NO874882L - PROCEDURE FOR CONCENTRATION OF FACTOR VIII. - Google Patents
PROCEDURE FOR CONCENTRATION OF FACTOR VIII.Info
- Publication number
- NO874882L NO874882L NO87874882A NO874882A NO874882L NO 874882 L NO874882 L NO 874882L NO 87874882 A NO87874882 A NO 87874882A NO 874882 A NO874882 A NO 874882A NO 874882 L NO874882 L NO 874882L
- Authority
- NO
- Norway
- Prior art keywords
- plasma
- factor viii
- filter
- blood
- container device
- Prior art date
Links
- 229960000301 factor viii Drugs 0.000 title claims description 37
- 238000000034 method Methods 0.000 title claims description 17
- 210000002381 plasma Anatomy 0.000 claims description 49
- 102000001690 Factor VIII Human genes 0.000 claims description 38
- 108010054218 Factor VIII Proteins 0.000 claims description 38
- 210000004369 blood Anatomy 0.000 claims description 14
- 239000008280 blood Substances 0.000 claims description 14
- 238000001816 cooling Methods 0.000 claims description 6
- 239000012465 retentate Substances 0.000 claims description 6
- 102000009027 Albumins Human genes 0.000 claims description 5
- 108010088751 Albumins Proteins 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 210000000601 blood cell Anatomy 0.000 claims description 4
- 208000015181 infectious disease Diseases 0.000 claims description 4
- 230000000717 retained effect Effects 0.000 claims description 3
- 230000002458 infectious effect Effects 0.000 claims description 2
- 210000003743 erythrocyte Anatomy 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 description 6
- 238000002955 isolation Methods 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 208000032843 Hemorrhage Diseases 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000012678 infectious agent Substances 0.000 description 3
- 201000003542 Factor VIII deficiency Diseases 0.000 description 2
- 238000002617 apheresis Methods 0.000 description 2
- 208000034158 bleeding Diseases 0.000 description 2
- 230000000740 bleeding effect Effects 0.000 description 2
- 229940105778 coagulation factor viii Drugs 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 206010019786 Hepatitis non-A non-B Diseases 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 208000031169 hemorrhagic disease Diseases 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
Metode for isolering av koagulasjonsfaktor VIII Method for isolation of coagulation factor VIII
Foreliggende oppfinnelse vedrører en fremgangsmåte for isolering av koagulasjonsfaktor VIII fra blod. The present invention relates to a method for isolating coagulation factor VIII from blood.
Hemofili A er en arvelig blødersykdom som skyldes at pasienten mangler evnen til å syntetisere et protein, Faktor VIII, som er nødvendig for at blod skal kunne koagulere. Det viktigste behandlingsprinsipp ved oppståtte blødninger hos hemofili A pasienter er tilførsel av Faktor VIII. Haemophilia A is a hereditary bleeding disorder caused by the patient lacking the ability to synthesize a protein, Factor VIII, which is necessary for blood to coagulate. The most important treatment principle in the event of bleeding in haemophilia A patients is the administration of Factor VIII.
Faktor VIII isoleres fra plasma fra friske blodgivere.Factor VIII is isolated from plasma from healthy blood donors.
Ved en vanlig blodtapping vil man få ca. 200 ml plasma. DenWith a normal blood donation, you will receive approx. 200 ml of plasma. It
lave konsentrasjonen i plasma gjør at Faktor VIII må oppkonsen-treres før den kan benyttes i behandlings-øyemed. Dette skjer ved kryopresipitering, det vil si at plasmaet fryses ned til - 70°C for minst et døgn og tines deretter langsomt. Ved denne prosessen vil en del plasmaproteiner felles ut og deriblandt Faktor VIII. På grunn av proteinets varmelabilitet vil imidlertid denne prosedyren medføre et betydelig tap av Faktor Vlll-aktivitet (50-70% tap). the low concentration in plasma means that Factor VIII must be concentrated before it can be used for treatment purposes. This happens by cryoprecipitation, which means that the plasma is frozen to - 70°C for at least a day and then thawed slowly. During this process, a number of plasma proteins will precipitate out, including Factor VIII. However, due to the protein's heat lability, this procedure will entail a significant loss of Factor VIII activity (50-70% loss).
For å fremstille en behandlingsenhet Faktor Vlll-konsentrat trengs plasma fra 5-6 blodgivere (vel 1 liter plasma). En pasient på 60 kg. trenger én Faktor VIII enhet ved en mindre blødning, mens det ved en større blødning eller en tyngre pasient trengs flere enheter. Pasientene vil dessuten etter hvert danne nøytraliserende antistoffer mot Faktor VIII - noe som medfører at behandlingsdosene måøkes. To produce one treatment unit of Factor Vlll concentrate, plasma from 5-6 blood donors is needed (about 1 liter of plasma). A patient weighing 60 kg. need one Factor VIII unit for a minor bleed, while for a larger bleed or a heavier patient, more units are needed. The patients will also eventually form neutralizing antibodies against Factor VIII - which means that the treatment doses must be increased.
På verdensbasis er Faktor Vlll-konsentrat mangelvare.On a worldwide basis, Factor Vlll concentrate is in short supply.
Et alvorlig problem i forbindelse med Faktor VIII behandling er risikoen for overføring av smittestoffer. Sjansen for at Faktor VIII konsentrat fremstilt etter dagens metoder skal inneholde vira som hepatitt B, non A non B hepatitt eller AIDS-virus er ikke ubetydelig. Tiltak som kan redusere smitte-risikoen har høy prioritet. Idag benyttes varmebehandling for å inaktivere virus, men denne prosessen medfører samtidig en betydelig ødeleggelse av Faktor VIII aktiviteten, slik at den totale gevinsten ved varmebehandling er usikker. A serious problem in connection with Factor VIII treatment is the risk of transmission of infectious agents. The chance that Factor VIII concentrate produced using current methods will contain viruses such as hepatitis B, non-A non-B hepatitis or the AIDS virus is not insignificant. Measures that can reduce the risk of infection have a high priority. Today, heat treatment is used to inactivate viruses, but this process also leads to a significant destruction of Factor VIII activity, so that the total gain from heat treatment is uncertain.
Halveringstiden for Faktor VIII er omkring 10 timer, og det betyr at hastigheten for nysyntese er tilsvarende stor. Økt blødningsrisiko hos giver oppstår ikke før Faktor VIII konsentrasjonen reduseres til under 10% av normalverdien. Dette innebærer at en blodgiver kan donere Faktor VIII mengder langt utover innholdet i 200 ml plasma som oppnås ved en vanlig blodgiving. The half-life of Factor VIII is around 10 hours, which means that the rate of new synthesis is correspondingly high. Increased risk of bleeding in the donor does not occur until the Factor VIII concentration is reduced to below 10% of the normal value. This means that a blood donor can donate quantities of Factor VIII far beyond the content of 200 ml of plasma that is obtained from a normal blood donation.
Mot denne bakgrunn vil man foreslå et nytt prinsipp for isolering av Faktor VIII som reduserer infeksjonsrisikoen og øker Faktor VIII mengden pr. donasjon. Prinsippet består i at man lar giverens blod passere et filter, plasmaseparator, som skiller blodceller fra plasma. Blodcellene føres tilbake til pasienten, mens plasmaet ledes via et filter 2, plasma filter, med en maskestørrelse som gjør at proteiner av størrelse som Faktor VIII (molekylvekt 180000) holdes tilbake. Andre plasmakomponenter som f.eks. albumin (65000) vil passere filteret og ledes tilbake til pasienten. Dersom filter 1, plasmaseparatoren, har en maskestørrelse som holder tilbake komponenter større enn f.eks. 20 nm, så vil viruspartikler ikke kunne passere membranen, og man vil derved forhindre at infeksiøst materiale overføres sammen med Faktor VIII. Against this background, a new principle for the isolation of Factor VIII will be proposed which reduces the risk of infection and increases the Factor VIII amount per donation. The principle consists in allowing the donor's blood to pass through a filter, plasma separator, which separates blood cells from plasma. The blood cells are returned to the patient, while the plasma is led via a filter 2, plasma filter, with a mesh size that means that proteins of a size such as Factor VIII (molecular weight 180,000) are retained. Other plasma components such as albumin (65000) will pass the filter and be returned to the patient. If filter 1, the plasma separator, has a mesh size that retains components larger than e.g. 20 nm, then virus particles will not be able to pass the membrane, and you will thereby prevent infectious material from being transferred together with Factor VIII.
På grunn av Faktor VIII's labile karakter vil det være en fordel om denne isoleringsprosedyren foregår ved lav temperatur. Due to Factor VIII's labile nature, it would be an advantage if this isolation procedure takes place at a low temperature.
Målet for foreliggende oppfinnelse er derfor å tilveiebringe en fremgangsmåte for isolering av Faktor VIII i blodplasma som fører til at man får en bedre utvinningsgrad enn tidligere og samtidig reduserer faren for å overføre smittestoffer til mottaker. The aim of the present invention is therefore to provide a method for isolating Factor VIII in blood plasma which leads to a better recovery rate than before and at the same time reduces the risk of transferring infectious agents to the recipient.
Dette oppnås ved fremgangsmåten ifølge foreliggende oppfinnelse ved at man fører blod som inneholdere Faktor VIII gjennom et ikke-ekskluderende første filter (plasmaseparator) This is achieved by the method according to the present invention by passing blood containing Factor VIII through a non-exclusive first filter (plasma separator)
som holder tilbake blodceller og eventuelle smittestoffer og videre fører plasmaet (filtratet) til et ekskluderende andre filter (plasmafilter) som holder Faktor VIII tilbake, men er permeabelt for mindre komponenter som f.eks. albumin, og samler opp det Faktor Vlll-anrikede retentat. which holds back blood cells and any infectious agents and further leads the plasma (filtrate) to an exclusionary second filter (plasma filter) which holds back Factor VIII, but is permeable to smaller components such as e.g. albumin, and collects the Factor VIII-enriched retentate.
Fortrinnsvis føres retentatet i resirkulasjon til plasma-strømmen mellom første og andre filter. Preferably, the retentate is recirculated to the plasma flow between the first and second filter.
Særlig vil man ifølge foreliggende oppfinnelse oppnå dette ved at plasmaet fra plasmaseparator føres inn i en avkjølt beholderanordning hvorfra plasma føres videre til et plasmafilter som i det vesentlige holder tilbake Faktor VIII men slipper gjennom mindre komponenter som albumin. Det tilbakeholdte plasmaet (retentatet) i plasmafilteret føres via et kjøle-element tilbake til beholderanordningen hvor plasmaet fra plasmaseparatoren først kommer og blandes med dette, som så igjen føres til plasmafilteret slik at plasmamengden som filtreres gjennom plasmaseparatoren i volum i det vesentlige tilsvarer filtratvolumet fra plasmafilteret, og etter en ønsket operasjonstid tappes plasmaet i resirkulasjonskretsen (beholderanordningen) som er sterkt anriket på Faktor VIII. In particular, according to the present invention, this will be achieved by the plasma from the plasma separator being fed into a cooled container device from which the plasma is fed on to a plasma filter which essentially retains Factor VIII but allows smaller components such as albumin to pass through. The retained plasma (retentate) in the plasma filter is fed via a cooling element back to the container device where the plasma from the plasma separator first comes and is mixed with this, which is then again fed to the plasma filter so that the volume of plasma that is filtered through the plasma separator in volume essentially corresponds to the filtrate volume from the plasma filter , and after a desired operating time the plasma is drained into the recirculation circuit (container device) which is highly enriched in Factor VIII.
Som et kjøle-element i dette sirkulasjons-systemet anvender man fortrinnsvis et peltierelement. Med fordel drives dette peltierelement slik at plasmaet har en temperatur på 3-5° C- A peltier element is preferably used as a cooling element in this circulation system. Advantageously, this peltier element is operated so that the plasma has a temperature of 3-5° C-
Også temperaturen i beholderanordningen holdes på 3-5° C under sirkuleringen i systemet. The temperature in the container device is also kept at 3-5° C during circulation in the system.
Med fordel kan plasmaet i resirkulasjonen kjøres med en hgastighet på ca. 500 ml/min. Advantageously, the plasma in the recirculation can be run at a speed of approx. 500 ml/min.
Filtratet fra plasmafilteret vil ledes via et varme-element slik at det antar en temperatur på 37° C før det returneres til blodgiver. The filtrate from the plasma filter will be led via a heating element so that it assumes a temperature of 37° C before being returned to the blood donor.
I Fabio's apheresesystem, Fasting Biotech A/S, Trondheim, Norge som er beskrevet i norsk patentsøknad 86.4728, benyttes et resirkulasjonssystem mellom filter 1, plasmaseparator, og filter 2, plasmafilter. Dette resirkulasjonsprinsippet hindrer tiltetting av filter 2. I resirkulasjonskretsen er det dessuten inkorporert et kjøleelement som gjør at man i re-sirkulas jonskretsen kan oppnå en temperatur på f.eks. 4°C. Dette vil virke stabiliserende på et varmelabilt protein som Faktor VIII. In Fabio's apheresis system, Fasting Biotech A/S, Trondheim, Norway, which is described in Norwegian patent application 86.4728, a recirculation system is used between filter 1, plasma separator, and filter 2, plasma filter. This recirculation principle prevents clogging of filter 2. In addition, a cooling element is incorporated in the recirculation circuit which means that in the recirculation circuit a temperature of e.g. 4°C. This will have a stabilizing effect on a heat-labile protein such as Factor VIII.
EksperimenteltExperimental
For å undersøke muligheten for isolering av Faktor VIII ved dobbelt membran-filtrering, benyttet man Fabio's apheresesystem Fasting Biotech A/S, Trondheim, Norge med ASAHI filter AP05H (ASAHI Medical Co., Tokyo, Japan) som filter 1 med cut off 3 x IO<6>og ASAHI filter AC 1760 som filter 2 med cut off 10s . I resirkulasjonskretsen var det innkoblet en bag med volum ca. 0,5 liter, slik at det totale plasmavolum i resirkulasjonskretsen var 0,7 liter. Faktor Vlll-aktiviteten ble målt med Cephotest (Nycomed A/S, Oslo, Norge) etter metode beskrevet av produsenten. To investigate the possibility of isolation of Factor VIII by double membrane filtration, Fabio's apheresis system Fasting Biotech A/S, Trondheim, Norway with ASAHI filter AP05H (ASAHI Medical Co., Tokyo, Japan) was used as filter 1 with cut off 3 x IO<6> and ASAHI filter AC 1760 as filter 2 with cut off 10s. A bag with a volume of approx. 0.5 litres, so that the total plasma volume in the recirculation circuit was 0.7 litres. Factor Vlll activity was measured with Cephotest (Nycomed A/S, Oslo, Norway) according to the method described by the manufacturer.
Faktor VTII-aktiviteten i plasma fra blodgiver var 0,9 enheter pr. ml. Faktor VTII-aktiviteten i resirkulasjonskretsen etter at 1,5 liter plasma hadde passert filter 1 var 1,8 The factor VTII activity in the plasma from the blood donor was 0.9 units per ml. The factor VTII activity in the recirculation circuit after 1.5 liters of plasma had passed filter 1 was 1.8
enheter pr. ml., mens konsentrasjonen av Faktor VIII etter at 3 liter plasma hadde passert filter 1 var 2,5 enheter pr. ml. units per ml., while the concentration of Factor VIII after 3 liters of plasma had passed filter 1 was 2.5 units per ml.
Med et antatt plasmavolum på 3 liter innebærer dette at man ved denne metoden har isolert ca. 65% av Faktor VIII aktiviteten. Analysene indikerer også at den biologiske aktivitet av Faktor VIII er besvart under isoleringsprosedyren. \ With an assumed plasma volume of 3 litres, this means that with this method, approx. 65% of Factor VIII activity. The analyzes also indicate that the biological activity of Factor VIII is answered during the isolation procedure. \
Claims (6)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NO87874882A NO874882L (en) | 1987-11-24 | 1987-11-24 | PROCEDURE FOR CONCENTRATION OF FACTOR VIII. |
| PCT/NO1988/000086 WO1989004840A1 (en) | 1987-11-24 | 1988-11-22 | Method of isolating coagulation factor viii |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NO87874882A NO874882L (en) | 1987-11-24 | 1987-11-24 | PROCEDURE FOR CONCENTRATION OF FACTOR VIII. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| NO874882D0 NO874882D0 (en) | 1987-11-24 |
| NO874882L true NO874882L (en) | 1989-05-25 |
Family
ID=19890424
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NO87874882A NO874882L (en) | 1987-11-24 | 1987-11-24 | PROCEDURE FOR CONCENTRATION OF FACTOR VIII. |
Country Status (2)
| Country | Link |
|---|---|
| NO (1) | NO874882L (en) |
| WO (1) | WO1989004840A1 (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2624373C2 (en) * | 1976-05-31 | 1983-02-03 | Arnold Dr. 8782 Karlstadt Seufert | Process for the production of sterile filtered cryoprecipitate with an enrichment of factor VIII |
| US4350156A (en) * | 1980-05-29 | 1982-09-21 | Japan Foundation For Artificial Organs | Method and apparatus for on-line filtration removal of macromolecules from a physiological fluid |
| JPS583705B2 (en) * | 1980-07-18 | 1983-01-22 | 川澄化学工業株式会社 | Double filtration plasma exchange device |
| JPS58155865A (en) * | 1982-03-12 | 1983-09-16 | 株式会社クラレ | Hollow yarn membrane for treating serum |
-
1987
- 1987-11-24 NO NO87874882A patent/NO874882L/en unknown
-
1988
- 1988-11-22 WO PCT/NO1988/000086 patent/WO1989004840A1/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| NO874882D0 (en) | 1987-11-24 |
| WO1989004840A1 (en) | 1989-06-01 |
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