JP2612851B2 - Sampler - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a blood sampler capable of effectively collecting plasma, particularly plasma containing blood coagulation factor VIII, from blood.

(Prior Art) For the treatment of hemophilia patients, administration of blood coagulation factor VIII is performed. Blood coagulation factor VIII is a protein with a molecular weight of about 1 million that is present in plasma. Normally, plasma components are separated from the blood of a healthy individual, and the precipitate formed by cooling is adsorbed to an aluminum hydroxide gel, cooled to 0 ° C, redissolved in glycine buffer, and treated with β-alanine buffer. To obtain a concentrated preparation of blood coagulation factor VIII. Over 90% of these plasma products are imported. At present, the procurement of plasma in Japan is a major issue because of the danger of AIDS caused by imported plasma products. For that purpose, it is desired to effectively separate plasma from blood.

In order to obtain plasma from blood, there is a method of centrifuging the collected whole blood. However, (1) a large amount of blood is required due to poor yield, and (2) a long time is required for centrifugation. There are drawbacks. In addition to centrifugation, there is a method using a sampler using a hollow fiber membrane. According to the method, blood is applied to a blood collecting device, a plasma component is collected through a hollow fiber membrane, and the concentrated blood having a high blood cell component concentration is returned to a blood donor. For example, a plasma separator used for treatment of rheumatic patients by extracorporeal blood circulation is diverted to such a blood sampling device. In the treatment of rheumatic patients, blood from the patient is applied to a plasma separator having a hollow fiber membrane (first filter), and the obtained plasma is applied to a second filter. The plasma from which globulin has been removed and the concentrated blood having a high blood cell component concentration after passing through the first filter are combined and returned to the patient's body again. When treating rheumatic patients, slowly observe the patient's condition (2
Because it is desirable to perform the treatment by separating the plasma (over 時間 3 hours), the rate of separation of the plasma by the plasma separator is relatively slow. As the hollow fiber membrane of such a plasma separator, for example, a hollow fiber membrane disclosed in JP-B-57-49248 (Example: film thickness of 95 μm, porosity of 62.5%, ultrafiltration coefficient of 1150 ml) / m ² · hr · mmHg are disclosed). Such a hollow fiber membrane has a relatively low plasma separation rate and is well permeable to the immunoglobulin having a molecular weight of about 160,000.

In contrast, when plasma is collected from healthy subjects,
It is not desirable to restrain the donor for as long as three hours, and it is desirable that the plasma be separated in as short a time as possible. In addition, the molecular weight of blood coagulation factor VIII is about 1 million, which is much larger than the immunoglobulin to be removed from the blood of rheumatic patients, about 160,000. Therefore, there is a disadvantage that it is difficult to be filtered through the membrane holes of the hollow fiber membrane of the conventional plasma separator. By increasing the pore size of these conventional hollow fiber membranes, it is thought that plasma containing blood coagulation factor VIII can be obtained effectively. However, in practice, there is a problem of clogging by macromolecules such as blood cells. At present, there is no plasma collector with a hollow fiber membrane that can effectively separate plasma containing factor VIII.

(Problems to be solved by the invention) The present invention is to solve the above-mentioned conventional problems, and an object of the present invention is to produce plasma containing a high rate of blood coagulation factor VIII in a short time. An object of the present invention is to provide a blood sampler that can be separated from blood. Another object of the present invention is to provide a blood sampling device using a hollow fiber membrane that can effectively pass plasma containing a high percentage of blood coagulation factor VIII.

(Means for Solving the Problems) The sampler according to the present invention comprises a module in which a plurality of hollow fiber membranes are loaded in a cylindrical container in parallel and both ends thereof are fixed to the container with a resin; Blood is supplied from one end opening of the hollow fiber membrane of the book, and the blood is separated into concentrated blood having a high blood cell component concentration discharged from the other end opening and plasma component permeating through the hollow fiber membrane. The hollow fiber membrane is a cellulose ester hollow fiber membrane having a thickness of 60 μm or less, and the porosity (x) of the hollow fiber membrane and the ultrafiltration coefficient (y Satisfies the following equation: y ≧ 48x−330 (where the unit of x is% and the unit of y is ml / m ² · hr · mmHg), and β-lipo of the hollow fiber membrane The protein sieve coefficient (SC) is 0.95 or more, thereby achieving the above object.

For the hollow fiber membrane used in the sampler of the present invention, a cellulose ester having excellent strength is used. Examples of the cellulose ester include cellulose diacetate, cellulose triacetate, and cellulose nitrate. The hollow fiber membrane that satisfies all of the above-mentioned conditions is used to effectively separate plasma containing blood coagulation factor VIII. If any one of these conditions deviates from the above range, sufficient performance cannot be obtained. For example,
If the film thickness exceeds 60 μm, clogging occurs due to giant particles such as blood cells in the early stage of plasma separation, and the plasma separation speed is stabilized at a very low level. If the relationship between the porosity and the ultrafiltration coefficient cannot be satisfied, the plasma separation rate decreases over time. For example, even if the porosity is the same, if there are many small holes, or if the holes that penetrate the membrane are not cylindrical but constricted at the center, this condition cannot be satisfied, The separation speed decreases over time. Β-lipoprotein has a molecular weight similar to that of blood coagulation factor VIII, and its sieving coefficient (SC) is an important factor indicating the membrane permeability of blood coagulation factor VIII. Hollow fiber membranes with a sieving coefficient of less than 0.95 for β-lipoprotein cannot penetrate blood coagulation factor VIII satisfactorily because the diameter of the holes is small on average.

The ultrafiltration coefficient of the hollow fiber membrane and the sieving coefficient of β-lipoprotein are measured as follows. As shown in FIG. 1, the ultrafiltration coefficient measuring apparatus has a thermostat 100, a water tank 1 and a hollow fiber membrane module 2 housed in the thermostat. Pressure measuring devices 31 and 32 are provided in the middle of the line connecting the water tank 1 and the hollow fiber membrane module, respectively. The hollow fiber membrane module consists of 1,700 hollow fiber membranes bundled and both ends fixed to a cylindrical container in a liquid-tight manner. The effective length of the hollow fiber membrane is 16.4 cm. 37 baths
Set to ± 1 ° C. The water from the water tank 1 is guided by the pump 12 to the water tank 312 of the pressure measuring device 31, and further to the inlet 21 of the hollow fiber membrane module 2. Water storage tank 312
The internal pressure P _Bi is measured by a pressure gauge 311. The water filtered by the hollow fiber membrane is discharged from the filtered water discharge port 23 of the hollow fiber membrane module 2 and guided to the storage tank 25. Water that has passed through the hollow fiber membrane without being filtered is sent to the outlet 22 through a pressure measurement device.
The water is returned to the water tank 1 via the 32 water storage tanks 322. The internal pressure P _Bo of the water storage tank 322 is measured by the pressure gauge 321. Pressure measuring device 32
The average transmembrane pressure difference (TMP) is adjusted to 30 mmHg by a flow control valve 33 provided between the water tank 1. TMP
Is the average value of the pressure P _Bi and pressure gauge 321P _Bo of the pressure gauge _{311 (P Bi> P Bo)} . At this time, the amount of water filtered out per hour (ml) is determined by the unit area (m ² ) of the hollow fiber membrane and TMP (mmH
g) corresponds to the ultrafiltration coefficient (ml / m ² · hr · mmHg).

As shown in FIG. 2, the β-lipoprotein sieving coefficient measuring device includes a β-lipoprotein solution supply tank 41, a pressure measuring device 61, a hollow fiber membrane module 5, and a pressure measuring device.
62 are sequentially connected in series. A physiological saline supply tank 42 is connected between the pressure measuring device 61 and the hollow fiber membrane module 4 via a three-way cock 300. The hollow fiber module bundles 42 hollow fiber membranes 51 in a U-shaped container 52,
Both ends are fixed to the container 52 with an adhesive,
The effective length of the hollow fiber membrane is 15cm. β-lipoprotein solution 411 stored in β-lipoptin solution supply tank 41
Is β-lipoprotein [Cohn Fraction 110-O; United
States Boochemical Corporation (Cleveland Ohio441
28) at a rate of 30 mg / dl in phosphate buffered saline (P
hosphate Blffer Saline). The measurement system is maintained at 37 ° C by a thermostat (not shown).

In order to measure the sieving coefficient of β-lipoprotein by this device, first, the three-way cock 300 is switched to transfer the physiological saline 421 contained in the physiological saline supply tank 42 to the hollow fiber membrane module 5 via the pump 422. Lead. This saline 421
Is discharged through the water storage tank 621 of the pressure measuring device 62.
Thus, after the inside of the system is replaced with the physiological saline 421 and completely degassed, the three-way cock 300 is switched, and the β-lipoprotein solution 411 is supplied to the heat converter 413 and the pressure measuring device 61 by the pump 412. After that, it is led to the hollow fiber membrane module 5. β-
The lipoprotein solution 411 is discharged from the hollow fiber membrane module 5 through the water reservoir 621 of the pressure measuring device 62. When the entire system is replaced with the β-lipoprotein solution, the flow rate of the β-lipoprotein solution flowing in the system is adjusted by the flow control valve 60, and the measured values by the pressure gauges 612 and 622 (P ₁ and P ₂ respectively)
Of 5 mmHg (however, P ₁ > P ₂ ). Adjustment of the flow rate by the flow rate control valve 60 is performed within 5 minutes after the replacement of the system with the β-lipoprotein solution. Flow control valve
After adjusting the flow rate (pressure) by 60, the solution (A) filtered through the pores of the hollow fiber membrane 51 after 15 minutes, the solution (C) discharged from the measurement system without being filtered, and the hollow fiber membrane Β-lipoprotein solution (B) supplied to module 5
Collect. The concentration of β-lipoprotein in each solution is measured by colorimetry and the sieving coefficient (SC) of β-lipoprotein is calculated by the following equation: Here, a is a solution (A) filtered through the membrane pores of the hollow fiber membrane 51, and b is β supplied to the hollow fiber membrane module 5.
-Lipoprotein solution (B) and c indicate the respective β-lipoprotein concentrations of the solution (C) discharged from the measurement system without being filtered.

The hollow fiber membrane that satisfies the above-mentioned conditions can be basically prepared by a usual hollow fiber membrane preparation method in which a spinning stock solution is discharged and solidified from a double tube nozzle. The spinning dope contains the cellulose ester, a solvent for dissolving the cellulose ester, and a swelling agent (non-solvent). As the solvent, an aprotic polar solvent that can dissolve the cellulose ester is used, and as the swelling agent, a non-solvent that does not dissolve the cellulose ester is used. The solvent has a boiling point of 150 ° C. or higher. Such solvents include N-methylpyrrolidone, dimethylformamide, dimethylacetamide, dimethylsulfoxide and the like. As the swelling agent, for example, ethylene glycol,
Examples include polyhydric alcohols such as triethylene glycol, polyethylene glycol, glycerin, and polypropylene glycol. The mixing ratio between the solvent and the swelling agent is usually in the range of 3: 7 to 8: 2 by weight. It is necessary that the concentration of the cellulose ester in the spinning dope containing the cellulose ester, the solvent and the swelling agent be relatively high, at least 20% by weight, preferably 22 to 30% by weight. If the amount is less than 20% by weight, the strength of the hollow fiber membrane obtained is insufficient.

As the inner solution and the coagulating solution for coagulating the spinning solution, an aqueous solution of the above-mentioned solvent-non-solvent mixture is used. The solvent-non-solvent ratio is in the range of 3: 7 to 8: 2 in the same weight ratio as above, and the solvent-non-solvent mixture is 50% by weight or more, preferably 55 to 70% by weight in the aqueous solution. It is contained in proportion. If it is less than 50% by weight, the resulting hollow fiber membrane has a low β-lipoprotein sieving coefficient (SC). The diameter of the hollow fiber membrane is not particularly limited, but usually the size of the nozzle for discharging the spinning solution is selected so that the inner diameter is 200 to 400 μm.

According to the hollow fiber membrane of the present invention, the spinning solution is simultaneously discharged from the outer nozzle of the double tube nozzle and the inner solution is simultaneously discharged from the inner nozzle, guided into a coagulation bath, and solidified. At this time,
The discharge temperature (T ₁ ) and the temperature of the coagulation bath (T ₂ ) are set to 0 <T ₁ −
It is important to set T ₂ ≦ 40 (° C.). T ₁ and T ₂
Is out of the above range, the ultrafiltration coefficient of the obtained hollow fiber membrane becomes low. When such a hollow fiber membrane is used, the separation rate of plasma decreases over time. The solidified hollow fiber membrane is washed with water and wound up. Heat treatment at 80 ° C. or higher can increase the strength of the hollow fiber membrane and impart heat resistance. It is recommended that heat treatment be performed at 115 ° C for 30 minutes, 121 ° C for 20 minutes, or 126 ° C for 15 minutes.

A plurality of the hollow fiber membranes thus obtained are loaded in parallel into, for example, a cylindrical container 72 shown in FIGS. 3 and 4, and both ends thereof are fixed to a case with a resin 73 in a liquid-tight manner. Thus, the sampler of the present invention is obtained. To fix the hollow fiber membrane,
For example, the ends of a plurality of hollow fiber membranes are cut out, put into a resin casting mold, and then poured with a casting resin. As such a resin, a resin such as a polyurethane resin in which a plasticizer is not eluted from the resin is used. The part fixed with the resin is called an adhesive part 74. In the bonding section 74, the fifth
As shown in the partial cross-sectional view of the figure, the hollow fiber membrane is fixed to each other by resin without being in close contact with each other.

Although the size of the sampler module is not particularly limited, usually, the inner diameter of the cylindrical container 72 is 15 to 60 mm, and the effective length 1 of the hollow fiber membrane contained therein is about 120 to 300 mm.
The loading amount of the hollow fiber membrane into the container is defined by the membrane area, and is usually 0.05 to 2.0 m ² .

In order to separate blood plasma from blood using this module, for example, the collected blood is supplied under pressure from the blood inlet 75 of the module shown in FIG. 4 and passed through the hollow fiber membrane 71 of the module. While the blood passes through the hollow fiber membrane 71, the blood plasma component containing the blood coagulation factor VIII is filtered through the hollow fiber membrane pores and discharged from the blood outlets 77 and 78. The concentrated blood having a high blood cell concentration after the plasma is filtered is discharged from the blood outlet 76 to the outside of the module. The blood flow through the module is 10-150 ml / min. 10ml /
If it is less than a minute, it takes a long time to separate the plasma, and if it exceeds 150 ml / min, the pressure loss increases and blood cells are often damaged. In order to set such a blood flow, the transmembrane pressure difference of the hollow fiber membrane is set to 100 mmHg or less, preferably 10 mmHg.
It is necessary to be ~ 50mmHg. The blood concentrated and discharged from the module is returned to the donor again.

(Operation) The hollow fiber membrane used in the sampler of the present invention has a high membrane strength because of the high cellulose ester concentration of the spinning stock solution. Set the solvent-swelling agent (non-solvent) concentration in the coagulating solution and solid solution to solidify the cellulose ester in the spinning stock solution high, and set the temperature difference between the discharge temperature and the coagulating bath to 40 ° C.
Because of the following settings, the cellulose ester solidifies slowly. As a result, a hollow fiber membrane having a relatively large pore diameter and having a pore size sufficient to allow blood coagulation factor VIII having a relatively large molecular weight to penetrate stably without clogging is obtained. Using such a blood sampler containing a hollow fiber membrane, plasma containing blood coagulation factor VIII is separated from blood at a stable and high speed.

Since plasma can be separated in a short time, blood is returned to the donor quickly, and the burden on the donor is small. Since the hollow fiber membrane of the blood sampling device of the present invention can effectively permeate blood coagulation factor VIII having a relatively high molecular weight, the blood plasma obtained using a conventional separation membrane is used for the blood coagulation factor VIII content. Much higher than when. When the obtained plasma is processed by a conventional method, a blood coagulation factor VIII preparation is prepared.

(Examples) Hereinafter, the present invention will be described with reference to examples.

Example 1 Cellulose triacetate was dissolved in a mixed solution consisting of 52.5 parts by weight of N-methylpyrrolidone (solvent) and 22.5 parts by weight of polyethylene glycol (swelling agent) to prepare a spinning dope having a cellulose triacetate concentration of 25% by weight.
As an inner solution for spinning, a mixed solution of 49% by weight of N-methylpyrrolidone, 21% by weight of polyethylene glycol and 30% by weight of water was prepared. The above spinning stock solution was simultaneously discharged from the outer nozzle of the double tube nozzle and the inner solution from the inner nozzle, and a hollow fiber membrane was prepared by a conventional method. The coagulation liquid in the coagulation bath is N
-Methylpyrrolidone 45% by weight, polyethylene glycol
A mixture of 19.3% by weight and 35.7% by weight of water was used. The discharge temperature of the spinning solution is 85 ° C, and the temperature of the coagulation bath is 75 ° C. The obtained hollow fiber membrane is washed with water and placed in an autoclave.
Heated at 1 ° C. for 20 minutes.

The thickness and the porosity of the hollow fiber membrane thus obtained were measured. Separately, using the device shown in FIG.
The ultrafiltration coefficient was measured. Further, bovine blood having a hematocrit value (Ht; blood cell content rate) of 40% was supplied at a flow rate of 50 ml / min instead of water using the apparatus shown in FIG. 1 (the membrane area of the module was 0.25 m ² ). The amount of plasma filtered for 5 minutes after the start of supply was measured, and the amount of filtered plasma per 1 mmHg of the average transmembrane pressure difference was calculated, and this was defined as the initial plasma separation rate. The same measurement was performed 30 minutes after the start of the supply, and the amount of filtered plasma per average transmembrane pressure difference was calculated. Next, the β-lipoprotein sieving coefficient of the hollow fiber membrane was measured using the apparatus shown in FIG. Further, bovine blood having a hematocrit value of 40% was supplied instead of the β-lipoprotein solution, and the sieving coefficient of blood coagulation factor VIII was calculated. The results are shown in the table below.
The results of Examples 2 and 3 are also shown in the table below.

Example 2 Example 1 was repeated except that a spinning dope having a cellulose triacetate concentration of 22% by weight was used and the temperature of the coagulation bath was 70 ° C.
Is the same as

Example 3 Example 1 was repeated except that a spinning dope having a cellulose triacetate concentration of 20% by weight was used and the temperature of the coagulation bath was set at 65 ° C.
Is the same as

Comparative Example 1 A hollow fiber membrane having a thickness of 95 μm was prepared by changing the thickness of the nozzle during spinning. Using this, the initial plasma separation rate was measured and calculated in the same manner as in Example 1, and the result was 0.33 ml / mi.
The level was as low as n · mmHg. This is considered to be due to clogging caused by blood cell components in the pores.

Comparative Example 2 The concentration of N-methylpyrrolidone contained in the coagulation solution was 35% by weight, and the concentration of polyethylene glycol was 13% by weight.
The same operation as in Example 1 was performed, except that The β-lipoprotein sieving coefficient and factor VIII sieving coefficient of the obtained hollow fiber membrane were as low as 0.78 and 0.70, respectively.

Comparative Example 3 The same operation as in Example 1 was performed except that the temperature of the coagulation bath was set to 40 ° C. The porosity of the obtained hollow fiber membrane was 63%, and the ultrafiltration coefficient was 2320 ml / m ² · hr · mmHg. By applying the porosity (x) of the hollow fiber membrane of this comparative example to the relational expression between the ultrafiltration coefficient (y) and the porosity (x) of the hollow fiber membrane of the present invention, 48x−330 = 48 × 63−330 = 2694, and y ≧ 2694 is derived. The ultrafiltration coefficient y of the hollow fiber membrane of this comparative example is 2320 as described above, and does not satisfy this relationship. Initial plasma separation rate of the hollow fiber membrane obtained in this comparative example is 0.50ml / m ² · h
The value was r · mmHg, but after 30 minutes it decreased to 0.31 ml / m ² · hr · mmHg. When a hollow fiber membrane that does not satisfy the above expression is used in this way, the plasma separation rate decreases over time.
β-lipoprotein sieving coefficient was 0.95.

(Effects of the Invention) According to the present invention, a sampler using a hollow fiber membrane that can effectively penetrate plasma containing blood coagulation factor VIII can be obtained. With this plasma collector, plasma containing blood coagulation factor VIII can be separated from blood in a short period of time, so that plasma can be collected without restricting the donor for a long time. Since the obtained plasma has a much higher factor VIII content than that obtained using a conventional blood sampling device, the factor VIII preparation can be effectively prepared.

[Brief description of the drawings]

FIG. 1 is a schematic view showing an apparatus for measuring an ultrafiltration coefficient of a hollow fiber membrane, FIG. 2 is a schematic view showing an apparatus for measuring a β-lipoprotein sieving coefficient of a hollow fiber membrane, and FIG. FIG. 4 is a perspective view showing an example of the vessel, FIG. 4 is a cross-sectional view thereof, and FIG. 5 is an enlarged cross-sectional view of an adhesive portion of the sampler of FIG. 2,5,7 …… Hollow fiber membrane module, 51,71 …… Hollow fiber membrane,
52: U-shaped container, 72: cylindrical container, 73: resin.

 ──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Hitoshi Ohno 2-1-1 Katata, Otsu City Inside Toyobo Co., Ltd. Research Institute (56) References JP-A-60-201253 (JP, A) JP-A Sho 60-11166 (JP, A) JP-A-60-7853 (JP, A)

Claims (1)

(57) [Claims] 1. A module in which a plurality of hollow fiber membranes are loaded in parallel in a cylindrical container and both ends of the hollow fiber membranes are fixed to the container with resin; blood is supplied from one end openings of the plurality of hollow fiber membranes. A blood sampler for supplying and separating the blood into a concentrated blood having a high blood cell component concentration discharged from the other end opening and a plasma component passing through the hollow fiber membrane, wherein the hollow fiber membrane has a thickness of 60 μm. The following cellulose ester-based hollow fiber membrane, wherein the porosity (x) of the hollow fiber membrane and the ultrafiltration coefficient (y) measured using water satisfy the following equation: y ≧ 48x− 330 (where the unit of x is% and the unit of y is ml / m ² · hr · mmHg), and the β-lipoprotein sieving coefficient (SC) of the hollow fiber membrane is 0.
A sampler that is 95 or more.