cyclosporine L, cyclosporine U and cyclosporine D is less than 0.05% by weight and its content of cyclosporine B and C is less than 0.02% by weight. 10. Postupak u industrijskom mjerilu za pročišćavanje ciklosporina A od ciklosporinskog kompleksa koji sadrži sirovi produkt s kromatografskom metodom na stupcu silikagela, naznačen time, što je kromatografija u više stupnjeva provedena na stupcu koji sadrži silikagel normalne faze sa smjesom otapala koja sadržava toluen kao glavni sastojak.10. Process on an industrial scale for the purification of cyclosporine A from the cyclosporine complex containing the crude product with the chromatographic method on a silica gel column, indicated by the fact that the multi-stage chromatography was carried out on a column containing normal phase silica gel with a solvent mixture containing toluene as the main ingredient . 11. Bilo koji postupak iz zahtjeva 1 ili 10, naznačen time, što se upotrebljeni ciklosporinski kompleks kao početna tvar u kromatografiji zagrije do 80-120°C prije kromatografiranj a.11. Any method from claim 1 or 10, characterized in that the cyclosporine complex used as starting substance in chromatography is heated to 80-120°C before chromatography. 12. Bilo koji postupak iz zahtjeva 1 ili 10, naznačen time, što upotrebljeni toluena sadži 10-30% acetona.12. Any process from claim 1 or 10, characterized in that the toluene used contains 10-30% acetone. 13. Bilo koji postupak iz zahtjeva 1 ili 10, naznačen time, što upotrebljeni toluena sadrži 10-35% vol. etilnog acetata.13. Any process from claim 1 or 10, characterized in that the toluene used contains 10-35% by volume of ethyl acetate.