JPH01192368A - Viral disease medical treatment system - 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 (Field of Industrial Application) The present invention relates to a viral disease treatment system that removes viruses from blood by a two-stage membrane filtration method.

The number of blood donors in Japan is approximately 8 million people (1982), and there is still not enough blood for medical purposes.

Particularly for plasma-derived preparations, it is supplemented by hyperemia and imported plasma. On the other hand, since the outbreak of the AIDS virus in 1978, the number of patients worldwide has increased exponentially, and in the United States, it is said to have exceeded 30,000 in February 1987. In Japan, as of March 12, 1987, there were 29 people, most of whom were hemophiliacs, infected by therapeutic blood products imported from the United States.

The number of hepatitis B carriers is estimated to be approximately 200 million worldwide, of which approximately 3 million are said to be in Japan. Like the AIDS virus, hepatitis B is transmitted through blood transfusions and can progress to serious diseases such as chronic hepatitis, liver cirrhosis, and liver cancer.

However, no fundamental treatment method for patients with AIDS or hepatitis B has yet been established.

(Prior Art) Hepatitis B and AIDS viruses are transmitted primarily through blood and saliva. For this reason, for example, heat sterilization is employed during the manufacturing process of plasma fraction preparations from pooled plasma. Other methods include a method of inactivating viruses using a chemical reaction, and a method of sterilizing with an alkaline aqueous solution or ultraviolet light. All of these methods are methods for inactivating viruses in blood products used for blood transfusions, and the current situation is that there are almost no treatments or preventive vaccines for patients who have developed AIDS or hepatitis B.

Nishihara et al. used DFP (double filtration) as an immunotherapeutic method for hepatitis B (chronic).
We attempted to remove HBV by plasmapheresis (Toshiharu Nishihara et al., Artificial Organs, 14(4), 17
62 (1985)). As a result, HBsAg was inhibited by 97%, and in the case of DNA-P (hepatitis B virus DNA synthase, present in zone particles), it was not detected in the filtrate compared to the concentration of the original solution. However, the permeability of proteins is low; for example, the permeability of γ-globulin and albumin is 60% and 71%, respectively.

(Problems to be Solved by the Invention) The present invention adopts a two-stage membrane filtration method, - Separates blood into blood cells and plasma by membrane formation, and removes viruses in the separated plasma by a secondary membrane. . Moreover, since the secondary membrane has little protein adsorption, the protein permeability is extremely high. An object of the present invention is to provide a closed extracorporeal circulation circuit treatment system that uses two stages of hollow fibers to remove viruses from the blood of people with viral illnesses and can be expected to have a life-prolonging effect.

(Means for Solving the Problems) The gist of the present invention is to provide a closed extracorporeal circulation circuit for removing viruses in blood, in which blood has an average pore diameter of 0.2 to 0.5 μm measured by the water filtration rate method, and a protein permeable rate is 9
5% or more of porous hollow fibers at a transmembrane pressure of 100 mmHg or less to separate plasma from blood. Average pore diameter by velocity method is 0.1 μm
and the minimum average pore diameter is 0.04 to 0.20. μm, the minimum in-plane porosity (Pre) is 0.1 to 0.5, it has a layered structure with 10 or more layers in the film thickness direction, and the film thickness of the hollow fiber is 10 μm or more, And virus removal rate is 90
% or more, and the treatment system for viral diseases by a two-stage membrane filtration method characterized by filtering through hollow fibers with a protein permeability of 85% or more. can be expected to have a life-prolonging effect.

The first feature of the present invention is that the secondary membrane is made of regenerated cellulose.

Porous hollow fibers made from regenerated cellulose are those in which pores are clearly seen when the inner and outer walls are observed using an electron microscope, and the pores account for 5% or more (area ratio). It means a hollow fiber whose material is composed of 90% or more of cellulose molecules.

Regenerated cellulose has less adsorption of proteins in blood.

When compared with the same membrane area, the amount of protein adsorbed is 1/3 or less compared to conventionally known materials such as polyolefins and cellulose esters. In addition, protein detachment after adsorption is extremely rapid; for example, when the hollow fiber of the present invention on which protein has been adsorbed is immersed in physiological saline, the protein detaches immediately. Therefore, clogging of the membrane by proteins is less likely to occur. However, on the contrary, the effect of removing viruses through the adsorption action of the membrane is hardly expected.

Among regenerated cellulose, cuprammonium regenerated cellulose is most desirable. Here, the cuprammonium regenerated cellulose means cellulose obtained from a cellulose cuprammonium solution. Regenerated cellulose produced by the cuprammonium method has significantly lower adsorption of proteins in aqueous solutions than other polymeric materials.

Therefore, the formation of a cake layer on the surface of the hollow fibers due to adsorption can be almost completely prevented with the cuprammonium regenerated cellulose, and the decrease in the filtration rate of the hollow fibers over time can be suppressed.

Although it is a regenerated cellulose, the cuprammonium regenerated cellulose porous hollow fiber has excellent mechanical properties. It also has high wood affinity and is suitable for filtration of aqueous solutions.

There are various methods for producing regenerated cellulose, such as the viscose method, the saponification method of cellulose ester, and the copper ammonia method. It is not something that can be discussed uniformly. In the cuprammonium method, the generation of fine pores accompanying the removal of copper is observed due to the acid treatment essential for regeneration, so the cuprammonium method regenerated cellulose hollow fibers also exhibit unique behavior in terms of protein permeability.

Regenerated cellulose is 0. It is desirable that the content of the NaOH aqueous solution be as small as possible. 40°C, 48 hours, 0. If the dissolved content is 1100 pp or less when immersed in an IN NaOH aqueous solution, this regenerated cellulose porous hollow fiber is most suitable for removing viruses from plasma.

Such regenerated cellulose is called "cellulose/alkaline aqueous solution regenerated cellulose."

In order to produce porous hollow fibers from cellulose as described above, regenerated cellulose is produced using a copper ammonia method using a stock solution of high-purity cellulose, or after the porous hollow fibers are produced, 0. It is sufficient to perform cleaning treatment with an IN NaOH aqueous solution for 72 hours or more. It is more preferable to use a high-purity cellulose raw material because the above-mentioned dissolved content is significantly reduced. Here, the term "high purity cellulose raw material" refers to cotton linters and wood pulps having an α-cellulose content of 95 wt% or more and a degree of polymerization of 500 or more.

For these raw materials, it is preferable to always use purified water in order to prevent decomposition and oxidation during bleaching and washing steps and to avoid contamination with impurities.

A second feature of the invention is the specified pore structure. That is, the point is to use porous hollow fibers produced by a microphase separation method having a specific average pore size range and in-plane porosity (Pre). The diameter of hepatitis B virus (HBg) is approximately 4
2nm, the diameter of the AIDS virus is approximately 1100n, adult T
Since the diameter of cellular leukemia virus is approximately 1100n,
It can be first inferred that the average pore diameter of the porous hollow fibers is 10' On m or less.

A particularly important feature of the secondary membrane of the present invention is the combination of the specified membrane structure and specified pore characteristics in order to increase the virus removal rate and the protein permeability.

That is, the average pore diameter determined by the water filtration rate method is 0.5 times or more and 1.0 times or less than the virus diameter, and the minimum average pore diameter is 0.0.
4 to 0.20 μm, minimal in-plane porosity (Pre) 0.1
~0.5, and is a porous hollow fiber having a layered structure with 10 or more layers in the thickness direction.

Here, hollow fibers with a layered structure in the film thickness direction are: ■ have a homogeneous structure in a plane parallel to the outer wall surface or inner wall surface, and ■ have a certain pore size distribution, average pore size, and Pre defined in each layer. .For planes at different distances from the membrane surface, the pore size distribution, average pore diameter, and Pre all change depending on the distance from the membrane surface, and . In either case, it means having a homogeneous structure.

A homogeneous structure is one in which the pores are arranged in a disorderly manner.If the average pore diameter at any two locations is determined to be 1Jll by an electron microscope, the tertiary average pore diameter calculated using equation (1) below is
This means that the difference between the values of 3 corresponds within 20% as a relative value. Further, the structure of a cross section perpendicular to the film surface is approximated by a deposit of particles with a diameter of 0.1 to 2 μm (particle diameter is 282 μm). The number of layers in the present invention refers to the film thickness T
Then, it is defined as T/4S2. In addition, the minimum average pore diameter means the minimum value of the distance dependence of 273 in the film thickness direction, and the minimum in-plane porosity (Pre) refers to the distance dependence of the in-plane porosity in the film thickness direction. It means the minimum value in terms of gender.

As the average pore size increases, the filtration rate increases. Therefore, the larger the average pore diameter, the better.

However, as the average pore size increases, the virus concentration in the filtrate increases, making it difficult to achieve a removal rate of 90% or more. If the average pore diameter is 0.5 to 1.0 times the virus diameter according to the water filtration rate method, the virus removal rate is 9.
It will be 0% or more. This removal efficiency increases rapidly when the number of layers in the film thickness direction increases to 10 or more. If the number of layers is 10 or more, the virus removal rate in the filtrate will be 90% or more even if the minimum average pore diameter is 1.0 times or more the diameter of the virus to be removed. As the number of layers increases, even if the average pore diameter measured by the water filtration rate method becomes larger, the above-mentioned virus removal ability is maintained. However, if the minimum average pore diameter exceeds four times the diameter of the virus to be removed, the above virus removal rate cannot be achieved even if the number of layers is 100. As the number of layers increases, the filtration rate decreases, so the number of layers cannot be increased significantly. In addition, under conditions where the number of layers and the ratio of the virus diameter to the minimum average pore size are constant, in order to further increase the virus removal rate and maintain a high filtration rate, the minimum average pore size should be 0.04~
It is necessary that the diameter is 0.2 μm and the minimum in-plane porosity is 0.1 to 0.5.

Generally, as the minimum in-plane porosity increases, the virus removal ability decreases. Therefore, it is preferable to select an appropriate combination of minimal in-plane porosity and film thickness. With conventional asymmetric membranes, it was believed that the thinner the membrane, the better. However, as mentioned above, in order to achieve a virus removal rate of 90% or higher and a protein permeability of 85% or higher, the membrane must be thinner. The thickness needs to be 10 μm or more. Further, from the viewpoint of mechanical properties, the larger the film thickness, the better. However, as the membrane thickness increases, the amount of protein adsorbed increases or the filtration rate decreases. The film thickness is preferably 100 μm or less in order to be able to fully exhibit its function as a layered structure and to facilitate the production of the layered structure.

The features of the present invention, namely, high virus removal rate, high filtration rate, high protein concentration in the filtrate, and little change in filtration properties over time, are achieved by providing a specific porous membrane shape. That is, the porous hollow fibers used in the present invention have a hollow fiber shape with a circular cross section with an inner diameter of 200 to 800 μm, and a film thickness of 20 to 100 μm.
It is preferable to design it to have a continuous penetration part and a length of 5 to 50 cm.

These specific shapes can prevent denaturation of coexisting proteins during ultrafiltration of viruses. This is probably because there is a correlation between the denaturation of proteins and the rate of friction applied to the solution before filtration, the smoothness of the solution flow (streamline), and the local rate of friction when passing through the pores. It will be done.

Proteins are also present in the virus-containing solution, so the viscosity of the solution increases with filtration time. When the length of the hollow fiber is long and the inner diameter is small, it is necessary to increase the pressure applied to the hollow fiber. Increasing the inner diameter reduces protein denaturation, but the loadable pressure rapidly decreases, the amount of solution remaining inside the hollow fiber increases, and the filtration device becomes larger with the same effective area. Therefore, the inner diameter of the hollow fibers is preferably designed to be 200 to 800 μm. The length of the hollow fiber should be varied depending on the inner diameter, but in the case of the above inner diameter range, an appropriate effective length is 5 to 50 cm. Under conditions of the specified inner diameter and film thickness, as Pre increases, the mechanical properties of the hollow fiber decrease.

In order to maintain a high ultrafiltration rate and high virus removal ability, the optimum range of Pre is preferably 0.15 to 0.45. Under these conditions, it is desirable to design the mechanical properties of the hollow fibers within a range that is sufficiently practical even under water.

As an example of the hollow fibers used in the present invention, in the process of spinning hollow fibers using a cuprate-ammonia solution with a cellulose concentration of 3 to 10 wt% as a spinning stock solution, microphase separation is slowly carried out from the inner and outer wall surfaces toward the inside. , can be produced by simultaneously generating and progressing within the same plane. The winding speed is set so that the hollow fibers are immersed in the coagulation bath for one minute or more. At this time, it is necessary to strictly control the temperature of the stock solution, hollow agent, and coagulant. Here, microphase separation means a state in which a concentrated phase or a dilute phase of a polymer is dispersed and stabilized as particles having a diameter of 0.02 to several μm in a solution. The occurrence of microphase separation can be observed directly with the naked eye by the devitrification phenomenon of the fibers during the spinning process, or by electron microscopic observation of the fibers after spinning, based on the presence of particles with a diameter of 0.02 to several μm. Next, the system of the present invention will be explained using the drawings.

FIG. 1 is a schematic diagram showing an example of a viral disease treatment system using two-stage membrane filtration according to the present invention. First, the virus-containing blood from the patient is passed through the blood inlet (1) and pumped (
Ml) and stored in the blood reservoir (2). The reservoir (2) is equipped with a pressure gauge (Pl) to monitor abnormally high pressure caused by clogging in the subsequent first filter (3). A known plasma separator can be used as this first filter (3). For example, the filter (3) is partitioned by a filtration membrane such as a cellulose acetate or polyvinyl alcohol membrane,
The virus-containing blood introduced from the blood reservoir (2) is transferred to the plasma components containing the virus through the filter membrane (5) by positive pressure from the pump (Ml) provided in the plasma derivation circuit (4). and blood components. The plasma component containing the virus separated here is transferred to the plasma extraction circuit (4).
through the plasma reservoir (6) provided in the second filter (
7). At this time, if the plasma pressure exceeds the set pressure, there is a risk of hemolysis, etc., so a pressure gauge (P2) is installed in the plasma reservoir (6) to monitor the pressure. .

A feature of the invention is that the second filter is partitioned by a filter membrane composed of regenerated cellulose porous hollow fibers with a specified pore structure. Second filter (7
The virus-containing plasma introduced into the pump (M
l) and the pump (M3) set in the discharge circuit (8) described below.
The positive pressure created by the flow rate difference between the blood and the virus separates the plasma and virus. The separated virus is discharged through the discharge circuit (9). At this time, pump (Ml) and pump (
If the flow rate of M3) becomes unbalanced, the second filter (7
) is applied with abnormal pressure, making stable filtration and separation impossible.

On the other hand, the plasma separated by the second filter is sent to the warmer (10) through the derivation circuit (8), and is sent from the first filter (3) to the derivation circuit (11). After combining with the blood components sent by the blood, it will be returned to the patient's body through the blood reservoir (13). In addition, in order to supplement the proteins in the plasma components removed in the second filter (7), a replacement fluid such as albumin or HES is sent from the replacement fluid container (12).

What has been described above is an example of the present invention. For example, the virus discharge circuit (9) of the second filter (7) is connected to the plasma inlet of the second filter (7), and the virus is circulated. It is also possible to use a system that filters while

Prior to detailed description of the present invention, definitions of technical terms used in this specification and methods for measuring the same are shown below.

[Extremely small average pore diameter, extremely small in-plane porosity] After embedding the hollow fibers in acrylic resin, an ultramicrotome (Ultradome Ul manufactured by LKB (Sweden)) was used.
Using a glass knife attached to a tratome I [8800 model], ultrathin sections with a thickness of about 1 μm are cut out at various positions from the inner wall surface to the outer wall surface of the hollow fiber parallel to the fiber axis direction. An electron micrograph of the sample section is taken. Each of the sample sections is at a different distance from the inner wall. Per 1 crn' of the section of interest, the hole radius is r''-r+dr
The number of holes present in is denoted as N (r)dr. The average pore radius 〒3 and the in-plane porosity Pre are each expressed by formula (1),
It is given by equation (2).

〒3 and Pre are measured as a function of the distance from the inner wall surface, and the respective minimum values are set as 73° and Preo. 2
r3° is the minimum average pore diameter, and Pre' corresponds to the minimum in-plane porosity.

[A porous hollow fiber module made of co-regenerated cellulose with an average pore diameter according to the water filtration rate method was prepared, and the water filtration rate Q (ml/ml/
m1n) was measured and substituted into equation (3) to calculate the average pore diameter.

d: Film thickness (μm), △P: Transmembrane pressure (mmHg) A
;Effective filtration area of module (G) Prρ;Porosity (-) μ;Viscosity of water (cP) Prρ is the apparent density when swollen with water, ρaw, and the density of cellulose solid is 1.561 g/crn'. It was calculated using equation (4).

Prp= (1-paw/1.561) −(4)
(Effects of the Invention) The regenerated cellulose porous hollow fibers used in the present invention have little protein adsorption, high recovery rate, and high virus removal rate.

The treatment system of the present invention, which uses a filter using hollow fibers and a plasma separator, has low adsorption of proteins within the system, a high recovery rate of effective proteins in the blood, and can remove viruses. Since the rate is also high, it can be expected to have a life-extending effect on people with viral illnesses.

(Example) (Example 1) Various concentrations (A-
F), and filtered and defoamed to obtain a spinning stock solution. While controlling this stock solution at a constant temperature of 25.0℃±0.1℃, 1.9m1
It was discharged at 7 min. On the other hand, as a hollow agent, acetone 44wt%, ammonia 0.60wt%, water 55.40wt%
wt% mixed solution at a temperature of 22.0°C ± 0.1°C, 2.2 mρ/m from the central spinning spout (outer diameter 0.6 mmφ)
It was discharged in the falling direction. The discharged liquid contains 44 wt% acetone, 0.58 wt% ammonia, and 55.42 wt% water.
wt% mixed solution (controlled at 25.0°C±0.1°C) and wound up at a speed of 3 to 6 m/min. It was observed with the naked eye that the transparent blue fibers immediately after being discharged gradually turned white as the spinning process progressed, and that a microphase layer was beginning to form.

After that, coagulation progresses, and in the winding process, the fiber becomes a bluish-white hollow fiber. This hollow fiber is heated at a constant length at 20°C.
of acetone/water (50150 weight ratio) for 1 hour. After that, it was regenerated into cellulose with a 2wt% sulfuric acid aqueous solution,
It was then washed with water. The water-washed hollow fibers were immersed in an acetone solution, and after the solvent was replaced with water and acetone, the acetone inside the hollow fibers was removed by air drying while being stretched by about 10%.

Table 1 shows the structural properties of the hollow fibers obtained.

(Left below) Table 1 Each of these hollow fibers has 100 pieces, length 20 cm.
They were bundled into 500 m pieces and molded into a cylindrical module. The module was steam sterilized before use for filtration, and then the inside of the hollow fiber was washed with PBS.

Colloidal silica with a diameter of 35 to 55 nm (manufactured by Catalysts Kasei Kogyo Co., Ltd., Cataloid, 5) was used instead of the virus.
1-45P) and 5 mg into ACDCD cluster blood.
/d IL and used this as a model of virus-mixed human blood. It is said that serum hepatitis virus has a diameter of 42 nm and retrovirus has a diameter of 1100 nm to 120 nm, and the colloidal silica used in this experiment has a particle diameter close to that of serum hepatitis virus. The first filter was made of cellulose diacetate hollow fiber with a maximum pore diameter of 0.2 μm, an inner diameter of 330 μm, and a membrane thickness of 75 μm.
A plasma separator (AP-058 manufactured by Asahi Medical Co., Ltd.) with a membrane area of 0.5rn" was used. In the system shown in Fig. 2, ACDCD group bovine blood 3I containing colloidal silica was used.
1. from the blood inlet (1) at 60mj2/min.
filter (3) from the inside of the hollow fiber (5) to the outside, and the filtrate is pumped (M2) to 30 mu
/min from the plasma extraction circuit (4) and introduced into the plasma reservoir (6). Thereafter, the mixture was introduced into a second filter (7) at 30 mu/min and separated into colloidal silica and plasma by vertical filtration while circulating for 5 hours.

The fluid in the blood reservoir (13) after 5 hours in Figure 2 was collected, and the colloidal silica concentration was measured by the absorption method, and the protein concentration was measured by the chrome cresol green method, and compared with the original solution. The protein transmittance and colloidal silica removal rate were determined.

Protein transmittance = (filtrate concentration / stock solution concentration) x 100
(%) Colloidal silica removal rate = ioo-(filtrate concentration/undiluted solution concentration) .

However, samples A and B are comparative examples. From the results in Table 2,
In sample CNF, the protein transmittance is 85% or more, and the model virus removal rate is 90% or more. Therefore, if the system of the present invention is used for the treatment of viral diseases, a life-prolonging effect can be expected. When used on the human body, it is preferable to employ parallel filtration in the second filter rather than vertical filtration as in the embodiment, and to use the system shown in FIG. 1 in which the liquid is discarded little by little and the liquid is replaced in the same amount.

Table 2

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram showing an example of the viral disease treatment system of the present invention. FIG. 2 is a schematic diagram showing a model experimental system used in the examples. 1. Blood inlet 11. Direct liquid lead-out circuit 2, blood reservoir 12. Replacement fluid container 3, first filter
13. Blood storage device 4, plasma derivation circuit Ml,
Blood pump 5 filtration membrane M2. Plasma pump 6, plasma reservoir M3. Plasma pump 7, second filter pt, pressure gauge 8 plasma discharge circuit
P2. Pressure gauge 9, virus discharge circuit P3. Pressure gauge 1
0, warmer

Claims (5)

[Claims] (1) In a closed extracorporeal circulation circuit that removes viruses from the blood, the average pore diameter of the blood as determined by the water filtration rate method is 0.2 to
A transmembrane pressure difference of 100 mm is applied to the hollow interior of a porous hollow fiber with a diameter of 0.5 μm and a protein permeability of 95% or more.
Plasma is separated from blood by flowing under Hg or less, and the separated plasma is made of regenerated cellulose porous hollow fibers with an average pore size of 0.1 μm or less as measured by the water filtration rate method and 0.1 μm or less of the virus diameter. 5 times or more and 1.0 times or less, the minimum average pore diameter is 0.04 to 0.20 μm, and the minimum in-plane porosity (P
re) is 0.1 to 0.5, has a layered structure with 10 or more layers in the thickness direction, and has a hollow fiber thickness of 10 μm
A system for treating viral diseases using a two-stage membrane filtration method, which is the above, and is characterized in that filtration is performed using hollow fibers having a virus removal rate of 90% or more and a protein permeability of 85% or more. (2) The hollow fibers that remove viruses in plasma are made of regenerated cellulose using the copper ammonia method, and have an average pore diameter of 0.02 to 0.04 μm and a minimal average pore diameter of 0.04 to 0.10 μm, determined by the water filtration rate method. , Pre is 0.10 to 0.50
Hepatitis B virus (H) according to claim 1, which is a hollow fiber of hepatitis B virus (H
BV) Sexual disease treatment system. (3) The hollow fibers that remove viruses in plasma are made of regenerated cellulose using the copper ammonia method, and have an average pore diameter of 0.04 to 0.08 μm and a minimum average pore diameter of 0.07 to 0.15 μm, determined by the water filtration rate method. , Pre is 0.25 to 0.50
AIDS virus (HI) according to claim 1, which is a hollow fiber of
V) Sexual disease treatment system. (4) The hollow fibers that remove viruses in plasma are made of regenerated cellulose using the copper ammonia method, and have an average pore diameter of 0.05 to 0.1 μm and a minimal average pore diameter of 0.
．． The adult T-cell leukemia virus (ATLV) disease treatment system according to claim 1, wherein the hollow fiber has a diameter of 0.09 to 0.17 μm and a pre of 0.25 to 0.50. (5) The viral disease treatment system according to any one of claims 1 to 4, wherein the hollow fibers that remove viruses in plasma are hollow fibers made of cellulose/alkaline aqueous solution regenerated cellulose.