HRP930277A2

HRP930277A2 - A method for isolating biologically active compounds

Info

Publication number: HRP930277A2
Application number: HRP-2105/90A
Authority: HR
Inventors: Per Kaersgaard
Original assignee: Novo Nordisk As
Priority date: 1989-11-09
Filing date: 1993-03-08
Publication date: 1996-10-31
Also published as: SI9012105A

Description

Oblast tehnike The field of technology

Ovaj izum se odnosi na postupak za izoliranje bioloških spojeva kao što su proteini, naročito koagulacijskog faktora VIII, iz tekućih tijela kao što je plazma, upotrebom gel filtracije kao prve faze u filtraciji. This invention relates to a process for isolating biological compounds such as proteins, especially coagulation factor VIII, from liquid bodies such as plasma, using gel filtration as the first stage in the filtration.

Definirani tehnički problem Defined technical problem

Predmet ovog izuma je da osigura postupak za izoliranje Faktora VIII direktno iz plazme, uz upotrebu gel filtracije. The object of this invention is to provide a process for isolating Factor VIII directly from plasma, using gel filtration.

Stanje tehnike State of the art

Faktor VIII koji je poznati kao antihemofiličini faktor A ili AHF je plazma protein koji sudjeluje u unutrašnjem putu koagulacije krvi. Factor VIII, also known as antihemophilic factor A or AHF, is a plasma protein that participates in the intrinsic pathway of blood coagulation.

Faktor VIII cirkulira u krvnoj plazmi u eksterno maloj koncentraciji u obliku ne-kovalentnog kompleksa dva proteina koji imaju Faktor VIII kao aktivni koagulator (Faktor VIII:C) i aktivni ristocetin kofaktor (von Willebrand Faktor (vWF); odnosno kompleks ima molekularnu težinu od 1-20 x 106D. Factor VIII circulates in the blood plasma in an externally low concentration in the form of a non-covalent complex of two proteins that have Factor VIII as an active coagulant (Factor VIII:C) and an active ristocetin cofactor (von Willebrand Factor (vWF); that is, the complex has a molecular weight of 1 -20 x 106D.

Faktor VIII:C nedostaje ili je nedovoljan kod pojedinaca koji boluju od bolesti krvarenja Hemofilija A koja se javlja kod 5 na 100.000 ljudi. Factor VIII:C is missing or insufficient in individuals suffering from the bleeding disease Hemophilia A, which occurs in 5 per 100,000 people.

von Willeband Faktor se veže za aktivne platelete na takav način da se povećava agregacija aktivnih plateleta. von Willeband Factor binds to active platelets in such a way that aggregation of active platelets increases.

Ovaj efekat se može otkriti in vitro agregacijom ristocetina induciranih plateleta. Zajedno sa Willebrandovom bolešću vidi se i produženo vrijeme krvarenja zbog nedostatka ili smanjenog nivoa bioloških aktivnosti Willebrand Faktora. This effect can be detected in vitro by ristocetin-induced platelet aggregation. Along with Willebrand's disease, prolonged bleeding time is also seen due to the lack or reduced level of biological activities of Willebrand Factor.

Hemofiličari koji pate od hemofilije A i pacijenti koji pate od različitih slučajeva von Willebrandove bolesti se danas liječe koncentratima koji obuhvaćaju Faktor VIII:C/vWF, liječenjem koje može da poboljša kvalitet života pacijenta i uzima u obzir ekonomsku mogućnost i koje pomaže u produženju života ovih pacijenata. Hemophiliacs suffering from hemophilia A and patients suffering from various cases of von Willebrand disease are now treated with concentrates that include Factor VIII:C/vWF, a treatment that can improve the patient's quality of life and takes into account the economic possibility and that helps to prolong the life of these patients.

Farmaceutski preparati koji sadrže faktor VIII (Faktor VIII:C i/ili vWF) mogu biti proizvedeni iz krvi ili krvne plazme. Proizvodnja ovakvih preparata može se izvoditi na nekoliko poznatih načina koje karakterizira mali prinos, naročito Faktora VIII:C. Zajedničko za skoro sve postupke je početna faza purifikacije koja obuhvaća kriotaloženje. Kriotalog smrznute plazme je otopljen na temperaturi od 0-4°C koja daje porast taloga koji obuhvaća Faktor VIII koji može biti sakupljen npr. centrifugiranjem. Iako je kriotaloženje relativno jedinstveno, ono je osnovni nedostatak zbog toga što kada je upotrebljeno u velikom opsegu, tj. bazen sa plazmom koji sadrži više od 5 kg, daje mali prinos Faktora VIII:C (30-45% sadržaja plazme) što znači da je finalni prinos mali bez obzira koja je faza purifikacije kasnije upotrebljena. Pharmaceutical preparations containing factor VIII (Factor VIII:C and/or vWF) can be produced from blood or blood plasma. The production of such preparations can be carried out in several known ways, which are characterized by a low yield, especially of Factor VIII:C. Common to almost all procedures is the initial stage of purification, which includes cryoprecipitation. The cryoprecipitate of frozen plasma is thawed at a temperature of 0-4°C, which gives rise to the precipitate containing Factor VIII, which can be collected, for example, by centrifugation. Although cryoprecipitation is relatively unique, it has a major drawback in that when used on a large scale, i.e. a pool of plasma containing more than 5 kg, it gives a low yield of Factor VIII:C (30-45% of the plasma content) which means that the final yield is small regardless of which stage of purification is used later.

Osim ovoga, generalno, faza umrtvljavanja virusa je obuhvaćena proizvodnjom preparata Faktora VIII. Faza umrtvljavanja virusa povećava sigurnost od virusa u preparatima, ali u mnogo slučajeva izaziva daljnjim smanjenjem prinosa Faktora VIII:C. Apart from this, in general, the virus killing phase is included in the production of Factor VIII preparations. The killing phase of the virus increases the safety of the preparations against the virus, but in many cases causes a further decrease in the yield of Factor VIII:C.

Vrlo mali opći prinos Faktora VIII dovodi do nestašice ovih preparata na nekoliko lokacija i zbog toga potrebni su novi postupci za purifikaciju Faktora VIII u visoki prinos radi ispunjavanja potrebe za Faktorom VIII u liječenju hemofiličara. The very low overall yield of Factor VIII leads to a shortage of these preparations in several locations, and because of this, new procedures are needed for the purification of Factor VIII to a high yield in order to meet the need for Factor VIII in the treatment of hemophiliacs.

Novi postupci za izoliranje Faktora VIII direktno iz plazme u visokom prinosu će biti od velikog interesa premda se čak do 70% sadržaja Faktora VIII u plazmi izgubi prilikom ili prije kriotaloženja. New procedures for isolating Factor VIII directly from plasma in high yield will be of great interest, although up to 70% of Factor VIII content in plasma is lost during or before cryoprecipitation.

Nedavno je pokušano da se izolira Faktor VIII direktno iz plazme upotrebom afinitet kromatografije (Thromb. Haemost., 61, (2), 234-237 (1989) ali dobijen je samo prinos manji od 60% i specifične aktivnosti od 1 IU Faktora VIII/mg protein izoliranog Faktora VIII koji sadrži frakcija. Recently, an attempt was made to isolate Factor VIII directly from plasma using affinity chromatography (Thromb. Haemost., 61, (2), 234-237 (1989), but only a yield of less than 60% and a specific activity of 1 IU of Factor VIII/ mg protein of isolated Factor VIII containing fraction.

Gel filtracija, nazvana i kromatografija zasićenja gela ili kromatografije ograničene veličine je široko kontroliran postupak koji se upotrebljava za separaciju otopine prema njihovoj veličini. Otopine su prošle kroz stup napunjen česticama inertnog poroznog gela koji ima pore veličine koje isključuju veće molekule, dok se manje molekule šire u stacionarnoj fazi unutar čestica gela. Na ovaj način, veće molekule koje bivaju potpuno isključene iz čestica gela su elutirani prvo sa "praznim volumenom", dok manje molekule provode duže vrijeme prolazeći stup i elutirani su u skladu sa smanjenjem veličine sa povećanjem "elutiranog volumena". Gel filtration, also called gel saturation chromatography or size restriction chromatography, is a widely controlled procedure used to separate solutions according to their size. The solutions were passed through a column filled with inert porous gel particles that have pore sizes that exclude larger molecules, while smaller molecules diffuse in the stationary phase within the gel particles. In this way, larger molecules that are completely excluded from the gel particles are eluted first with the "void volume", while smaller molecules spend a longer time passing the column and are eluted according to decreasing size with increasing "eluted volume".

Gel filtracija može biti izvedena na dva različita načina: Gel filtration can be performed in two different ways:

Mod grupne separacije Group separation mode

U modu grupne separacije otopine su separirane u dvije grupe koje imaju velike razlike u njihovim molekularnim veličinama, jedna grupa je elutirana sa "praznim volumenom", a druga je zatim elutirana sa mnogo većim elutiranim volumenom, često blizu ukupnog "nataloženog volumena"; ovaj postupak je primarno upotrebljen za separaciju proteina iz otopljenih soli, ili za promjenu pufera i odnosi se na "desalinizaciju". In group separation mode, solutions are separated into two groups that have large differences in their molecular sizes, one group is eluted with a "void volume" and the other is then eluted with a much larger eluted volume, often close to the total "deposited volume"; this process is primarily used to separate proteins from dissolved salts, or to change buffers and is referred to as "desalination".

Za desalinzaciju je upotrebljen čvrsti gel koji ima pore male veličine i postupak može biti izveden upotrebom velike količine materijala (uzorak volumena se sastoji od 20-30% nataloženog volumena) i upotrebom velike protočne brzine (oko jednog nataloženog volumena pufera na sat); na ovaj način kapacitet stupa će biti veći. A solid gel with small pores is used for desalination and the procedure can be performed using a large amount of material (the sample volume consists of 20-30% of the deposited volume) and using a high flow rate (about one deposited buffer volume per hour); in this way, the capacity of the column will be higher.

Mod djeljenja na frakcije Mode of division into factions

U ovom modu, otopine koje imaju slične molekularne veličine su separirane; ovaj postupak se često upotrebljava za separiranje proteina. Za ovu svrhu upotrebljene su čestice gela sa većim porama i sredina gel filtracije je odabrana tako da osigura da se proteini eluiraju između "praznog volumena" i elutiranog volumena koji odgovara ukupno "nataloženom volumenu". Supstance su elutirane mnogo gušće nego kada se upotrebljavaju uvjeti grupne separacije i mogu se preklapati. Velike brzine protoka nisu poželjne jer ne dozvoljavaju efikasnu separaciju proteina, i punjenje stupa mora da se drži nisko da bi se dobila prihvatljiva separacija pojedinih proteina. Na ovaj način, gel filtracija upotrebljena u modu djeljenja na frakcije može biti preporučena samo za separaciju proteina kao posljednja faza poliranja gdje je mali volumen koji treba frakcionirati (Jagschies, Ullmanns Encyclopedia of Industrial Chemistry, vol B3(10), 1988 i Bio/Technol., 4, 954-58 (1986). In this mode, solutions having similar molecular sizes are separated; this procedure is often used to separate proteins. For this purpose, gel particles with larger pores were used and the gel filtration medium was chosen to ensure that the proteins eluted between the "void volume" and the eluted volume corresponding to the total "precipitated volume". Substances are eluted much more densely than when group separation conditions are used and may overlap. High flow rates are not desirable because they do not allow efficient protein separation, and the column loading must be kept low to obtain acceptable separation of individual proteins. In this way, gel filtration used in fractionation mode can only be recommended for protein separation as a final polishing step where the volume to be fractionated is small (Jagschies, Ullmanns Encyclopedia of Industrial Chemistry, vol B3(10), 1988 and Bio/Technol ., 4, 954-58 (1986).

Ovo su bili pokušaji izoliranja Faktora VIII iz plazme upotrebom gel filtracije (J.Lab.Clin.Med., 72, (6), 1968, 1007-1008 i J.Clin.Invest., 48, 1969, 957-962). Velika purifikacija je pronađena za vrijeme eksperimenata, ali su prinosi samo oko 40-50%. Pronađeno je da čistoća dobijenog Faktora VIII koji sadrži frakcije zavisi od početne plazme, kako veliki sadržaj lipida i hilomikrona podiže zamućenost frakcije Faktora VIII koja ima nižu specifičnu aktivnost. These were attempts to isolate Factor VIII from plasma using gel filtration (J.Lab.Clin.Med., 72, (6), 1968, 1007-1008 and J.Clin.Invest., 48, 1969, 957-962). Great purification was found during the experiments, but the yields were only about 40-50%. It was found that the purity of the obtained Factor VIII containing fractions depends on the initial plasma, as the high content of lipids and chylomicrons increases the turbidity of the Factor VIII fraction that has a lower specific activity.

Zabilježeno je da tehnika gel filtracije koja je upotrebljena ne dozvoljava rukovanje velikim količinama plazme, iako uvodni eksperimenti pokazuju da gel filtracija mnogo koncentriranije Cohn frakcije i izgleda moguća. It is noted that the gel filtration technique used does not allow the handling of large quantities of plasma, although preliminary experiments show that gel filtration of the much more concentrated Cohn fraction seems possible.

Osim ovoga, Paulssen et. al. (Thromb. Diathes. Haemorr., 22, 1969, 577-583) je našao da Faktor VIII može biti separiran iz drugih proteina plazme upotrebom gel sredine Sefaroze 6B, ali kromatografija upotrebom gel filtracije, samo izgleda moguća u većem opsegu, kada je upotrebljen otopljen kriotalog, kao početni materijal. In addition to this, Paulssen et. al. (Thromb. Diathes. Haemorr., 22, 1969, 577-583) found that Factor VIII could be separated from other plasma proteins using Sepharose 6B gel medium, but chromatography using gel filtration only seemed possible on a larger scale when used melted cryo precipitate, as a starting material.

US patent br. 3,637,489 otkriva postupak za separaciju komponenti krvi koji koristi gel filtraciju i koristi porozne staklene kuglice. Postupak je specijalno određen za separaciju imunološki aktivnih materija iz drugih sastava, u serumu ili plazmi. US patent no. 3,637,489 discloses a process for the separation of blood components that uses gel filtration and uses porous glass beads. The procedure is specially designed for the separation of immunologically active substances from other compositions, in serum or plasma.

Od tada je izvedeno nekoliko pokušaja da se gel fitracija upotrebi za purifikaciju Faktora VIII, ali su pokušaji koncentrirani na gel filtraciju djelomično purificirane frakcije plazme (otopljen kriotalog i njegove purificirane frakcije). Svi pokušaji su izvedeni upotrebom malog punjenja stupova i/ili malih protočnih brzina ili i jednog i drugog zajedno. Since then, several attempts have been made to use gel filtration for the purification of Factor VIII, but the attempts have concentrated on gel filtration of the partially purified plasma fraction (dissolved cryoprecipitate and its purified fractions). All experiments were performed using low column loading and/or low flow rates or both.

Gel filtracija kao postupak za proteinsko dijeljenje na frakcije je poznata još od 1959. g. i bila je u širokoj upotrebi u biokemijskim laboratorijskim istraživanjima kao postupak za karakterizaciju proteina i za purifikaciju proteina iz uzoraka sa malim volumenima, npr. manjim od 1 litar. Gel filtracija, do ovog izuma, nije bila u velikoj upotrebi u dijeljenju plazme na frakcije za separaciju proteina, jedina upotreba je bila desalinacija etanola i soli iz albumin otopina. Tako, kao što se može naći u referentnim knjigama: "Glavni razlog zašto gel filtracija nije postala glavnom tehnikom u dijeljenju na frakcije plazme je protok proteina po volumenu stupa". (J.H.Berglöf: "Fractionation by Gel Filtration", str. 163-173 u J.M.Curling (Ed.): "Methods of Plasma Protein Fractionation", Academic Press, London 1980) i "Nažalost, proteinske mase kojima se može rukovati sa razumnim veličinama stupova su male, i razblaživanje uzorka ne može biti zanemareno. Zbog toga, postupak nije mnogo u upotrebi u dijeljenju plazme na frakcije (J.J.Morgenthaler et al.: "Preservation of structure and function during isolation of human plasma proteins", st. 127-138 u Smit Sibinga et al. (Eds.): "Plasma Fractionation and Blood Transfusion", Martinus Nijhoff Publishers, Bost, 1985). Gel filtration as a procedure for protein fractionation has been known since 1959 and was widely used in biochemical laboratory research as a procedure for protein characterization and protein purification from samples with small volumes, eg less than 1 liter. Gel filtration, until this invention, was not widely used in dividing plasma into fractions for protein separation, the only use being the desalination of ethanol and salts from albumin solutions. Thus, as can be found in reference books: "The main reason why gel filtration has not become the main technique in plasma fractionation is the flow of proteins per column volume". (J.H.Berglöf: "Fractionation by Gel Filtration", pp. 163-173 in J.M.Curling (Ed.): "Methods of Plasma Protein Fractionation", Academic Press, London 1980) and "Unfortunately, protein masses that can be handled with reasonable column sizes are small, and dilution of the sample cannot be neglected. Therefore, the procedure is not widely used in plasma fractionation (J.J. Morgenthaler et al.: "Preservation of structure and function during isolation of human plasma proteins", p. 127 -138 in Smit Sibinga et al.(Eds.): "Plasma Fractionation and Blood Transfusion", Martinus Nijhoff Publishers, Bost, 1985).

US patent br. 4,675,385 otkriva postupak za izolaciju Faktora VIII prokoagulacijskog proteina iz preparata plazme koji obuhvaćaju Faktor VIII, sastave visoke molekularne težine i sastave male molekularne težine nizom kromatografije ograničene veličine visoke performance pripremanjem, u prvoj fazi, puferiranog vodenog sastava preparata plazme i separacijom sastava male molekularne težine unošenjem sastava kromatografskog stupa porone tekuće kromatografije slojeva visoke performance koji ima veličine od oko 13 mikrona do oko 35 mikrona i elutira stup puferiranim vodenim eluatom. Da bismo imali dobru separaciju US patent br. 4,675,385 uči upotrebi stupova koji imaju jedan aspekt odnosa ne manji od između 10 i 40 koji smanjuje kapacitet, ali ne osigurava dobru separaciju proteina koji pokazuju aktivnost Faktora VIII:C iz drugih sastava preparata plazme iz preparata plazme koji se dobija izvođenjem druge HPLC. US patent no. 4,675,385 discloses a process for the isolation of Factor VIII procoagulation protein from plasma preparations comprising Factor VIII, high molecular weight compositions and low molecular weight compositions by high performance size-limited array chromatography by preparing, in the first stage, a buffered aqueous composition of the plasma preparation and separating the low molecular weight composition by introducing the composition of the chromatographic column of porona liquid chromatography of high performance layers, which has sizes from about 13 microns to about 35 microns and elutes the column with a buffered aqueous eluate. In order to have a good separation US patent no. 4,675,385 teaches the use of columns having an aspect ratio of not less than between 10 and 40 which reduces capacity but does not ensure good separation of proteins exhibiting Factor VIII:C activity from other compositions of the plasma preparation from the plasma preparation obtained by performing the second HPLC.

Tako je sve do ovog izuma generalno prihvaćena činjenica da gel filtracija nije pogodan postupak za separaciju proteina u dijeljenju na frakcije plazme, kada se rukuje većim volumenima, npr. iznad 5 litara. Thus, until this invention, it was generally accepted that gel filtration is not a suitable procedure for protein separation in plasma fractionation, when handling larger volumes, eg above 5 liters.

Iznenađujuće je saznanje da je kada je odabrani materijal za gel filtraciju predviđen za velike protočne brzine, moguće izolirati Faktor VIII u obliku čiste frakcije i velikog prinosa direktno iz plazme vrlo pažljivom mehaničkom separacijom, bez oslanjanja na normalno početno kriotaloženje. It is surprising to learn that when the selected gel filtration material is designed for high flow rates, it is possible to isolate Factor VIII in the form of a pure fraction and in high yield directly from plasma by very careful mechanical separation, without relying on normal initial cryoprecipitation.

Opis rješenja tehničkog problema Description of the solution to the technical problem

Predmet ovog izuma je postupak za izoliranje Faktora VIII u obliku kompleksa Faktora VIII:C i vWF iz drugih proteina u krvnoj plazmi upotrebom gel filtracije. The subject of this invention is a process for isolating Factor VIII in the form of a complex of Factor VIII:C and vWF from other proteins in blood plasma using gel filtration.

Postupak prema ovom izumu je karakterističan po tome što je izolirana plazma ili otopljena svježe smrznuta plazma podvrgnuta gel filtraciji pod uvjetima grupne separacije upotrebom visokog punjenja i velike protočne brzine, sredina gel filtracije postaje konstituirana od čestica koje su inertne za Faktor VIII i imaju opseg djeljenja na frakcije u intervalu od 1 x 103 do 1 x 108, ili još poželjnije od 1 x 104 od 8 x 107. Opseg djeljenja na frakcije može biti npr. u intervalu od 5 x 104 do 4 x 107. The method according to the present invention is characterized by the fact that the isolated plasma or thawed fresh frozen plasma is subjected to gel filtration under conditions of group separation using high loading and high flow rate, the gel filtration medium becomes constituted by particles that are inert to Factor VIII and have a division range of fractions in the interval from 1 x 103 to 1 x 108, or even more preferably from 1 x 104 to 8 x 107. The range of division into fractions can be, for example, in the interval from 5 x 104 to 4 x 107.

U poželjnom ostvarenju volumena plazme koja je dodana je najmanje 5% od nataloženog volumena. Količina plazme koja je dodana je poželjno 15-40% od nataloženog volumena. In a preferred embodiment, the volume of plasma added is at least 5% of the deposited volume. The amount of plasma added is preferably 15-40% of the deposited volume.

Postupak prema izumu je poželjno izveden upotrebom protočne brzine od najmanje 0,3 nataloženog volumena na sat, najpoželjnije 0,5-2 nataloženog volumena na sat. The process according to the invention is preferably carried out using a flow rate of at least 0.3 deposited volume per hour, most preferably 0.5-2 deposited volume per hour.

Za potrebe ovog izuma upotrebljena je sredina gel filtracije koja ima čvrstoću koja dozvoljava brzu elutaciju. Osim ovoga, gel mora biti kemijski i imunološki inertan za Faktor VIII za vrijeme gel filtracije. Eksperimenti će pokazati da postupak prema ovom izumu može biti izveden upotrebom komercijalnih gela kao što su: Sefaroza CL-4B, Sefaroza CL-6B, Sefaroza 4FF, Sefaroza 6FF, Sefakril S-400, Sefakril S-500, Fraktogel TSK HW-65(F) i Matreks Celufin CGL 2000 koji su određeni za svrhu ovog izuma. Ovi gelovi su generalno veličine čestice u intervalu od oko 32 µm do oko 200 µm. For the purposes of this invention, a gel filtration medium is used that has a strength that allows rapid elution. In addition, the gel must be chemically and immunologically inert to Factor VIII during gel filtration. Experiments will show that the procedure according to this invention can be performed using commercial gels such as: Sepharose CL-4B, Sepharose CL-6B, Sepharose 4FF, Sepharose 6FF, Sefakril S-400, Sefakril S-500, Fraktogel TSK HW-65( F) and Matreks Celufin CGL 2000 which are determined for the purpose of this invention. These gels generally range in particle size from about 32 µm to about 200 µm.

Prema jednom od ostvarenja postupka prema ovom izumu, smrznuta plazma je otopljena i to osigurava da su svi Faktori VIII otopljeni, nakon čega se poželjno dodaje otopljena plazma stupu odmah nakon što su svi Faktori VIII otopljeni. Temperatura ne smije da raste previše da bi se izbjegla ekstenziona degradacija Faktora VIII. Plazmi može biti prethodno dodan npr. heparin, citrat, saharoza, amino kiseline, soli ili drugi stabilizatori i poželjno može biti isfiltrirana, centrifugirana, koncentrirana ultrafiltracijom prethodno nataložena upotrebom uobičajenih sredstava za taloženje, ili može biti pripremljena na neki drugi način prije svog dodavanja stupu i to onoliko dugo da ne bi došlo do utjecaja na sadržaj Faktora VIII iz plazme. According to one embodiment of the process according to the present invention, the frozen plasma is thawed and this ensures that all Factor VIIIs are dissolved, after which the thawed plasma is preferably added to the column immediately after all Factors VIII are dissolved. The temperature must not rise too high to avoid extensive degradation of Factor VIII. The plasma may be pre-added, for example, to heparin, citrate, sucrose, amino acids, salts or other stabilizers, and may preferably be filtered, centrifuged, concentrated by ultrafiltration, pre-precipitated using conventional precipitating agents, or otherwise prepared prior to addition to the column. and for as long as there is no influence on the content of Factor VIII in the plasma.

Postupak prema ovom izumu pokazuje da daje visok prinos Faktora VIII. Tipično, iznad 70% sadržaja iz Faktora VIII iz plazme je nadoknađeno u proizvodu, i postupak daje porast do vrlo čistih proizvoda koji imaju specifičnu aktivnost od 1 do 4 IU Faktor VIII:C/mg proteina. Ovo se može djelomično pripisati činjenici da je upotrebom postupka prema ovom izumu Faktor VIII također separiran iz proteolitičkih enzima, koji normalno mogu izazvati propast Faktora VIII:C vrlo rano u postupku izolacije. The process according to the present invention has been shown to give a high yield of Factor VIII. Typically, over 70% of the plasma Factor VIII content is recovered in the product, and the process yields highly pure products that have a specific activity of 1 to 4 IU Factor VIII:C/mg protein. This can be attributed in part to the fact that using the process of the present invention, Factor VIII is also separated from proteolytic enzymes, which normally can cause the destruction of Factor VIII:C very early in the isolation process.

Postupak prema ovom izumu omogućuje tretman velikih količina plazme pod uvjetima gdje je plazma primijenjena upotrebom visokih punjenja i velikih protočnih brzina u gel filtraciji, dajući porast do vrlo visokih nadoknada Faktora VIII visoke čistoće. Na ovaj način je ponuđen vrlo efikasan postupak za industrijsku primjenu. The process of the present invention enables the treatment of large volumes of plasma under conditions where the plasma is applied using high loadings and high flow rates in gel filtration, giving rise to very high recoveries of high purity Factor VIII. In this way, a very efficient procedure for industrial application is offered.

Faktor VIII koji sadrži frakcije iz gel filtracije ili kombinirane frakcije iz više gel filtracija može biti koncentriran i zatim purificiran upotrebom tehnika poznatih per se, kao što su ultrafiltracija, taloženje, izmjena iona, kromatografija afiniteta ili slično. Factor VIII containing fractions from gel filtration or combined fractions from multiple gel filtrations can be concentrated and then purified using techniques known per se, such as ultrafiltration, precipitation, ion exchange, affinity chromatography or the like.

Preostali plazma proteini, kao što su albumin, imunoglobulini, protrombin kompleks, antitrombin III i drugi mogu također biti izolirani iz kasnijih frakcija gel filtracije upotrebom tehnika poznatih per se, kao što su taloženje sa alkoholom, PEG-taloženje, kromatografija ili slične. Remaining plasma proteins, such as albumin, immunoglobulins, prothrombin complex, antithrombin III and others can also be isolated from subsequent gel filtration fractions using techniques known per se, such as alcohol precipitation, PEG-precipitation, chromatography or the like.

Određivanje aktivnosti Faktora VIII može biti izvedeno upotrebom jedne od dvofazne ili jednofazne analize. Determination of Factor VIII activity can be performed using one of the two-phase or one-phase assays.

Utvrđeno je da jednofazna i dvofazna analiza mogu dati različite rezultate u određivanju aktivnosti Faktora VIII:C u uzorku. Osim ovoga, poznato je da ponovljena određivanja upotrebom iste analize na istom uzorku može dati porast varijacija u određivanju aktivnosti Faktora VIII:C. It was found that one-phase and two-phase analysis can give different results in the determination of Factor VIII:C activity in a sample. In addition, it is known that repeated determinations using the same assay on the same sample may give rise to variation in the determination of Factor VIII:C activity.

Kako je ovdje upotrebljen izraz "plazma" on predstavlja krv iz koje su sve krvne korpuskule i platelete uklonjene npr. centrifugiranjem. As the term "plasma" is used here, it represents blood from which all blood corpuscles and platelets have been removed, for example by centrifugation.

"Faktor VII:Ag" označuje Faktor VIII u vezi sa antigenom, i "vWF-Ag" von Willebrand Faktor u vezi sa antigenom. "Factor VII:Ag" refers to antigen-related Factor VIII, and "vWF-Ag" to von Willebrand Antigen-related Factor.

"Nataloženi volumen" je definiran kao napunjena gel sredina i intermedijarna tekućina. "Prazni volumen" je definiran kao volumen pufera između gel čestica, i "elutiran volumen" je volumen pufera koji je upotrebljen za elutiranje specifičnog materijala. Izraz "napunjen stup" je upotrebljen da označi volumen materijala dodan sloju kalkuliran kao postotak nataloženog volumena. Izraz "volumen dijeljenja na frakcije" označuje volumen molekularnih težina (granula) proteina ili većih molekula za koje se preporuča gel materijal. "Deposited volume" is defined as the filled gel medium and intermediate liquid. "Void volume" is defined as the volume of buffer between the gel particles, and "eluted volume" is the volume of buffer that was used to elute the specific material. The term "filled column" is used to denote the volume of material added to the layer calculated as a percentage of the deposited volume. The term "fractionation volume" refers to the volume of molecular weights (granules) of proteins or larger molecules for which the gel material is recommended.

Postupak prema ovom izumu je, nadalje, objašnjen odgovarajućim crtežima i primjerima objašnjenih ostvarenja ovog izuma. Primjeri su samo za ilustraciju i ne mogu biti shvaćeni kao ograničavajući prikaz izuma koji je definiran priloženim zahtjevima. The process according to this invention is further explained with corresponding drawings and examples of explained embodiments of this invention. The examples are for illustration only and cannot be understood as a limiting representation of the invention defined by the appended claims.

Ovaj izum je ilustriran odgovarajućim priloženim crtežima u kojima: This invention is illustrated by the corresponding attached drawings in which:

Crtež 1 prikazuje grafički prikaz elutiranja Faktora VIII gel filtracijom u skladu sa ovim izumom određenim različitim analizama, i Figure 1 shows a graphical representation of the elution of Factor VIII by gel filtration in accordance with the present invention determined by various assays, and

Crtež 2 ilustrira poredak elutiranja različitih plazma proteina gel filtracijom u skladu sa ovim izumom. Figure 2 illustrates the order of elution of various plasma proteins by gel filtration in accordance with the present invention.

Primjer 1 Example 1

Gel filtracija plazme radi izoliranja Faktora VIII. Gel filtration of plasma to isolate Factor VIII.

Stup od Ø 2,6 cm je napunjen Sefarozom CL-4B do konačne visine od 60 cm. A Ø 2.6 cm column is filled with Sepharose CL-4B to a final height of 60 cm.

Smrznuta plazma iz danske banke krvi je otopljena na 25°C u vodenoj kupaoni. Nakon dodavanja 1 IU heparina/ml 50 ml (16% nataloženog volumena) plazme je dodano stupu upotrebom protoka od 100 ml/hr, nakon čega je stup elutiran koristeći pojačani protok od 200 ml/ht pufera, što odgovara brzini od 0,63 nataloženog volumena na sat. Uravnoteženje stupa i elutiranje je izvedeno upotrebom pufera, 0,02 M citrata, 0,15 M NaCI, pH 7,4. Puferu je zatim dodano 2,55 ml 1 M CaCl2 po litri dajući slobodan kalcijev ion (Ca2+) koncentraciju na oko 7 x 10-5M. Koncentracija slobodnog kalcijevog iona je provjerena upotrebom kalcij selektivne elektrode od Ingold GmbH (Frankfurt/Main, FRG). Frakcije su sakupljene, i za svaku frakciju OD28O su određeni Faktor VIII:C (upotrebom i jednofazne koagulacijske analize i dvofazne kromogeničke analize), Faktor VIII:Ag, Albumin, IgG, fibrinogen, IgM, Alfa-2-makroglobulinglobulin, Faktor IX, Faktor X, protein C i antitrombin III. Kao što je protein izmjeren sa OD28O elutiran sa malim maksimumom na praznom volumenu (frakcije 10-18) praćen veoma širokim maksimumom (frakcije 19-50 cfcrt. 1). 82% aktivnosti Faktora VIII:C koje je određeno dvofaznom i 91% aktivnosti faktora VIII:C koje je određeno jednofaznom koagulacijskom analizom je elutirano u jednom maksimumu agregata sa praznim volumenom (Faktor VIII-glavna frakcija) zajedno sa ranijim malim protein maksimumom. Ostatak Faktora VIII je elutiran u manje važnom slijedećem maksimumu odmah do Faktora VIII-glavne frakcije (frakcije 19-26). Faktor VIII:Ag i vWF:Ag su elutirani zajedno sa Faktorom VIII:C, ali imaju nešto malo širi slijedeći maksimum. Frozen plasma from a Danish blood bank was thawed at 25°C in a water bath. After the addition of 1 IU heparin/ml, 50 ml (16% of the deposited volume) of plasma was added to the column using a flow rate of 100 ml/hr, after which the column was eluted using an enhanced flow rate of 200 ml/hr of buffer, corresponding to a rate of 0.63 of the deposited volume per hour. Column equilibration and elution was performed using buffer, 0.02 M citrate, 0.15 M NaCl, pH 7.4. 2.55 ml of 1 M CaCl2 per liter was then added to the buffer giving a free calcium ion (Ca2+) concentration of about 7 x 10-5M. The free calcium ion concentration was checked using a calcium selective electrode from Ingold GmbH (Frankfurt/Main, FRG). Fractions were collected, and for each fraction OD28O were determined Factor VIII:C (using both one-phase coagulation analysis and two-phase chromogenic analysis), Factor VIII:Ag, Albumin, IgG, fibrinogen, IgM, Alpha-2-macroglobulinglobulin, Factor IX, Factor X, protein C and antithrombin III. As measured by OD28O, the protein eluted with a small maximum at void volume (fractions 10-18) followed by a very broad maximum (fractions 19-50 cfcrt. 1). 82% of the Factor VIII:C activity determined by the biphasic and 91% of the Factor VIII:C activity determined by the one-phase coagulation assay was eluted in a single peak of the void volume aggregate (Factor VIII-major fraction) together with the earlier small protein peak. The rest of the Factor VIII was eluted in the minor next peak immediately after the Factor VIII-major fraction (fractions 19-26). Factor VIII:Ag and vWF:Ag are eluted together with Factor VIII:C, but have a slightly broader following peak.

Svi drugi određeni plazma proteini su elutirani u frakciju nakon Faktora VIII-glavne frakcije zajedno sa velikom širinom protein maksimuma (vidi crt. 2). Plazma proteini separirani iz Faktora VIII:C su slijedećih molekularnih težina: All other determined plasma proteins were eluted in the post-Factor VIII-major fraction along with a broad protein peak (see Fig. 2). Plasma proteins separated from Factor VIII:C have the following molecular weights:

[image] Određivanje aktivnosti Faktora VIII:C upotrebom dvofazne kromogeničke analize je izvedeno korištenjem kromogene substrat metode (KABI Coatest Faktor VIII), originalni metod koji koristi test cijevi na 37°C je modificiran da se izvodi korištenjem mikrolitarskih ploča sa smanjenom upotrebom reagensa. [image] Determination of Factor VIII:C activity using a biphasic chromogenic assay was performed using the chromogenic substrate method (KABI Coatest Factor VIII), the original method using a tube test at 37°C was modified to be performed using microliter plates with reduced reagent usage.

50 mikrolitarski uzorak ili standardni je upotrebljen, i on se nakon miješanja sa 75 mikrolitarskom otopinom fosfolipida, Faktora IXa, Faktora X i CaCl2 inkubira na 37°C za 15 minuta, nakon čega je dodano 50 mikrolitara substrata. Nakon dodatnih 20 minuta inkubacije na 37°C, reakcija je ohlađena sa 50 mikrolitara 1 M limunske kiseline. Razvoj boje se čita na 405 nm referentna je na 492 nm. A 50 microliter sample or standard was used, and after mixing with a 75 microliter solution of phospholipids, Factor IXa, Factor X, and CaCl2, it was incubated at 37°C for 15 minutes, after which 50 microliters of substrate was added. After an additional 20 minutes of incubation at 37°C, the reaction was quenched with 50 microliters of 1 M citric acid. Color development is read at 405 nm, the reference is at 492 nm.

Određivanje aktivnosti Faktora VIII:C upotrebom jednofazne analize je izvedeno upotrebom APTT-postupka (Activated Partial Thromboplastin Time). 100 mikrolitara uzorka ili standardnog je pipetirano u kuvete, nakon čega je dodano 100 mikrolitara nepotpune plazme (Faktor VIII nepotpune plazme, generalno dijagnostički), i otopina je držana na 37°C za 5 minuta. Nakon dodavanja 100 mikrolitara 0,03 M CaCl2, određeno je vrijeme koagulacije otopine. Determination of Factor VIII:C activity using a single-phase analysis was performed using the APTT-procedure (Activated Partial Thromboplastin Time). 100 microliters of sample or standard was pipetted into cuvettes, after which 100 microliters of incomplete plasma (Factor VIII incomplete plasma, general diagnostic) was added, and the solution was kept at 37°C for 5 minutes. After adding 100 microliters of 0.03 M CaCl2, the coagulation time of the solution was determined.

Za brojna određivanja napravljene su kalibrirane krivulje koje su kalibrirane na bazi serije razblaživanja prema WHO standardu (3rd Int. Standard FVIII, ljudska plazma, 3,9 lU/ml). Dvofazna analiza ovdje opisana, ima nižu (oko 10 puta) granicu detekcije od jednofazne analize. Dodavanjem heparina unapređuje se upotreba dvofazne analize, kao što se mogući utjecaj heparina na analizu može ukloniti jednostavnim razblaživanjem. For numerous determinations, calibrated curves were made that were calibrated on the basis of a dilution series according to the WHO standard (3rd Int. Standard FVIII, human plasma, 3.9 lU/ml). The two-phase analysis described here has a lower (about 10 times) detection limit than the one-phase analysis. The addition of heparin improves the use of biphasic analysis, as the possible influence of heparin on the analysis can be removed by simple dilution.

Faktor VIII:Ag je određen upotrebom ELISA koji koristi antitijela iz Faktora VIII inhibitor pacijenta koji upotrebljava materijal za presvlačenje mikrotitet ploče (Nune, Kamstrup, 4000 Roskilde, Danska) i upotrebljava peroksidazu obilježenih F(AB')2 fragmenata iz istog inhibotor pacijenta za određivanje veze faktora VIII (Thromb. Haemost., 53 (3), 1985, 346-350). Standardne krivulje su proizvedene upotrebom normalne plazme kalibrirane prema WHO standardu (1. IRP, osnovan 1982). Factor VIII:Ag was determined using an ELISA using antibodies from a Factor VIII inhibitor patient using microtite plate coating material (Nune, Kamstrup, 4000 Roskilde, Denmark) and using peroxidase-labeled F(AB')2 fragments from the same inhibitor patient to determine factor VIII binding (Thromb. Haemost., 53 (3), 1985, 346-350). Standard curves were produced using normal plasma calibrated according to the WHO standard (1st IRP, established in 1982).

vWF:Ag je također određen korištenjem ELISA, ali upotrebom zečijeg anti-čovječjeg vWF (DAKO, Danska) kao materijal za presvlačenje i peroksidaze obilježenog zečjeg antičovječjeg vWF (DAKO, Danska) za određivanje veze vWF. Standardna krivulje su proizvedene upotrebom iste normalne plazme kao kod Faktora VIII:Ag ELISA. vWF:Ag was also determined using ELISA, but using rabbit anti-human vWF (DAKO, Denmark) as a coating material and peroxidase-labeled rabbit anti-human vWF (DAKO, Denmark) to determine vWF binding. Standard curves were produced using the same normal plasma as in the Factor VIII:Ag ELISA.

Faktor IX je određen jednofaznom koagulacijskom analizom, na analogni način kao Faktor VIII:C samo koristeći Faktor IX nepotpune plazme. IgG je određen upotrebom radijalne imunodifuzije (Imunochemistry, 2, 1965, 235-254) i albumin, fibrinogen, alfa-2-makroglobulin, Faktor X, protein C i anti-trombin III su određeni upotrebom imunoelektroforeze (Anal.Biochem., 15, 1966, 45-52). OD280 je određen upotrebom spektrofotometra (Spectronic 601 od Milton Roy Company), i protein, upotrebom Kjeldahl, je određen prema Ph. Fur. 2. Bd., I, V. 3.5.2 bez taloženja sa TCA. Specifična aktivnost purificiranih frakcija je izračunata kao odnos koncentracije Faktora VIII:C prema OD28O ili prema koncentraciji proteina određenog upotrebom Kjeldahl. Kada koristimo OD28O za računanje specifične aktivnosti, rezultati različitih eksperimenata nisu direktno uspoređeni, osim kada upotrebljavamo istu početnu plazmu, pošto je frakcija koja sadrži Faktor VIII često mutna, vidi rezultate Ratnoff et al. (J.Clin.Invest., 48, 1969, 957-962). Factor IX was determined by a single-phase coagulation assay, in an analogous manner to Factor VIII:C only using incomplete plasma Factor IX. IgG was determined using radial immunodiffusion (Immunochemistry, 2, 1965, 235-254) and albumin, fibrinogen, alpha-2-macroglobulin, Factor X, protein C and anti-thrombin III were determined using immunoelectrophoresis (Anal.Biochem., 15, 1966, 45-52). OD280 was determined using a spectrophotometer (Spectronic 601 from Milton Roy Company), and protein, using Kjeldahl, was determined according to Ph. Cool. 2. Bd., I, V. 3.5.2 without precipitation with TCA. The specific activity of the purified fractions was calculated as the ratio of Factor VIII:C concentration to OD28O or to protein concentration determined using Kjeldahl. When we use OD28O to calculate specific activity, the results of different experiments are not directly compared, except when we use the same starting plasma, since the fraction containing Factor VIII is often turbid, see the results of Ratnoff et al. (J. Clin. Invest., 48, 1969, 957-962).

Različita sredstva gel filtracije upotrebljena u eksperimentima su od slijedećih izvora: The various gel filtration media used in the experiments are from the following sources:

Sefaroza CL-6B, Sefaroza CL-4B, Sefaroza CL-2B, Sefaroza 6FF, Sefaroza 4FF, Sefakril S-400, i Sefakril S-500 koji su iz farmacije (HILLERØD, Danska), Biogel A-5m, Fine je iz BioRad (Bie i Berntsen, RØDOVRE, Danska), Fraktogel TSK HW-65(F) je iz Merek (Struers, RØDOVRE, Danska) i Matreks Celufin GCL 2000 je iz Kimicon (Helsingborg, Švedska). Sepharose CL-6B, Sepharose CL-4B, Sepharose CL-2B, Sepharose 6FF, Sepharose 4FF, Sefakril S-400, and Sefakril S-500 which are from a pharmacy (HILLERØD, Denmark), Biogel A-5m, Fine is from BioRad (Bie and Berntsen, RØDOVRE, Denmark), Fraktogel TSK HW-65(F) was from Merek (Struers, RØDOVRE, Denmark) and Matreks Celufin GCL 2000 was from Kimicon (Helsingborg, Sweden).

Primjer 2 Example 2

Gel filtracija plazme do izoliranog faktora VIII upotrebom različitih sredstava gel filtracije. Gel filtration of plasma to isolated factor VIII using different gel filtration media.

Stup Ø 2,6 cm je napunjen različitim sredstvima gel filtracije koji imaju različiti opseg djeljenja na frakcije i različite strukture. U svim slučajevima finalna visina napunjenog taloga je 60 cm. Plazma je otopljena kao što je opisano u Primjeru 1 i dodan je 1 IU heparina na ml. Za svako sredstvo gel filtracije stup je napunjen sa 50 ml plazme (16% nataloženog volumena). Punjenje stupa i elutiranje sa puferom je izvedeno kako je opisano u Primjeru 1. Protočna brzina je u svim slučajevima ista kao u Primjeru 1, osim za eksperiment koji koristi Biogel A-5m, gdje je protočna brzina niža od 50 ml/sat zbog porasta povratnog pritiska. Frakcije su sakupljene i za svaku frakciju OD28O i Faktor VIII:C, upotrebom koatesta, su određene. Specifična aktivnost Faktora VIII-glavne frakcije je izračunata kao odnos Faktora VIII:C/ml sa OD280. Eksperimenti su ponovljeni n puta upotrebom različitih plazmi za svako punjenje i izračunate su srednje vrijednosti prinosa i specifične aktivnosti. Faktor VIII-glavne frakcije je odabran na isti način kao u Primjeru 1, i prinos je sadržaj Faktora VIII:C u Faktoru VIII-glavne frakcije kao postotni sadržaj faktora VIII:C dodane plazme. A Ø 2.6 cm column is filled with different gel filtration media that have different extents of division into fractions and different structures. In all cases, the final height of the filled sediment is 60 cm. Plasma was thawed as described in Example 1 and 1 IU of heparin per ml was added. For each gel filtration agent, the column was filled with 50 ml of plasma (16% of the deposited volume). Column loading and elution with buffer was performed as described in Example 1. The flow rate is in all cases the same as in Example 1, except for the experiment using Biogel A-5m, where the flow rate is lower than 50 ml/hour due to the increase in reflux pressure. Fractions were collected and for each fraction OD28O and Factor VIII:C, using the co-test, were determined. The specific activity of Factor VIII-major fraction was calculated as the ratio of Factor VIII:C/ml with OD280. The experiments were repeated n times using different plasmas for each charge and the mean yield and specific activity values were calculated. Factor VIII-Major Fraction is selected in the same manner as in Example 1, and the yield is the Factor VIII:C content of Factor VIII-Major Fraction as a percentage of the Factor VIII:C content of added plasma.

Volumen djelovanja na frakcije i veličine čestica za različita sredstva i izračunate srednje vrijednosti se nalaze u Tabeli 1. The volume of action on fractions and particle sizes for different agents and the calculated mean values are found in Table 1.

Tabela 1 Table 1

[image] [image]

A: Sefaroza CL-6B; B: Sefaroza CL-4B; C: Sefaroza CL-2B; A: Sepharose CL-6B; B: Sepharose CL-4B; C: Sepharose CL-2B;

D: Sefaroza 6FF; E: Sefaroza 4FF; F: Sefakril S-500; D: Sepharose 6FF; E: Sepharose 4FF; F: Sefacryl S-500;

G: Biogel A-5m Fino; H: Fraktogel TSK HW-65 (F); G: Biogel A-5m Fine; H: Fraktogel TSK HW-65 (F);

I: Natreks Celufin GCL 2000; J: Sefakril S-400 I: Natrex Celufin GCL 2000; J: Sefacryl S-400

Primjer 3 Example 3

Gel filtracija plazme do izolacije Faktora VIII upotrebom različitih punjenja stupa. Gel filtration of plasma to isolation of Factor VIII using different column loadings.

Stup Ø 2,6 je napunjen Sefarozom 4FF do finalne visine od 60 cm. Plazma je otopljena kao u Primjeru 1. Nakon otapanja, pH plazme je podešeno do 7,0 upotrebom 0,5 M HCl, 1 IU heparina po ml je dodano i plazma je isfiltrirana kroz 10 mikrometarski najlonski filter. Stupu je dodano 30 ml, 40 ml, 50 ml, 60 ml i 70 ml plazme. Dodatak, protok i elutiranje upotrebom pufera je izvedeno kao što je opisano u Primjeru 1, iako je pufer podešen na 7,0. Frakcije su sakupljene, i za svaku frakciju OD28O i Faktor VIII:C upotrebom koatesta su određeni. Specifična aktivnost Faktora VIII-glavne frakcije je izračunata kao odnos Faktora VIII:c/ml sa OD280. Eksperimenti su ponovljeni tri puta upotrebom tri različite plazme za svako punjenje i srednje vrijednoti (x) su izračunate. Volumen Faktora VIII-glavne frakcije (ml) je voljem frakcije koja sadrži faktor VIII, koji može biti sakupljen prije širokog proteinskog maksimuma (OD280) je elutiran i odabran na isti način kao što je opisano u Primjeru 1. Prinos je sadržaj Faktora VIII:C u Faktoru VIII-glavne frakcije kao postotni sadržaj Faktora VIII:C dodane plazme. Column Ø 2.6 is filled with Sepharose 4FF to a final height of 60 cm. Plasma was thawed as in Example 1. After thawing, the pH of the plasma was adjusted to 7.0 using 0.5 M HCl, 1 IU of heparin per ml was added and the plasma was filtered through a 10 micrometer nylon filter. 30 ml, 40 ml, 50 ml, 60 ml and 70 ml of plasma were added to the column. Addition, flow-through and elution using buffer was performed as described in Example 1, although the buffer was adjusted to 7.0. Fractions were collected, and for each fraction OD28O and Factor VIII:C were determined using the co-assay. The specific activity of Factor VIII-main fraction was calculated as the ratio of Factor VIII:c/ml with OD280. Experiments were repeated three times using three different plasmas for each charge and mean values (x) were calculated. The volume of the Factor VIII-major fraction (ml) is the volume of the fraction containing Factor VIII, which can be collected before the broad protein maximum (OD280) is eluted and selected in the same way as described in Example 1. The yield is the content of Factor VIII:C in Factor VIII-major fractions as a percentage of Factor VIII:C added plasma.

Dobijeni rezultati se nalaze u Tabeli II. The obtained results can be found in Table II.

Tabela II Table II

[image] [image]

Primjer 4 Example 4

Gel filtracija plazme do izolacije Faktora VIII upotrebom različitih vrijednosti elutiranja. Gel filtration of plasma to isolate Factor VIII using different elution values.

Istom stupu koji je upotrebljen u Primjeru 3 je dodano 50 ml plazme otpljene kao što je prethodno opisano. Nakon otapanja, pH plazme je podešeno na 7,0 upotrebom 0,5 M HCl, 1 IU heparin/ml je dodano, i plazma je isfiltrirana kroz 10 mikrometarski najlonski filter. Upotrebom tri različita dijela plazme protočne brzine od 100, 200 i 300 ml/sat su isprane. Isti protok je korišten za vrijeme dodavanja plazme i pratećeg elutiranja. Elutiranje je izvedeno upotrebom istog pufera kao što je opisano u Primjeru 3. Frakcije su sakupljene i za svaku frakciju OD28O i Faktor VIII:C upotrebom koatesta su određeni. Specifična aktivnost i prinos u Faktoru VIII-glavne frakcije su izračunate kao u Primjeru 3. Srednje vrijednosti (x) volumena Faktora VIII-glavne frakcije, prinos i specifična aktivnost su izračunate. Rezultati se nalaze u Tabeli III. To the same column used in Example 3 was added 50 ml of plasma thawed as previously described. After thawing, the plasma pH was adjusted to 7.0 using 0.5 M HCl, 1 IU heparin/ml was added, and the plasma was filtered through a 10 micrometer nylon filter. Using three different parts of plasma flow rates of 100, 200 and 300 ml/hour were washed. The same flow rate was used during plasma addition and subsequent elution. Elution was performed using the same buffer as described in Example 3. Fractions were collected and for each fraction OD28O and Factor VIII:C were determined using the co-assay. Specific activity and yield in Factor VIII-major fraction were calculated as in Example 3. Mean values (x) of Factor VIII-major fraction volume, yield and specific activity were calculated. The results are in Table III.

Tabela III Table III

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Primjer 5 Example 5

Gel filtracija plazme do izoliranja Faktora VIII upotrebom različitih punjenja stupa. Gel filtration of plasma to isolate Factor VIII using different column loadings.

Stup Ø 10 cm je napunjen Sefarozom 4FF do finalne visine od 60 cm. Smrznuta plazma iz danske banke krvi je otopljena na 30°C u vodenoj kupaoni. 925 grama, odnosno 1497 grama i 2000 grama je dodano stupu. Protok se drži na oko 4200 ml/sat za vrijeme dodavanja i elutiranja plazme upotrebom Masterfleks pumpe (Buch i Holm, Herlev, Danska) što je odgovarajuće za 0,89 nataloženog volumena na sat. Za elutiranje je upotrebljen isti pufer kao što je opisano u Primjeru 1. OD28O je praćen kontinualno upotrebom Farmacija monitor UV-1. Kada OD28O počinje da raste na prazni volumen, sakupljanje Faktora VIII-glavne frakcije počinje i to prestaje kada OD28O pokazuje da veliki proteinski maksimum počinje da se elutira. U Faktoru VIII-glavne frakcije sadržaj Faktora VIII:C je određen upotrebom jednofazne koagulacijske analize, i protein je određen upotrebom Kjeldahl. Specifična aktivnost je izračunata kao odnos ukupnog broja jedinica Faktora VIII:C u Faktoru VIII-glavne frackije sa ukupnim brojem miligrama proteina u Faktoru VIII-glavne frakcije. Prinos Faktora VIII:C je izračunat kao sadržaj Faktora VIII:C u Faktoru VIII-glavne frakcije u postocima sadržaja Faktora VIII:c u dodanoj plazmi. A Ø 10 cm column is filled with Sepharose 4FF to a final height of 60 cm. Frozen plasma from a Danish blood bank was thawed at 30°C in a water bath. 925 grams, i.e. 1497 grams and 2000 grams were added to the column. The flow rate is maintained at about 4200 ml/hr during plasma addition and elution using a Masterfleks pump (Buch and Holm, Herlev, Denmark) which corresponds to 0.89 deposited volume per hour. The same buffer as described in Example 1 was used for elution. OD28O was monitored continuously using Farmacija monitor UV-1. When the OD28O begins to rise to the void volume, the collection of Factor VIII-major fraction begins and this stops when the OD28O shows that a large protein peak begins to elute. In the Factor VIII-major fraction, the Factor VIII:C content was determined using a single-phase coagulation assay, and the protein was determined using Kjeldahl. Specific activity was calculated as the ratio of the total number of Factor VIII:C units in the Factor VIII-major fraction to the total number of milligrams of protein in the Factor VIII-major fraction. The Factor VIII:C yield was calculated as the Factor VIII:C content in the Factor VIII-major fraction as a percentage of the Factor VIII:c content in the added plasma.

Rezultati se nalaze u Tabeli IV. The results can be found in Table IV.

Tabela IV Table IV

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Primjer 6 Example 6

Gel filtracija plazme do izoliranja Faktora VIII upotrebom stupa industrijskih razmjera. Gel filtration of plasma to isolate Factor VIII using an industrial scale column.

Stup Ø 29 cm je napunjen Sefarozom 4FF. Nakon uravnoteženja upotrebom istog pufera kao što je u Primjeru 1 visina gela je 53 cm. 10 kilograma smrznute plazme iz danske banke krvi je otopljeno i zagrijano do 30°C, nakon čega je dodano stupu. Dodana količina se sastoji od 28,8% nataloženog volumena. Upotrebom Masterfleks pumpe, protok od 30 litara na sat, što odgovara za 0,86 nataloženog volumena na sat, se održava za vrijeme dodavanja i elutiranja upotrebom ravnotežnog pufera. OD28O jepromatran kontinualno upotrebom farmacija monitor UV-1, i u vrijeme prve indikacije porasta u OD28O na prazni volumen, Faktor VIII-glavne frakcije je sakupljen. Ukupno 11,73 kilograma Faktora VIII-glavne frakcije je sakupljeno. Zatim je sadržaj Faktora VIII:C određen upotrebom jednofazne koagulacijske analize i protein je određen upotrebom Kjeldahl. Ukupno 8798 IU Faktora VIII:C i manje od 2346 miligrama proteina je nađeno u Faktoru VIII-glavne frakcije dajući prinos Faktora VIII:C od 880 lU/kilogram plazme što odgovara za 88% prinosa u obliku proizvoda koji ima specifičnu aktivnost više od 3,75 IU/kilogram proteina. Column Ø 29 cm is filled with Sepharose 4FF. After equilibration using the same buffer as in Example 1, the height of the gel is 53 cm. 10 kilograms of frozen plasma from a Danish blood bank was thawed and heated to 30°C, after which it was added to the column. The added amount consists of 28.8% of the deposited volume. Using the Masterflex pump, a flow rate of 30 liters per hour, corresponding to 0.86 of the deposited volume per hour, is maintained during addition and elution using the equilibration buffer. OD28O was monitored continuously using a pharmacy monitor UV-1, and at the time of the first indication of an increase in OD28O on empty volume, Factor VIII-major fraction was collected. A total of 11.73 kilograms of Factor VIII-major fraction was collected. Then, Factor VIII:C content was determined using a single-phase coagulation assay and protein was determined using Kjeldahl. A total of 8798 IU of Factor VIII:C and less than 2346 milligrams of protein were found in the Factor VIII-major fraction giving a Factor VIII:C yield of 880 lU/kilogram of plasma corresponding to an 88% yield in the form of a product having a specific activity greater than 3, 75 IU/kilogram of protein.

Faktor VIII-glavne frakcije dobiven u skladu sa postupkom prema izumu može zatim biti očišćen na način analogan konvencionalnoj purifikaciji otopljenog kriotaloga koji obuhvaća npr. kromatografsku purifikaciju i liofilizaciju, upotrebom uobičajenog vezivnog sredstva, radi stvaranja stabilnog preparata. Preparat je rekonstruiran prije upotrebe, korištenjem pogodnog konvencionalnog prenositelja. The factor VIII-major fraction obtained according to the method according to the invention can then be purified in a manner analogous to the conventional purification of dissolved cryoprecipitate, which includes, for example, chromatographic purification and lyophilization, using a common binding agent, in order to create a stable preparation. The preparation is reconstituted before use, using a suitable conventional carrier.

Claims

1. Procedure for isolating factor VIII from other proteins in blood plasma using a gel filtration agent, indicated by the fact that the isolated plasma or thawed freshly frozen plasma, which was optionally pre-treated, as long as such possible pre-treatment does not have a significant effect on the factor content VIII plasma, is subjected directly to gel filtration under group filtration conditions where the volume of added plasma is at least 5% of the deposited volume and where the flow rate is at least 0.3 deposited volume per hour, whereby the gel filtration agent consists of particles that are inert in relation to the Factor VIII have a range of fractionation in the interval from 1 x 103 to 1 x 108, and the main fraction of Factor VIII is collected.

2. The method according to Patent Claim 1, characterized by the fact that the gel filtration agent is composed of a gel material whose extent of division into fractions is in the interval from 1 x 104 to 8 x 107.

3. The method according to Patent Claim 2, characterized in that the gel filtration agent is composed of a gel material whose extent of division into fractions is in the interval from 5 x 104 to 4 x 107.

4. The method according to any one of patent claims 1 to 3, characterized in that the volume of added plasma is 15-4-% of the deposited volume.

5. The method according to any one of patent claims 1 to 4, characterized in that the flow rate is 0.5-2 deposited volumes per hour.

6. The method according to any of the patent claims 1-5, characterized in that the obtained Factor VIII is further purified in a manner analogous to the conventional purification of re-dissolved cryoprecipitates (precipitates) which includes, for example, further purification such as ultrafiltration, precipitation, ion changes, affinity chromatography or the like, and lyophilization using the usual excipients to obtain a stable preparation.