WO1989004840A1

WO1989004840A1 - Method of isolating coagulation factor viii

Info

Publication number: WO1989004840A1
Application number: PCT/NO1988/000086
Authority: WO
Inventors: Ole-Jan Iversen; Kåre BERGH; Størker JØRSTAD; Gøril FASTING
Original assignee: Fasting Biotech A.S.
Priority date: 1987-11-24
Filing date: 1988-11-22
Publication date: 1989-06-01
Also published as: NO874882D0; NO874882L

Abstract

Process of concentrating Factor VIII, passing blood containing Factor VIII through a non-excluding first filter of Factor VIII and subsequently to an excluding second filter retaining Factor VIII. Preferably the retentate from the second filter is recirculated to the plasma stream between the first and the second filter.

Description

Method of isolating- coagulation Factor VIII

This invention relates to a process of isolating coagulatio Factor VIII from blood.

Hemophili A is a hereditary bleeding disease due to the subject's lack of ability to synthesize a protein, Factor VIII, which is required for the coagulation of blood. The most important treating principle by bleedings occured with hemophili A subjects is the supplement of Factor VIII.

Factor VIII is isolated from plasma of healthy blood donators. By a usual blood donation about 200 ml plasma is obtained. The low concentration in the plasma makes a concen¬ tration of Factor VIII necessary before it can be used for treatment purposes. This is accomplished by cryopreεipitation, i.e. the plasma is frozen down to -70°C for at least 24 hours and is subsequently slowly thawed. By this process part of the plasma proteins are presipitated and among them Factor VIII. Due to the heat lability of the protein, this procedure will, however, result in a substantial loss of Factor VIII activity (50-70% loss) .

To prepare one treatment unit of Factor VIII , concentrate plasma from 5-6 blood donators is required (approximately 1 liter plasma) . A subject of 60 kg requires one Factor VIII unit by a minor bleeding, whereas several units are required by an increased bleeding or a heavier patient. The patients will further by and by produce neutralizing antibodies against Factor VIII requiring increased treatment doses. On a world¬ wide basis Factor VIII concentrate is in short supply.

A serious problem in connection with Factor VIII treatment is the risk of transmitting infections. The chance that a Factor VIII concentrate prepared by the present methods may comprise virus such as hepatitis B, non A non B hepatitis or AIDS-virus is not to be overlooked. Measures to reduce infection risks have high priority. Today heat treatment is used to inactivate virus, but this process also results in a substantial destruction of Factor VIII activity, rendering the total yield of a heat treatment doubtful.

The half life of Factor VIII is about 10 hours, and this means that the velocity of new synthesis is correspondingly high. An increased risk of bleeding at the donator only occurs when the Factor VIII concentration is reduced to below 10% of the normal value. The consequence is that a blood donator may donate Factor VIII quantities far beyond the contents of 200 ml plasma obtained by an ordinary blood donation.

In view of this a new principle of isolating Factor VIII is suggested, reducing the infection risk and increasing the quantity of Factor VIII per donation. The principle resides in that the blood of the donator is passed through a filter, plasma separator, separating blood cells from plasma. The blood cells are returned to the patient, whereas the plasma is passed through a filter 2, plasma filter, having a size of porousity retaining proteins of such size as Factor VIII ( oleculear weight 180.000). Other plasma components such as e.g. albumin (65.000) will pass the filter and be returned to the patient. If filter l, the plasma separator, has a porousity size retaining larger components than e.g. 20 nm, virus particles will not be able to pass the membrane, thereby preventing transmission of infection material with Factor VIII.

Due to the unstable character of Factor VIII it will be beneficial to perform this isolation procedure at a low temperature.

The aim of the present invention is thus to provide a process of isolating Factor VIII in blood plasma resulting in a better degree of recovery than previously and simultaneously reduce the risk of transmitting infections to the recipient.

According to the present invention process this is obtained by passing blood containing Factor VIII through a non- excluding first filter (plasma separator) retaining blood cells and possible infectious agents and further passing the plasma (filtrate) to an excluding second filter (plasma filter) retaining Factor VIII but being permeable to smaller components as e.g. albumin and collecting the Factor VIII enriched retentate.

Preferably the retentate is recirculated to the plasma stream between the first and second filter.

This is particularly achieved by the present invention by leading the plasma from the plasma separator into a cooled container means from which the plasma is passed to a plasma filter which substantially retains Factor VIII but letting smaller components such as albumin penetrate. The retained plasma (retentate) of the plasma filter is passed through a cooling device back to the container means wherein the plasma from the plasma separated initially arrives and is mixed with this, and is then passed to the plasma filter, the amount of plasma filtered through the plasma separator is substantially in volume corresponding to the filtrate volume from the plasma filter and subsequently after a desired time of operation the plasma of the recirculation circuit (the container means) being highly enriched of Factor VIII, is drained off.

As a cooling member in this circulation system a peltier element is preferably used. Preferably this peltier element is run maintaining the plasma at a temperature of 3-5°C.

Also the temperature of the container means is kept at 3- 5°C during circulation in the system.

Perferably the plasma of the recirculation is run at a velocity of appr. 500 ml/min.

The filtrate from the plasma filter is passed through a heating element thereby obtaining a temperature of 37°C before being returned to the blood donator. In the Fabio's apherese system, Fasting Biotech A/S, Trondheim, Norway, being disclosed by the 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 the filter 2. Also a cooling member is incorporated in the recirculation circuit enabling a temperature of e.g. 4°C in the recirculation circuit. This will have a stabilizing effect on a heat labile protein such as Factor VIII.

Experimental part

To study the possibility of isolating the Factor VIII by double membrane filtering, Fabio's apherese system, Fasting Biotech A/S, Trondheim, Norway was used having ASAHI filter AP05H (ASAHI Medical Co., Tokyo, Japan) as filter I having a cut-off 3 x 10⁶ and ASAHI filter AC 1760 as filter II having a cut-off 10⁵. A - bag having a volume of appr. 0,5 1 was incorporated in the recirculation circuit making a total plasma volume of the recirculation circuit of 0.7 liters. The Factor VIII activity was measured by a Cephoteεt (Nycomed A/S, Oslo, Norway) according to the method described by the producer.

The Factor VIII activity of plasma from a donator was 0.9 units/ml. The Factor VIII activity of the recirculation circuit after 1,5 liter plasma having passed filter I was 1,8 units per ml, whereas the concentration of Factor VIII after 3 liter plasma having passed filter I was 2,5 units per ml. With an estimated plasma volume of 3 liter this means that about 65% of the Factor VIII activity is isolated by this method. The analyses also indicate that the biological activity of Factor VIII is maintained during the isolation procedure.

Claims

C l a i m s

1. Process of concentrating Factor VIII, characterized in that blood containing Factor VIII is passed through a non- exlucing first filter (plasma separator) retaining red blood cells and further passing the plasma (filtrate) to an excluding second filter retaining Factor VIII, but is permeable to albumin, and collecting the Factor VIII enriched retentate.

2. The process of claim 1, characterized in that the retentate is recirculated to the plasma stream between the first and the second filter.

3. The process of claim 1 and 2, of concentrating Factor VIII from blood, characterized in that the blood is passed through a first membrane filter (plasma separator) retaining blood corpuscles and infectious agents, the filtrate being passed into a cooled container means, preferably having a drain off means, further as plasma from said container means to a second membrane filter substantially retaining Factor VIII, but permeable to albumin, having a moleculear weight of about 65.000, whereas the retained stream (retentate) in the membrane filter is returned through a cooling device to the container means to which the plasma from the first membrane filter first arrives, being mixed with this and again being passed with this to the plasma filter, and from this recirculation system blood plasma enriched of Factor VIII is drained off, preferably from the container means.

4. The process of claim 1 - 3, characterized in that a peltier element is used as cooling member.

5. The process of claim 1 - 3, characterized in that the cooling member is run at a temperature down to 3-5°C.

6. The process of claims 1-3, characterized in that the temperature of the container means is kept down to 3-5°C during the circulation of the system.