JPS61168367A - Hepatitis virus removing filter - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a filter for removing hepatitis viruses from protein solutions such as serum proteins.

[Prior art]

Although it is strongly desired to remove harmful hepatitis viruses from plasma protein and other protein solutions for the purpose of preventing the risk of viral infection, no satisfactory removal method has been known so far.

For example, so-called microfiltration membranes permeate plasma proteins, but they also permeate small-sized viruses, so they cannot be used to remove viruses from plasma.

In addition, although there are so-called ultrafiltration membranes that can block viruses, they are insufficient for plasma protein permeation. Since these methods are incompatible, there has been no successful case yet, even though there is an extremely high need for the prevention of serum hepatitis.

In addition, in order to remove hepatitis B virus from plasma or serum, research is being conducted on a method to selectively adsorb and remove the virus by passing the plasma through a column immobilized with anti-HBs antibodies (Meng Oshiro et al. , Artificial Organ Yu 108
(1983)), there are still many problems in the practical application of L. Moreover, this column cannot remove viruses other than hepatitis B virus.

〔problem〕

To provide a filter that can easily remove hepatitis virus from a protein solution such as plasma without changing its composition.

[Solution]

The above problem is solved by forming one or two open adhesive ends formed by bonding the ends of a bundle consisting of a large number of porous hollow fiber membranes with both ends of each porous hollow fiber membrane open. A filter that is partitioned and has an inlet for a stock solution and an outlet for a filtrate, wherein the porous hollow fiber membrane has an effective membrane thickness of 5 μm or more, and has a rejection rate of 60% for polystyrene latex with a particle size of 0.091 μm. The above problems are solved by a hepatitis virus removal filter which is characterized by having a blocking rate of 95% or more for polystyrene latex with a particle size of 0.173 μm and a transmittance of bovine serum T-globulin of 80% or more.

The effective thickness of the hollow fiber membrane is 5 μm or more, preferably 10 μm or more. Here, the effective membrane thickness refers to the membrane thickness of the porous structure directly involved in protein permeation or blocking function. In other words, it is difficult to achieve both virus removal and high protein permeability with membranes with asymmetric structures such as conventional ultrafiltration membranes, so in the present invention, membranes with a relatively uniform structure in the thickness direction are used. A porous membrane having the following properties is used. If the effective film thickness is less than 5 μm, the purpose of the present invention cannot be achieved due to insufficient prevention of viruses such as hepatitis.

If the inhibition rate of the polystyrene latex of each of the above average particle diameters of the hollow fiber membrane (measurement method will be described later) is less than each of the above percentages, the removal of hepatitis virus will be insufficient.

Furthermore, if the bovine serum γ-globulin permeability of the hollow fiber membrane (measuring method will be described later) is less than 80%, the loss of proteins such as plasma will be large, making it impossible to achieve the object of the invention.

Furthermore, it is preferable that the hollow fiber membrane has a rejection rate of 50% or more for silica sol having a particle size of 0.045 μm.

This blocking function can further increase the rate of hepatitis virus removal.

As serum hepatitis viruses, hepatitis B virus (zonal particles) and non-A, non-B hepatitis virus are known. Among these, the lowest infection value for hepatitis B is 103 to 10.
It is said to be about 4 viruses/11β. Therefore, the removal rate of hepatitis B virus by the filter of the present invention is 99.
．． If it is 9% or more, preferably 99.99% or more, the risk of infection is significantly reduced.

Conventionally, it was thought that it was impossible to remove hepatitis viruses using porous membranes, but it is revolutionary that the present invention has successfully achieved both virus removal and protein permeation.

The porous membrane having the above characteristics used in the present invention can be made of any material such as cellulose acetate, polymethyl methacrylate, polyacrylonitrile, polysulfone, or polyolefin. Membranes are preferred. Particularly preferred polyolefin membranes are polyethylene membranes or polypropylene membranes having the following properties, with polyethylene membranes being the best.

That is, a particularly preferable polyolefin membrane has a porosity of 40 to 90% by volume and a membrane thickness of 5 to 100 itm.
The micropores in the membrane are like a stack of many strip-shaped micropores formed by microfibrils arranged in the stretching direction of the membrane and knots connected at almost right angles to the microfibrils. These micropores are interconnected from one side of the membrane to the other.

If the porosity is less than 40% by volume, it is difficult to obtain a membrane that is highly permeable to protein solutions and inhibits viruses;
If it exceeds 0% by volume, it has the disadvantage that the strength decreases. Even if the membrane thickness exceeds 100 μm, the filtration effect is not improved, the membrane structure tends to be non-uniform, and the membrane area that can be accommodated in a unit volume is reduced. A membrane with micropores in which strip-shaped micropores formed from microfibrils and nodes are stacked has micropores (through-holes) with a constant diameter extending from one surface of the membrane to the other. ) It has the characteristics of a higher rejection rate and a larger liquid permeation flow rate than a so-called straight bore type membrane with holes. Although the mechanism is unknown, membranes with these characteristics block small viruses and allow proteins such as T-globulin to pass through well.

The average width of the rectangular micropores in the porous polyolefin is preferably 0.05 to 0.35 μm. If the average width is less than 0.05 μm, protein permeation will be insufficient, and if it exceeds 0.635 μm, virus inhibition will be insufficient. The average width of the strip-shaped micropores can be determined by reading a scanning electron micrograph of the membrane surface, and is expressed as the average width of 50 pores.

A polyethylene hollow fiber having a particularly preferable membrane structure as described above can be produced, for example, as follows. That is, essentially unbranched high-density polyethylene having a melt index value of 1 to 15 and a density of at least 6955 g/crl is melt-spun using a hollow fiber manufacturing nozzle at a relatively low temperature and with a relatively high draft;
The resulting highly oriented crystalline unstretched hollow fibers are annealed at a temperature below the melting point of the polymer, if necessary, and then stretched at a stretching rate of 5.
~200% cold stretching, then 1
It is manufactured by performing heat stretching in stages or in multiple stages, and then heat setting as required.

[Rejection rate of polystyrene latex or silica sol particles]

This will be explained with reference to FIG. A module (5) with a filtration area of about 10 cta is prepared using a hollow fiber membrane (4), and its particle rejection rate is measured. A 0.1% aqueous dispersion of polystyrene latex or silica sol is sucked by a pump (13) and filtered from the outside to the inside of the hollow fiber membrane at a rate of 8IIIl/lll1n, filtering a total of 40 mj2. 2ml of filtrate
Aliquot it into test tubes (14), dilute it to a concentration of 25%, and measure the absorbance.

The rejection rate is calculated using the following formula.

The rejection rate of each component is determined, and the value therein is taken as the rejection rate of the membrane.

The absorbance measurement wavelength was 280 nm for 0.091 μm polystyrene (manufactured by Dow Chemical Company) and 400 μm for 0.173 μm polystyrene (manufactured by Dow Chemical Company).

0.095 μm silica sol (Catalyst Chemical Co., Ltd.)
oilSI-45P) uses 250 tlm.

[Transmittance of bovine serum γ-globulin]

Does the permeability of bovine serum T-globulin depend on the effective length of hollow fiber membranes? When a physiological saline solution of 0.1% bovine serum γ-globulin (Fraction I, manufactured by Sigma) was filtered using a CIl hollow fiber membrane module under conditions of a transmembrane pressure difference of 50 msHg, the filtrate contained The concentration of bovine serum γ-globulin is determined from absorbance measurement at 280 nm, and is calculated from the following formula.

Next, a module is produced using a plurality of the above hollow fiber membranes. The module may have two open adhesive ends (3) as shown in FIG. 1, or one open adhesive end (3) as shown in FIG. The protein solution may enter through the opening (2), be filtered from the outside to the inside of the hollow fiber membrane, and exit through the opening (1), or enter through the opening (1) and be filtered from the inside to the outside of the hollow fiber membrane. It may also be made to exit from the opening (2).

When using the hepatitis virus removal filter of the present invention for plasma transfusion, it is desirable to use a small module with a membrane area of 0.1 to 0.5M. In this case, the simple format of the module is one having one open adhesive end (3) as shown in FIG.

Moreover, the filtration method in this case is more preferably a method in which the opening (2) is used as the inlet, the opening (1) is used as the outlet, and the hollow fiber membrane is filtered from the outside to the inside. By incorporating such a filter into a blood transfusion circuit, it is possible to prevent the occurrence of serum hepatitis.

In recent years, plasmapheresis therapy has been used to treat autoimmune diseases, liver failure, malignant tumors, etc., and has achieved good results, but one of its biggest problems is the occurrence of serum hepatitis. In this treatment, blood cells and plasma are separated using a filter or centrifuge, and the separated plasma is discarded and fresh frozen plasma (FFP) is supplied as a replacement fluid.
A fairly high incidence of hepatitis has been observed due to P. Recently F
A double filtration method that does not use FP has also been adopted, but there are many diseases for which double filtration is not effective. FIG. 3 shows one example of the usage of the present invention, which includes an arterial blood supply circuit (6), a venous return circuit (7), a pump (10, 11, 12), a plasma separation filter (8), It consists of an FFP bag (9) and a hepatitis virus removal filter (5). Such a device allows safe plasma exchange therapy to be performed.

Grand opening The present invention will be specifically described below with reference to Examples.

Example 1 Micropores are interconnected from the inner surface to the outer surface of the membrane,
The micropores have a structure in which a large number of strip-shaped micropores are stacked, which are formed by microfibrils arranged in the stretching direction and knots connected at almost right angles to the microfibrils. rate of 66% by volume, film thickness of 55 μm, bovine serum T-globulin permeability of 99%, rejection rate of 0.091 μm polystyrene latex (manufactured by Dow Chemical Company): 9
0%, rejection rate of 0.173 μm polystyrene latex 100%, rejection rate of 0.045 nm silica sol 55
% polyethylene hollow fiber membranes were bundled into a loop, and with both ends of each hollow fiber membrane open, the ends were adhered with polyurethane resin to produce a module with an effective length of 6 cm and a membrane area of 0.05 rd. After making it hydrophilic with ethanol, fresh human plasma positive for HBs antigen was passed through the outside of the hollow fiber of this module at a flow rate of 5+wIl/+sin and filtered. Analysis of the filtrate revealed that the permeability of albumin was 100% and the permeability of γ-globulin was 96%. In addition, 100 ml each of plasma and filtrate before filtration was centrifuged at 2,000 G for 1 hour, the supernatant was centrifuged at 300,000 G for 18 hours, and the pellet was dissolved in 101111 physiological saline. Next, the mixture was treated with amylase, lipase, trypsin, neuraminidase, phospholipase C, and pronase to decompose mixed plasma components. Then 5 ephad
Gel filtration was performed using exG-200, and the first peak was collected. This was purified by a sucrose density gradient method, and then ultracentrifuged at 222,000G for 40 hours in a cesium chloride solution to extract d=1.21 (HB antigen band). When this was observed using an electron microscope, it was found that the plasma before filtration contained spherical particles with a diameter of 2.2 nm.
The presence of zone particles with a diameter of about 42 nm was observed mixed with the 230 nm tubular particles. On the other hand, no zone particles were observed in the plasma after filtration.

Comparative Example Polyacrylonitrile ultrafiltration membrane (molecular weight cut off: 13,
000, effective film thickness 0.5 μm, polystyrene latex rejection rate 99.9%, bovine serum T-globulin permeability rate 5
When plasma similar to that used in Example 1 was filtered using a module of %), almost neither albumin nor T-globulin passed through the filter, and the object of the present invention could not be achieved.

Comparative Example 2 Membrane filter manufactured by Plans Wisok Co., Ltd. (pore size 0.
As a result of filtering the same plasma as in Example 1 using a polystyrene latex with an effective membrane thickness of 2 μm, a particle size of 0.085 μm, a blocking rate of 20%, and a bovine serum γ-globulin permeability of 99%, albumin and γ-globulin were filtered. - The transmission of globulin was more than 90%, but it was not possible to remove zone particles.

Example 2 Porosity: 67%, membrane pressure: 53μ, bovine serum T-globulin permeability: 100%, rejection rate of 0.091 nm polystyrene latex: 100%, rejection rate of 0.173 nm latex: 100%, and The same polystyrene hollow fiber membrane as in Example 1 was used except that the rejection rate of 045 μm silica sol was 90%, and the filtration area was 0.1 in the same manner as in Example 1.
A module with M and an effective length of 7- was created and subjected to hydrophilic treatment with ethanol.

Fresh human plasma positive for HBs antigen is applied at 10 nu/min to the outside of the hollow fiber membrane of this module using a tube pump.
and filtered. Analysis of the filtered plasma revealed that the albumin permeability was 100% and the T-globulin permeability was 98%.

In addition, plasma before and after filtration was purified in the same manner as in Example 1,
When examined using an electron microscope, zonal particles were observed in the plasma before filtration, but no zonal particles were observed in the plasma after filtration.

Example 3 In the plasmapheresis treatment apparatus shown in FIG. 3, the same filter (5) as in Example 1 was used as a hepatitis virus removal filter. As an in vitro experiment, fresh bovine blood was supplied through the opening (1), blood cells and plasma were separated using a filter (8), and the plasma was discarded using a pump (11). On the other hand, as a hepatitis virus model, the 5abin Type 1 poliovirus vaccine strain was propagated using epithelial cells of the normal kidneys of African Mitris monkeys, and the virus propagation solution was
Centrifugation was performed for 00 Orp, and the supernatant was collected. 106TCID of virus stock solution in serum 0% medium. /ml, put this in the FFP bar knob (9),
It was filtered through a filter (5) at a speed of 10 m (1/min). The filtrate 50II11 was collected and subjected to virus quantification.

Virus quantification was performed using a measurement method based on CPE observation. As a result, the virus concentration in the filtrate was 10''TCIDs.
o/m A, and poliovirus is 1/1000
It was found that the value had decreased to below 0.

〔Effect of the invention〕

With the simple hepatitis virus removal filter of the present invention, it has become possible to remove hepatitis B virus from protein solutions such as plasma. Hepatitis B virus in transfused plasma is checked by testing for HBs antigen, but hepatitis virus may be present even in HBs antigen negative blood that is below the detection limit for HBs antigen, and this is a major cause of hepatitis B onset after blood transfusion. considered to be the cause. It is believed that the hepatitis removal filter of the present invention can solve these problems.

Furthermore, although the onset of non-A, non-B hepatitis due to post-transfusion inflammation is currently a serious problem, there is still no testing method (or countermeasures) for non-A, non-B hepatitis. According to the invention, since it is possible to remove poliovirus, which is said to be one of the smallest viruses, it is thought that non-A, non-B hepatitis virus can also be removed, and its significance is extremely large.

[Brief explanation of drawings]

1 and 2 are explanatory diagrams of embodiments of the hepatitis virus removal filter of the present invention, FIG. 3 is a system diagram of a plasma exchange treatment device equipped with the hepatitis virus removal filter of the present invention, and FIG. The figure is an explanatory diagram of a filtrate separation device for measuring the rejection rate. 1.2... Inlet or outlet of protein solution, 3...
・Open adhesive end of hollow fiber membrane, 4...Hollow fiber membrane, 5...
・Hepatitis virus removal filter, 6...Arterial blood supply circuit, 7...Return circuit to vein, 8...Plasma separation filter, 9...FFP bar knob, 10.11.12゜13...・Pump, 14...test tube.

Claims (1)

[Claims] 1. One or two open adhesives formed by bonding the ends of a bundle consisting of a large number of porous hollow fiber membranes with both ends of each porous hollow fiber membrane open. It is divided at the end,
and a filter having an inlet for a stock solution and an outlet for a filtrate, wherein the porous hollow fiber membrane has an effective membrane thickness of 5 μm or more, a rejection rate of 60% or more for polystyrene latex with a particle size of 0.091 μm, and a particle size of 0. A hepatitis virus removal filter characterized by having a blocking rate of 95% or more for polystyrene latex of 173 μm and a transmittance of bovine serum γ-globulin of 80% or more. 2. The hepatitis virus removal filter according to claim 1, wherein the porous hollow fiber membrane has a rejection rate of 50% or more for silica sol having a particle size of 0.045 μm. 3. The hepatitis virus removal filter according to claim 1, which is inserted into the fresh frozen plasma supply route in a plasma exchange device.