JPS6267456A - Separation of plasma - Google Patents

Abstract

PURPOSE:To separate plasma component free from no microorganism particle such as virus without being heated, by performing an ultra filtration of the plasma component simultaneously with a centrifugal separation by a cupro- ammonium cellulose hollow system. CONSTITUTION:A porous cupro-ammonium cellulose porous hollow fiber with the average pore size of 0.02mum to 0.2mum exceeding 10% in the surface vacancy rate is arranged to be aligned in the fiber axial direction with the direction of a centrifugal force and blood components are separated and fractioned by the centrifugal force as driving force while being filled with a fluid to be filtered or already filled therewith. To be more specific, cupro-ammonium cellulose hollow fibers are bundled and bonded inside a container (cylindrical) with an adhesive to form a liquid separator (module). Then, the module thus obtained is so set in a centrifugal machine that a centrifugal force is applied in the axial direction of the hollow fiber. In the module, inflow and outflow ports of a liquid to be filtered lead to the inside of the hollow fiber (hollow section) and a blood is made to flow in at the inflow port to be given the centrifugal force. Then, a filtrate is recovered from a container provided at the outflow port.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a plasma separation method. More specifically, it relates to a method for separating plasma components from blood that do not contain microbial particles such as viruses, and in the process of separating blood into blood cell components and plasma components by centrifugation,
The present invention relates to a method for separating plasma components without contaminating microbial particles such as viruses by ultrafiltering the plasma components through copper ammonium cellulose hollow fibers at the same time as this separation.

Plasma obtained by removing blood cell components from the blood of animals including humans can be used as raw material for plasma exchange therapy, plasma preparations for component transfusion, and plasma fractionation preparations (in the case of human blood, i.
It is used in tissue culture fluid (animal blood) or reagents used in genetic engineering (animal blood). When using these materials, it is often impossible to sterilize them by heating. In this case, it is required to remove microbial particles such as viruses from the plasma.

2. Description of the Related Art Conventionally, centrifugal separation and a transient method using a polymer membrane have been employed as methods for separating plasma from blood. Centrifugal separation requires large equipment and is expensive for industrial purposes (such as for plasma fraction preparations).

Contamination with serum hepatitis virus can be completely prevented.

In addition, this method may cause platelet contamination.

On the other hand, separation using polymer membranes is expected to have a compact device, the possibility of separation at the site of blood collection, the possibility of recovering all kinds of molecular weight fraction components, and the removal of viruses. However, in reality, the protein recovery rate is low and the filtration rate rapidly decreases during use due to clogging. Particularly in the recovery of coagulation factors, it is necessary to completely avoid contamination with microorganisms, but this requirement is not completely satisfied by conventional filtration methods using polymer membranes. Filtration aimed at separating and removing viruses has a low filtration rate and is not practical. Therefore, blood filtration using polymer membranes is not used industrially.

As a method for removing viruses mixed in plasma components,
The above-mentioned membrane separation, precipitation method in which a metal-virus complex is formed, adsorption method using activated carbon, and gel filtration method are known. The precipitation method requires a step for recovering the precipitate, and furthermore, continuous removal is difficult. The range of viruses to which the adsorption method and gel filtration method can be applied is limited, and it is difficult to apply the method when there are a large number of contaminated virus particles.

Problems to be Solved by the Invention The present invention is a plasma separation method that has the advantages of both centrifugation and ultrafiltration, and eliminates the drawbacks of each method. contains no virus particles and therefore does not require heat sterilization. In particular, a method for obtaining plasma for separation and recovery of coagulation factors 8 and 9 is provided.

Means to Solve the Problems The present invention provides a method for separating cellulose by centrifugation, in which copper ammonium cellulose porous hollow fibers having an average pore diameter of 0.02 μm to 0.2 μm and an in-plane porosity of 10 μm or more are used. A plasma separation method characterized in that the axial direction' is aligned with the direction of centrifugal force, and the liquid components are separated and fractionated using centrifugal force as a driving force while filling hollow fibers with the fluid to be filtered or in the filled state. /:!
It is A.

The plasma separation/fractionation method of the present invention involves applying centrifugal force to the above-mentioned porous hollow fibers of cuprammonium cellulose (abbreviated as copper ammonium cellulose) having a specific average pore diameter range and specific in-plane porosity. It is constituted by a method of separating and fractionating the liquid components while filling the inside of the hollow fiber with blood in the same direction, or in a filled state, using centrifugal force as a driving force.

Here, porous hollow fibers are defined by scanning electron microscopy.
It is a hollow fiber with clearly visible pores on either side of the outer wall.

Separation methods that are inferior to the method of the present invention include separation of blood cell components and plasma components by centrifugation, and microbial particles such as viruses by ultrafiltration using porous hollow fibers using centrifugal force applied to the fluid as hydrostatic pressure. It is characterized by the fact that both the separation and removal of blood from plasma components are carried out at the same time.

One of the features of the present invention is that copper ammonium cellulose is employed as the material polymer. As a result of examining the correlation between the adsorption properties of proteins and polymeric materials, it became clear that hydrophilic materials generally have low adsorption properties for proteins. Cellulose is an excellent hydrophilic material. Here, cellulose means so-called regenerated cellulose. Porous hollow fibers made of regenerated cellulose are produced by saponifying porous hollow fibers of a cellulose derivative (usually cellulose acetate) or by directly spinning hollow fibers using the copper ammonium method.

A comparison of protein adsorption properties between these two types of regenerated cellulose porous hollow fibers revealed that the cellulose derivative porous hollow fibers subjected to saponification treatment had higher adsorption properties. In addition, copper ammonium-based regenerated cellulose has excellent biocompatibility and mechanical toughness, and also has a high ultrafiltration rate when compared at the same porosity. Also, the change in average pore diameter due to steam sterilization is small. The viscosity average molecular weight of ammonium cellulose is preferably 7 x 10' or more. 0 again
, IN NaOH aqueous solution contains fewer components, the better.
When immersed in an OH aqueous solution, this dissolved component was 0.001
% or less, it is optimal for removing microorganisms from plasma. This kind of copper ammonium cellulose hollow fiber.

When preparing a copper ammonium cellulose solution from a high-purity cellulose raw material, care must be taken such as using ultrapure water to suppress chemical decomposition of cellulose and prevent contamination of foreign substances.

The second feature of the method of the present invention is that the average pore diameter [fourth-order average pore radius zr4 (F, the fourth-order average pore radius is abbreviated as average pore diameter in the present invention)] is 0.02 to 0.2 μm. The point is that porous hollow fibers having an in-plane porosity of 10% or more are used.

The porous hollow fiber as used in the present invention means that when the wall thickness of the hollow fiber is observed with an electron microscope, there is no
．． It means a hollow fiber in which pores of 0.02 μm or more are observed.

If the average pore diameter is less than 0.02 μm, the ultrafiltration rate is extremely low.

Moreover, the recovery rate of the protein dissolved in the aqueous solution as a t-F solution decreases significantly. For example, the recovery rate for serum albumin is less than 5%. Therefore.

The average pore size is preferably as large as possible to remove microbial particles, but in order to avoid contamination by microbial particles from the supply circuit of the fluid to be filtered during ultrafiltration, it is 0.2 μm.
It is necessary that the following is true.

If there is a possibility of the presence of hepatitis B virus, the average pore size is 0.04 μm or less, and if there is a possibility of the presence of ATL virus, the average pore size is 0.1 μm or less. Further, when the purpose is to separate and remove microorganisms, the in-plane porosity of the porous hollow fibers is required to be 10% or more.If the in-plane porosity is less than 10%, the ultrafiltration rate will decrease rapidly. Preferably 3
It is 0% or more. The influence of in-plane porosity on ultra-overspeed is approximately 5th power of in-plane porosity when it is less than 10%, 10~
At 30%, the ultrafiltration rate increases in proportion to approximately the second power, and at 30% or more, the ultrafiltration rate increases in proportion to approximately the first power. On the other hand, when the in-plane porosity exceeds 80, the mechanical properties of the hollow fibers are significantly reduced.

Defects such as pinholes may occur. As a result of examining the influence of pore size distribution on ultrafiltration rate with the same average pore diameter and in-plane porosity, we found that the smaller the value of 74/Te3 (rg is the tertiary average pore radius, which will be described later), the better the ultrafiltration rate. The speed increases. Moreover, when 〒,/〒3≦1.3, even if the fluid to be filtered is ultrafiltrated (abbreviated as vertical filtration) under static conditions using hollow fibers with an average pore diameter of 1.1 times the diameter of microbial particles, F Almost no microbial particles can be observed in the liquid. Furthermore, if ultra-filtration (abbreviated as parallel filtration) is performed under the flow of the fluid to be filtered, A4/
If ≦1.3, when hollow fibers with an average pore diameter of 1.2 times the diameter of microbial particles are used, almost no microbial particles can be observed in the obtained F solution. By eliminating the skirting in the large pore diameter portion of the pore size distribution, γ4/γ can be rapidly reduced.

The third feature of the method of the present invention is that ultrafiltration is performed using centrifugal force as the driving force for substance separation. Particle components of various sizes exist in the fluid to be filtered inside the hollow fibers. The density of the particles is generally large compared to the density of the medium (water). Therefore, at the beginning of applying centrifugal force, particle components with high density and large mass selectively move within the hollow portion of the hollow fiber along the direction of centrifugal force. This increases the moving speed and makes the particles hollow and the hollow fibers even more moving.

In order to reduce the probability of collision with the inner wall forming the section,
It is necessary to align the fiber axis direction of the hollow fibers with the direction of centrifugal force. If the parallelism between the two directions is not sufficient, not only will the movement speed of the particles decrease as described above, but the damage to the particle components will be significant (hemolysis in the case of blood, etc.). Characteristics of filtration under moving conditions due to centrifugal force (ie, parallel filtration) appear. As particle separation by centrifugal force progresses, the contribution of vertical filtration increases. Since all two types of filtration methods are employed, clogging of the pores by particles on the inner wall of the hollow fibers or by high molecular weight substances (proteins when blood is used as the fluid to be filtered) dissolved in the fluid to be filtered can be prevented.

In a configuration that can exhibit the effects of parallel filtration and vertical filtration, porous hollow fibers are arranged almost radially, and the fluid to be filtered (e.g., blood) is supplied from the center of the circle, and the periphery of the circle at the other end of the hollow fibers. Plasma for transfusion or fractionation can be collected by separating the proteins in the plasma components using hollow fibers and collecting them in the absence of microbial particles while storing blood components in the blood compartment. In this case, the viscosity is high and there is a limit value of the pressure that can be applied by centrifugal force or hydrostatic pressure.

Therefore, in blood filtration using hollow fibers, the hollow fibers used have an inner diameter of 200 μrrL~13 and a wall thickness of 5~13 μrrL.
50μm, if the length of the hollow fiber is 5~20crn,
It is preferable from the viewpoint of increasing the filtration rate and preventing hemolysis. At this time, the number of hollow fibers is 10 to 20,000.
It is more preferable to produce a plasma preparation for transfusion if it is constructed of assembled units (modules) made up of bundled books, and the amount of blood processed by each assembly is 200 to 5,007. By connecting two of these modules in series, it is also possible to change the component composition of the liquid obtained by ultrafiltration in each module.

The hollow fibers used in the method of the present invention have a degree of orientation of cellulose molecular chains constituting the hollow fibers in the fiber axis direction of 60q.
It is preferable that it is more than b. If this degree of orientation is 60-
The regenerated cellulose hollow fibers swell in the blood,
As a result, the hollow fibers are deformed, which disrupts the flow of the fluid to be filtered, making clogging more likely to occur.

Average pore radius of porous hollow fibers employed in the method of the present invention 7
4. The in-plane porosity s'F, /'F and the degree of orientation are determined by the following methods.

<Average pore radius γ4>, in-plane porosity> and <γ4/
rs>: Observe the cross-section of the wall of the hollow fiber with a scanning electron microscope, and determine the portion with the smallest pore diameter within a range of 10 inches of error relative to the wall thickness. An ultra-thin section with a thickness of about 0.1 μm is made parallel to the fiber axis direction of the hollow fiber through this smallest portion, and an electron micrograph of this section is taken. Nωdr is the number of holes whose hole diameter is γ to γ + dr
Then, the third-order and fourth-order average pore radii (γ3 and ra, respectively) and in-plane porosity Pr are defined by the following equations.

Pr (% display) = glo γ” N (1) dr
(3) γ4/γ3 is γ3 obtained from equations (1) and (2). It is directly calculated from γ4. The average pore size is given by 2γ4. The pore size distribution function Nω is determined from an electron micrograph of an ultrathin section as follows.

That is, take a scanning electron micrograph of the part where you want to find the pore size distribution and take it to an appropriate size, for example, 20 cm x 20 t.
20 test lines (straight lines) are drawn at equal intervals on the resulting photograph. The straight line of the valley crosses numerous holes. Measure the length of the straight line that exists within the hole when it crosses the hole, and find this frequency distribution function. Using this frequency distribution function, Nω is determined, for example, by the method of stereology (for example, "Quantitative Morphology 1" by Norio Suwa, published by Iwanami Shoten).

Degree of orientation: Rigaku Denki MX-ray generator (RU-200PL) and goniometer (SG-9R), scintillation counter for the counter, pulse height analyzer (PHA) e for the calculation section, monochromatic with nickel filter. It was then measured using the symmetrical transmission method using 4 lines (wavelength λ = 0.1542 nm) on 2Cu-.

The hollow fiber samples were bundled in parallel and fixed to a fiber sample measuring device (rotating sample stage) manufactured by Rigaku Denki Co., Ltd. at right angles to the X-ray incident direction. Scanning speed 4°/min, chart speed 1tW1/
minutes, time constant 1-2 seconds, dipersient slit 2Nφ, receiving slit vertical width 1.9m+g
X horizontal @ 3.511121 S 194 degrees Under the conditions of 30℃ and relative humidity 5o%, a constant diffraction angle (diffraction angle 2θ = 21
．． The X-ray diffraction intensity (m) in the azimuth direction was measured over a range of 180° from the meridian through the equator line and back to the meridian at an angle of 5° (corresponding to the diffraction angle of the (002) plane). Half width H of this curve
(in degrees) and substitute this value into equation (4) to calculate the degree of orientation.

Orientation degree (indication) = (180-H)/180X100
(4) Examples The present invention will be specifically explained in the following examples.

Example L Cellulose copper ammonia stock solution is mixed with acetone/ammonia/
Method of discharging into a coagulation bath composed of water (Japanese Patent Laid-Open No. 59
-204912) average pore diameter of 0.063
/jm% in-plane porosity 36%, γ4/γ3 = 1.22
An ammonium cellulose hollow fiber having an inner diameter of 300 μm and a wall thickness of 20 μm was prepared. The degree of orientation of the hollow fibers was 65 degrees. 100 of these fibers were bundled and adhered inside a polycarbonate container (cylindrical) using a urethane adhesive using a known method to create a liquid separator (module) similar to a dialysis-type artificial kidney with a length of 20 ctn. . The module was placed in a centrifuge so that centrifugal force was applied in the fiber axis direction of the hollow fibers of the module. The module has an inlet and an outlet for the liquid to be filtered, and both outlets communicate with the interior of the hollow fiber (hollow section). Blood flows in from one inlet. This inlet is located near the axis of rotation that provides the centrifugal force. A tube or container having a preset volume is connected to the other outlet. The volume of this tube or container is (0,008Ht) v, where the hematocrit value of blood is Ht%, the filling volume of the module is V (1 nt), and the volume of blood to be filtered is Va (d).
, ~(0,015Ht) V, (IP!ri +7
) between vchru. Va=200m/(Ht
=40%), and the volume of the container is set to 80-. 5
Bovine blood is allowed to flow through the inlet of the module under rotation of less than 00 rpm. After the inflow of 200 rpm, the rotation speed of the centrifuge was set to 400 rpm, and filtration was continued for about 30 minutes.

In the filtrate (part of plasma) obtained at the beginning of the inflow of the two fluids, the concentrations of both globulin and albumin were lower than the average concentration of plasma. After that, their concentrations gradually increased. In particular, the rate of increase in globulin concentration was large. It was also confirmed using an electron microscope that no microbial particles were present in the furnace fluid.

On the other hand, the module was subjected to pressurized heating steam sterilization for 12112.15 minutes. 5. Physiological food in water. OXlo'
Pieces/-Nomtte University P & Bacteria 77-di (IFO20004)
was dispersed and ultrafiltered at 20° C. under a pressure of 0.3 atm. The number of phage in the obtained furnace solution was evaluated by a plaque formation method using an agar overlay method. As a result, the number of plaques was zero, and the phage inhibition rate of this module was 99,999999.
It is 9 inches or more.

Example 2 Ammonium ammonium cellulose hollow fibers were produced in the same manner as in Example 1. The average pore diameter of the hollow fibers is 0.15 μm, and the porosity is 56.
Chi, r4/r3= 1.21, inner diameter 300 I'm
On the other hand, commercially available cellulose acetate hollow fiber (nominal average pore diameter 0.2 μm 1 in-plane porosity 1
Example 1
A plasma separation experiment was carried out in the same manner as described above. As a result, the filtration rate was approximately twice as high with ammonium cellulose hollow fibers, the protein concentration in the p-liquid was also lower than that with cellulose acetate hollow fibers.

Effects of the Invention According to the plasma separation method of the present invention, the following remarkable effects can be obtained.

(b) Since there are no virus particles in the collected plasma proteins, no heat sterilization is required.

(b) Parallel filtration and vertical filtration Since the type 02 filtration method is adopted, clogging of the pores by particles on the inner wall of the hollow fiber or by high molecular weight substances dissolved in the fluid to be filtered can be prevented.

By connecting modules in series, the P component composition in each module can be changed.

) High protein recovery rate.

Claims (1)

[Claims] In the plasma separation method using centrifugation, when the average pore size is 0.
The fiber axis direction of ammonium ammonium cellulose porous hollow fibers having a size of 02 to 0.2 μm and an in-plane porosity of 10% or more is aligned in the direction of centrifugal force, and the hollow fibers are filled with the fluid to be filtered, or in a state where the hollow fibers are filled with the fluid to be filtered. A plasma separation method characterized by separating and fractionating blood components using centrifugal force as a driving force.