JPH034230B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a plasma fractionation processing device, and more specifically, when removing high molecular weight proteins from plasma,
The present invention relates to a device that can maintain high plasma processing capacity and separation performance during plasma processing. In recent years, he has been diagnosed with nephritis, Gutsudpaschia syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, rheumatism,
For the treatment of various diseases thought to be caused by immune system abnormalities, abnormal metabolites, and increases in toxic substances, such as hypergammaglobulinemia, cancer, diabetes, hyperlipidemia, Raynaud's disease, drug addiction, and liver failure, plasma Exchange therapy is used. In such diseases,
For example, in glomerulonephritis, immune complexes in the circulating blood deposit in the kidneys and cause damage, so it is effective to remove antibodies in the blood, immune complexes, and intermediate products such as fibrinogen associated with inflammatory reactions. It is considered. Also, in Gutsudpasture syndrome, anti-inflammatory
In myasthenia gravis, it is desirable to reduce blood levels of GBM antibodies and remove complement and coagulation factors in plasma.In myasthenia gravis, it is desirable to remove antibodies against acetylcholine receptors at nerve junctions, that is, immunoglobulins, and in hyperlipidemic plasma, Symptom improvement and therapeutic effects have been seen by removing low-density riboprotein in the blood, fibrinogen and macroglobulin in Raynaud's syndrome, and rheumatoid factor and cryoprotein in rheumatoid arthritis. The pathogenic or harmful substances in the blood in these diseases are protein sources and play important functions in the body, such as maintaining blood osmotic pressure and transporting ions and substances, and account for 60 to 80% of plasma proteins. It is said that there are many substances with a larger molecular weight than albumin, which has a molecular weight of 66,000 (molecular size 38 x 150 Å), such as immunoglobulin, fibrinogen,
α ₂ -macroglobulin, immunoglobulin and antigenic substance, complement binding substance, i.e., immune complex,
It is desired to remove substances with a molecular weight of at least one million or more, such as low-density riboproteins and cryoproteins, and preferably γ-globulin with a molecular weight of around 160,000. In plasma exchange therapy, blood taken from a patient is separated into blood cell components and plasma using a centrifuge or membrane plasma separator, and all of the plasma, including albumin and other unnecessary components such as pathogenic or harmful substances, is removed. Then, approximately equal amounts of normal plasma are exchanged and returned to the patient along with blood cell components. Such separate exchange of plasma requires frequent exchange of a large amount of plasma in a short period of time, and usually as many as 3 to 6 plasmas are exchanged in 1.5 to 3 hours. Such plasma exchange therapy requires a large amount of fresh plasma, whose main component is albumin, but fresh plasma is extremely expensive, has limited supply, and is associated with risks such as hepatitis infection. There are many problems that impede the development of such treatments. Therefore, in order to solve these problems, a method has been considered in which only unnecessary components in autologous plasma are separated and removed, and useful components such as albumin are recovered without being discarded and returned to the body. Therefore, the therapeutic effect can be further promoted without using expensive fresh blood plasma from another person and without the risk of hepatitis. One such method is to treat two or more substances of different sizes in plasma in a flat membrane or tube shape, which is the most economical, easy, safe, and continuous method for processing a large amount of plasma. It is generally known that substances can be separated and removed using various membranes such as membranes and hollow fiber membranes. In particular, hollow fiber membranes are widely used in blood and body fluid processing devices such as artificial kidneys, plasma separators, and ascites treatment devices.These hollow fiber membranes have many advantages, such as a small priming volume and excellent pressure resistance. Furthermore, it is known that blood and body fluids can flow evenly along each membrane surface compared to flat membranes and tube-shaped membranes, resulting in higher dialysis or separation performance. There is. On the other hand, one of the problems with hollow fiber membranes is that in the case of artificial kidneys, the inner diameter of the hollow fiber membrane cannot be significantly reduced due to the problem of residual blood due to blood coagulation, and the normal diameter is 200 to 300μ. Blood coagulants are also used more often than with flat membranes or tubed membranes. When treating blood or body fluids using hollow fiber membranes, flowing the treated blood or body fluids inside the hollow fiber membranes is necessary for the following reasons:
This method has become widely established as a conventional method for treating blood and body fluids. That is, (1) an even distributed flow can be supplied to all the effective membrane surfaces; (2) A large linear velocity can be easily obtained, suppressing the formation of a polarization concentration layer, and contributing to maintaining overperformance and fractionation performance. (3) partial retention for uniform flow;
Stagnation is less likely to occur, and in the case of blood, coagulation is less likely to occur.
(4) The priming volume is relatively small. On the other hand, the method of passing from the outside to the inside is also easily considered, and is partially adopted in industrial ultrafiltration, but in this case, the apparent effective membrane area is calculated only by the ratio of the outer diameter to the inner diameter of the hollow fiber. However, the following disadvantages also occur regarding the treatment of blood and body fluids. In other words, (1) modules using hollow fiber membrane bundles result in non-uniform flow, resulting in coagulation and
Protein precipitation occurs. (2) Utilization rate of effective membrane area decreases due to non-uniform flow. (3) Since hydrostatic pressure is generated when the linear velocity is 0, the overperformance and deterioration of fractionation performance are significant due to the accumulation of blood cell components, proteins, and gel layers. From this point of view, when treating blood, body fluids, etc., a system that uses the inside is adopted, and all devices currently on the market are implemented using the inside method. By the way, the present inventors have been conducting extensive research on a device that uses a membrane to purify only plasma components, which do not contain blood cell components, instead of blood. In the course of this research, the present inventors naturally adopted the conventional method of passing from the inside to the outside of the hollow fiber membrane. Surprisingly, however, when we reversed the process and flowed plasma from the outside to the inside of the hollow fiber membrane, we discovered an unexpected phenomenon that wiped out the aforementioned disadvantages of flowing from the outside for some hollow fiber membranes. The results were obtained. In other words, when we compared the performance of the inner method and the outer method, we found that even after taking into account the difference in apparent membrane area mentioned above, there are differences in terms of overperformance retention, fractionation performance, fractionation performance retention, etc. was discovered. In addition to these completely new facts obtained by supplying plasma to the outside of such a hollow fiber membrane, the advantage is that, unlike in the case of blood, for example, the inner diameter can be made as small as possible and miniaturization is possible. Furthermore, it is easy to improve the hollow fiber membrane by chemically and physically treating the outer surface of the membrane. It is highly safe because it is taken out from inside the hollow fiber membrane and re-injected into the body, and excess residue may be easily removed by cleaning the outside of the hollow fiber membrane, that is, the outer wall. Can be mentioned. As mentioned above, the present invention intentionally applies to plasma the filtration from the outside of the hollow fiber membrane, which has not been used at all in blood and body fluid treatment, and therefore, contrary to expectations, the ultimate overpressure retention rate of plasma has been improved. This is based on the discovery that the ultra-excess retention rate and fractional retention capacity are greatly improved, and that the hollow fiber membrane exhibits anisotropic permselectivity. That is, in the present invention, plasma to be fractionated is supplied to the outside of an anisotropic permselective hollow fiber membrane in which the outside pore diameter of the hollow fiber membrane is smaller than the inside pore diameter, and the plasma is separated from the inside of the hollow fiber membrane. The present invention relates to a plasma fractionation processing method and apparatus for draining the fraction. The details of the apparatus of the present invention will be explained below with reference to the drawings and specific examples. FIG. 1 is an explanatory diagram showing one embodiment of the apparatus of the present invention. Blood collected from the human body is, for example, manufactured by the product name Celltrifuge (manufactured by Travenol).
Blood cells and plasma are separated using a centrifugal separator such as the above or a cellulose acetate hollow fiber membrane separator (product name: Plasma Flow, manufactured by Asahi Medical Co., Ltd.) with a pore size of 0.2μ, and then the separated plasma is passed through the plasma introduction section. 1
from there to the inlet 3 of the device 2 of the present invention. Device 2
contains a large number of hollow fiber membranes 4, both ends of which are adhesively fixed to the device 2 with an adhesive 5 such as polyurethane, one end 6 of the hollow fiber membrane has an opening closed with adhesive, and the other end 7. is open. The other end of the opening is connected to a nozzle 9 having a liquid discharge port 8 and is fixed with a cap 10, and one end is fixed with a cap 11. The plasma supplied to the outside of the hollow fiber membrane 4 of the device 2 becomes a fractionated liquid from the inside 12 of the hollow fiber membrane and is sent out from the liquid outlet 8 to the outside. On the other hand, the excess plasma that has not passed through the membrane is discharged from the excess plasma outlet 13 to the outside 14. At this time, part or all of the excess plasma is passed through the circulation system line 15 and the circulation pump 16 shown by dotted lines. It may then be returned to the inlet of the device. Further, when excess residual plasma is not discharged to the outside or recirculated, ie, it may be used completely, in which case the valve 17 is closed. In the device of the present invention, the liquid outlet 8, the plasma inlet 3, and the excess plasma outlet 13 may be one or more. The residual plasma outlet 13 may not be provided. Next, a specific example of the anisotropic permselective hollow fiber membrane defined in the present invention is shown below. 1.6 based on diameter and inner diameter membrane area
Create hollow fiber membrane modules of 2 types A and B of m ² ,
Module A is the first module that allows plasma to flow outside the hollow fiber membrane.
The device shown in the figure is used, and module B has a liquid outlet 8 for the conventional method of flowing plasma from inside the hollow fiber membrane.
The plasma introducing section 1 was connected to the device. Bovine plasma having a protein concentration of 5% was continuously supplied to each of the devices in a total pass-through manner at 30° C. at a supply rate of 30 ml/min for 3 hours. At this time, the ultra-overpressure (mmHg) of the module, the ultra-excess amount of plasma (), the membrane permeability (%) of albumin and high molecular weight protein components with a molecular weight of 1 million or more in plasma, and the recovery rate (%) are calculated. It was determined over time and shown in Figures 3-6. The pressure at the plasma introduction part of the module was used for the ultra-overpressure, and the transmittance and recovery rate of each protein component were measured using liquid chromatography HLC-801A (Column SW-3000X1 manufactured by Toyo Soda Co., Ltd.).
This was analyzed using a chromatograph (using phosphate buffer as the eluent). That is, the solid line in FIG. 2 is a liquid chromatography pattern of a 100-fold dilution of bovine plasma 5% solution (mother liquor), and the dotted line is a pattern of a 100-fold dilution of liquid plasma. Now, let the liquid chromatography peak areas of albumin and high-molecular protein in the mother liquor and solution at time t after the start be A _A , A _HMW , A _A ′, A′ _HMW , respectively, and the plasma supply rate to the module to be Q _P (ml/min). ), plasma hypervelocity is Q _F (ml/min), then albumin and high molecular protein permeability S _A (%), S _HMW (%)
is, S _A =Q _F・A′ _A /Q _P・A _A ×100% ………(1) S _HMW =Q _F・A′ _HMW /Q _P・A _HMW ×100%……(
2) Also, the recovery rate R _A (%) and R _HMW up to that point
(%) is R _A = _t 〓 ^O Q _F ×A′ _A ／Q _P ×A _A ×100% ………(3) R _HMW = _t 〓 ^O Q _F ×A′ _HMW ／Q _P ×A _HMW ×100% ......Calculated from (4). In Figures 3 to 6, the solid line shows the results when plasma is supplied to the outside of the hollow fiber membrane made of module A, and the dotted line shows the result when plasma is supplied to the inside of the hollow fiber membrane made of module B. For example, as shown in these figures, when plasma is supplied and separated from the outside to the inside of a hollow fiber membrane, contrary to conventional predictions, the rise in ultra-overpressure is slower, that is, membrane clogging is more likely to occur. It is unlikely to occur, the amount of ultraviolet excess is large, and the difference in permeability and recovery rate between albumin and high-molecular protein is large, so the fractionation of these substances is excellent. As is clear from these facts, there is a surprising fact that there are huge differences in properties between the inside and outside of hollow fiber membranes, that is, the properties and performance of hollow fiber membranes are anisotropic. I found out. In addition, the molecular weight cutoff of the membrane that regulates the passage of such plasma proteins is usually determined by passing dilute solutions of globular proteins of various molecular weights, but the display may differ slightly depending on the measurement conditions, analysis method, etc. The present inventors used standard substances such as Titochrome C (molecular weight 12400), serum albumin (67000), α-globulin (16000), catalase (232000), ferritin (440000), thyroglobulin (669000) and Blue dextran (2,000,000 linear polysaccharide) was selected, and the fractional molecular weight was determined from the rejection rate when each solute was passed through the membrane (Fumiji Hagiwara, Koichi Hashimoto, eds., "Separation Methods Using Membranes," Kodansha Scientific Photography, (see 1974). These proteins were dissolved in a pharmacopoeial saline solution to a concentration of 0.025%, and the total pressure was 200 mmHg.
The protein concentration of the solution obtained after 30 minutes is determined by the lorry method using spectrophotometry at a wavelength of 750 nm, and the rejection rate is determined. Blue dextran can be quantitatively analyzed directly in liquid form at a wavelength of 280 nm. Once the rejection rates of various molecular weight proteins are known, they are blotted onto a semi-log graph with the molecular weight on a logarithmic scale, and from the graph, the molecular weight corresponding to a rejection rate of 95% is defined as the molecular weight cutoff of the membrane. However, in the case of plasma actually used for membrane separation, the protein concentration is high and the solution is nearly 100 times more concentrated than a dilute solution, and when such a concentrated solution is used, the molecular weight cutoff generally decreases. The membrane used in the apparatus of the present invention is a membrane that cuts high molecular weight substances with a molecular weight of 1 million or more, and has an upper limit of 2 million to 5 million in terms of molecular weight cutoff. On the other hand, a membrane with a small pore diameter that cuts out polymeric substances of 200,000 or less improves the blocking rate of polymeric proteins, but the recovery rate of albumin is low, which is not preferable. Membrane materials include cellulose-based membranes such as cellulose acetate, and synthetic polymers such as polyvinyl alcohol, ethylene vinyl alcohol, polymethyl methacrylate, polyacrylonitrile, polycarbonate, polysulfone, polyvinylidene fluoride, polyethylene, polypropylene, polyamide, and polyester. It is a porous membrane. These porous membranes can be obtained by already known techniques, but for example, as a typical example,
No. 131028 and JP-A No. 52-84183. The hollow fiber membrane has a pore size large enough to prevent blood cell components from leaking out, satisfies the above-mentioned range of molecular weight cutoff, and the pore size on the outside of the hollow fiber membrane can be determined by observation with a microscope such as a scanning electron microscope. It is smaller than the inner pore diameter. The inner diameter of such a hollow fiber membrane is preferably small in order to downsize the priming volume and improve pressure resistance, and is in the range of 10 to 1,500μ, preferably 30 to 800μ, and the membrane thickness is in the range of 5 to 1,000μ, preferably 10μ. ~500μ. Furthermore, if the space outside the hollow fiber membrane is too large, the flow of plasma will not be smooth and stagnation will occur, which is undesirable. Therefore, the inner priming volume (Vi) and the outer priming volume (Vo) of the hollow fiber membrane
The ratio Vo/Vi is 1.0 to 5.0, preferably 1.1 to 3.0. As described above, as shown in the present invention, by using a plasma fractionation processing apparatus that supplies plasma to be fractionated to the outside of an anisotropic permselective hollow fiber membrane and flows out the fractionated liquid from the inside of the hollow fiber membrane. It has high ultrafiltration performance, reduces membrane clogging, and prevents a sudden rise in ultraoverpressure, significantly improving the albumin recovery rate (R _A ) and the separation of albumin and high-molecular proteins. I understand. Hereinafter, the present invention will be explained by giving examples. Cellulose acetate (CA- manufactured by Eastman)
394−45) 16.8%, acetone 34.7% and methanol 8.4% as solvents, and calcium chloride 2
A stock solution consisting of 13.7% aqueous salt and 26.4% cyclohexanol was discharged from an annular spinneret by the method disclosed in JP-A-52-84183, with an inner diameter of 350 μm and a film thickness of 160 μm.
A hollow fiber membrane was obtained. The obtained hollow fiber membrane was an anisotropic membrane in which the pore diameter on the outer surface was smaller than the pore diameter on the inner surface. We bundled 4,200 of these hollow fiber membranes and fixed both ends with urethane to create module 2 shown in Figure 1 with an effective length of 18 cm and an effective membrane area of 1.6 m ² based on the outer diameter. was assembled into the first apparatus shown in FIG. On the other hand, in order to use a conventional device that flows plasma from the inside of a hollow fiber membrane as a comparative example,
A module having the same form as shown in FIG. 12 was prepared, with 8000 hollow fiber membranes having the same anisotropy as above, an effective length of 18 cm, and an effective membrane area of 1.6 m ² based on the inner diameter. In this module, a second device was assembled in which the plasma inlet 1 was connected to the liquid outlet 8 of the module, the residual plasma outlet 13 was used as the liquid outlet, and the plasma inlet 3 was closed. Next, hematocrit is 45% and heparin is 10,000 units.
Added fresh bovine blood was added to a cellulose acetate ^hollow fiber plasma separator (trade name,
The sample was supplied to Plasma Flow Hi-05 (manufactured by Asahi Medical Co., Ltd.) at a flow rate of 100 ml/min, and plasma was separated at an ultra-overpressure of 80 mmHg to prepare plasma with a protein concentration of 5%. For example, in the case of the first device described above, the valve 17 is closed, that is, in a total pass-through mode, and in the second device, the same condition is applied continuously at 30° C. and a rate of 30 ml/min for 3 hours. Supply processed. At this time, the recovery rate of albumin and high molecular protein components with a molecular weight of 1 million or more in plasma was determined using the aforementioned Toyo Soda liquid chromatography HLC-801A (column SW-3000 x 1
Analysis was performed using equations (3) and (4) from a chromatograph using phosphate buffer as the eluent. In this case, these values were measured using a pool of liquid plasma up to 3 hours after the start. In addition, plasma total protein concentration was measured by the Biuretz method. Results first
Shown in the table. In Table 1, QFT is the ultraviolet amount of total plasma (), P is the ultimate pressure of the excess device (mmH
g), _IRA -R _HMW I is a measure that indicates fractionation by the difference in recovery rates of albumin and high molecular weight protein.

[Table] From the results, it can be seen that in the comparative example, the pressure of the filtration device suddenly rose above the supply capacity pressure of the pump, resulting in a decrease in processing capacity and poor separation performance. In contrast, it was found that the apparatus of the present invention had a good albumin recovery rate, dramatically improved separability, and had less clogging. Furthermore, the above-mentioned device also produced similar excellent effects on cryo-plasma cooled to 4°C.

[Brief explanation of the drawing]

Fig. 1 is an explanatory diagram showing one embodiment of the device of the present invention, Fig. 2 is a liquid chromatography pattern of bovine plasma and liquid plasma, and Figs. 3 to 6 show bovine plasma supplied to the device of the present invention and a conventional system. It is a graph showing ultra-overpressure, ultra-overflow, permeability, and recovery rate in the case of 1...Plasma introduction part, 2...The device of the present invention, 3...
Introduction port, 4...Hollow fiber membrane, 5...Adhesive, 6...
One end of the hollow fiber, 7...other end of the hollow fiber, 8...liquid outlet, 9...nozzle, 10, 11...cap, 12...inside of the hollow fiber membrane, 13...excess plasma outlet, 14...External, 15...Circulatory system tract, 16
...Circulation pump, 17...Valve.

Claims (1)

[Claims] 1. The plasma to be fractionated is supplied to the outside of an anisotropic permselective hollow fiber membrane in which the outside pore diameter of the hollow fiber membrane is smaller than the inside pore diameter, and the fractionated filtrate is flowed out from the inside of the hollow fiber membrane. plasma fractionation processing device.