JPS6099266A - Removal of protein in body fluids and apparatus used therein - 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 (1) Background of the Invention The present invention relates to a method for removing large molecular weight proteins present in body fluids and an apparatus used therefor.

In recent years, it has been used as a treatment for various collagen diseases, immune diseases, cancer, etc.
Plasma purification therapy, which separates plasma from a patient's blood, removes pathogenic substances present in the plasma, and reuses the purified plasma as a stimulant, has been developed and is now in clinical use. Although there are still many unknowns regarding pathogenic substances in plasma, Ill disease,
In immune diseases, large molecular weight proteins such as immune complexes, immunoglobulin, and fibrinogen are considered to be the main players, and plasma purification is performed by removing these from the plasma. The method and device of the present invention are effectively used for such plasma purification therapy.

In general, the method and apparatus of the present invention are used to purify plasma by removing source substances from Ff and raw plasma during the production of plasma preparations.

Current plasma purification methods include removing large molecular weight proteins using membranes with smaller pores than plasma separation membranes, or using the proteins as adsorbents. Adsorption methods are known, and the former has already been used clinically with some success and is establishing itself as a treatment method.The latter is undergoing basic research. The horn method is still not sufficient, and the development of a more effective and efficient method is desired.

11. Purpose of the Invention The present invention provides a method for safely removing large molecular weight proteins from body fluids such as mucus plasma and 11y water for the production of plasma preparations, and an apparatus used therefor. The purpose is to

In order to achieve the above object, the present invention consists of the following configuration.

(1) Salting out the proteins in the body fluid by adding an amount of alkali metal chloride in excess of the amount necessary to precipitate the proteins in the body fluid, separating the protein precipitating agent from the liquid, and dialysis treatment A method for removing proteins from body fluids, characterized by removing alkali metal chlorides from clear body fluids.

(2) The protein removal method according to item 1, wherein the body fluid is plasma.

(3) The method for protein removal according to item 1 or item s2, wherein the alkali metal chloride is sodium chloride or potassium chloride.

(4) A container having at least one body fluid inlet and containing an alkali metal chloride, a means for isolating the salted-out protein precipitate and the supernatant fluid, and a means for circulating the isolated supernatant fluid. a dialyzer for removing the alkali metal chloride (alkali gold) iA fl ide from the body fluid contained in the body fluid in an amount greater than that necessary to precipitate the proteins contained therein. protein removal equipment.

(5) The means for collapsing is Δ1(
5. The protein removal device according to item 4, which is a material.

(6) The protein removal device according to item 4, wherein the isolation means is a second container communicating with the upper part of the container.

(7) The protein removal device according to item 4, wherein the isolation means is a filter.

l■1 Detailed Description of the Invention Plasma contains large molecular weight proteins such as immune complexes, immunoglobulin, and fibrinogen, as well as relatively small proteins such as albumin. In plasma purification therapy, as described above, it is desirable to selectively precipitate and remove only large molecular weight proteins, while leaving the white matter that interacts with small molecules such as albumin in the plasma. The inventors of the present invention are very knowledgeable about protein precipitating agents in plasma.As a result of repeated research, we have found that alkali metal chlorides, such as sodium chloride, have a relatively weak salting-out effect and low solubility, so that they cannot be chlorinated in plasma in excess of the saturation amount. I learned that even when thorium is added, albumin hardly precipitates, and large molecules such as fibrinogen and immunoglobulin specifically precipitate only proteins.Therefore, the amount of sodium chloride added must be strictly controlled. It is not necessary, and it is sufficient if the amount is above saturation.On the other hand, with other precipitants, such as ethanol and ammonium sulfate, albumin will start to precipitate when the concentration exceeds a certain level, so it is important to strictly control the amount added. Furthermore, when adding ethanol or ammonium sulfate, there is a risk of heat generation or localized high concentrations, which may denature proteins in plasma. There is no need to worry about this.Therefore, unlike conventional methods, there is no need to introduce a precipitant into the plasma collection container before adding a precipitating agent. Since it is possible to contain chloride (chloride), it is possible to introduce plasma into it, and in this case, there is no change in the precipitation effect or composition of the precipitate.
Protein denaturation does not occur, and only large molecular weight proteins in plasma can be precipitated with an extremely simple operation.

The present invention was completed based on such knowledge.

The present invention first involves adding alkali metal chloride to a body fluid in an amount greater than the amount necessary to precipitate the proteins in the body fluid, salting out the proteins in the body fluid, separating the protein precipitate from the supernatant body fluid, and dialysis. It consists of a method of removing proteins in body fluids by removing alkali metal chlorides from supernatant body fluids through treatment.

In the method of the present invention, examples of body fluids include plasma, l1l (
Examples include water, lymph fluid, etc., and plasma is particularly preferably used. Examples of alkali metals are 1!
Sodium chloride, potassium chloride, etc. are used, especially sodium chloride.

In the method of the present invention, body fluid is introduced into a container containing in advance an amount of alkali metal chloride greater than the amount necessary to precipitate proteins in the body fluid, and the large molecules Ji in the body fluid are salted out.
3. Separate the proteinaceous fluid from the supernatant body fluid by appropriate means, and dialyze the supernatant body fluid against a dialysate etc. This is done by removing alkali metal chlorides. When m+ plasma is used as the body fluid and thorium chloride is used as the alkali metal chloride, the Jj of thorium chloride stored in the container is 20 g#f-1 or more with respect to the collected plasma.
8,9! Preferably, it is 25 to 35 g#ll. Currently -・
In plasma exchange therapy, which is commonly used, the amount of one exchange is 2
~39. Or SV average at most 4 sentences 4 ~! Since it is a melon, for example, if the capacity of the container is 4500 ml and the amount of thorium chloride is 900 g, it can be used in most cases. The optimal amount of plasma to be collected at this time is 2.5 to 3.5 tons. If the amount of collected carbon increases beyond this value, the effect of precipitated C will decrease slightly. Even if the amount collected is less than the above amount, there is no change in the composition of the precipitate produced. The precipitate is removed by filtration, etc., and the supernatant plasma is dialyzed to reduce the concentration of sodium chloride in the plasma to a physiological concentration to obtain purified plasma.

Through this purification treatment, more than 70% of plasma proteins such as fibrinogen and immunoglobulins are separated as precipitates.
be done. Diseases requiring plasmapheresis treatment, pathogenic factors of Tomoe,
Most of the harmful substances are contained in this part. -Benoy J
Only 7 to 8% of albumin, a component of the product, precipitates. The purified plasma obtained is returned to the person from whom it was collected as a replenishment fluid at the time of the next plasmapheresis treatment, either as is or with the protein concentration corrected if necessary.

A second aspect of the present invention is an apparatus suitably used in the above-mentioned method for removing proteins in body fluids, which has at least one body fluid inflow and outflow.
means for isolating a container containing an alkali gold 1 chloride therein, a salted-out protein precipitate, and a -L clear liquid;
and a dialyzer that circulates the isolated supernatant body fluid to remove the alkali metal chloride, and the alkali metal chloride described in n1j is necessary for precipitating proteins in the body fluid contained in the container described in 9j or Jj. It consists of a device that removes proteins in body fluids that are in excess of a certain amount. In the apparatus of the present invention, l) θ container I
It has at least one body fluid inlet/outlet 1-1 and contains an alkali metal chloride therein. The body fluid inlet is used to introduce body fluids such as plasma into the container, and to remove protein precipitates and deposits.
This is for taking out the supernatant body fluid from the container after the 11-day treatment, and one unit may be used for both purposes, or an outflow 1 and an inflow 1j may be provided separately. The alkali metal chloride contained in the container is sufficient to precipitate the protein contained in body fluids by salting out, and the amount of the alkali metal chloride is sufficient to precipitate the protein contained in the liquid introduced into the container. necessary I
It is jll of 11 or more 1-. Alkali metal chlorides saturate body fluids; precipitation occurs even when present in large excess
Horn! 11. The protein content will not increase any further and the protein content will not decrease more than necessary. The container is proteinaceous V.
It is desirable to have flexibility for convenience when isolating body fluids, and hangs made of soft polymers such as polyethylene and vinyl chloride are suitable for Ilf.

A known means can be used to separate the protein precipitate and the supernatant body fluid. Preferably, the boundary between the protein precipitate layer and the supernatant fluid layer is squeezed from the outside of the container using a pressure tool, or the lower part of the container is gently pressed to collect the supernatant fluid from the container. Filter the liquid inside.

The supernatant fluid separated from the protein precipitate is dialyzed using a dialyzer.
Alkali metal chlorides are removed. The dialyzer is placed in said container or in a second container containing the isolated liquid.
Can be installed to form a closed circuit. Next, the present invention will be explained in more detail with reference to an embodiment shown in FIG.

FIG. 1 is a block diagram of an apparatus for removing protein from plasma according to an embodiment of the present invention.

In FIG. 1, the apparatus of the present invention includes a flexible container 4 having a plasma inlet 1 and a plasma outlet 2'I in the upper part and containing sodium chloride 3 inside, and a dialyzer 5 connected to the container 4. The formed closed circuit communicates with the plasma population 1 through the sterile joint 6 to chlorinate the container. 1-jIi.

To remove proteins in blood using this device, the second
As shown in the figure, plasma is supplied from the ml QR separator 7 to the container 4, mixed with thorium chloride in the container, and left to stand for a while to precipitate the proteins in the plasma. The boundary between the V layer and the upper 1σ plasma is compressed with a crimp 8 to separate the protein precipitate and the J: clear plasma.

The container 4 is made of a flexible material so that the plasma precipitate V and the supernatant plasma can be separated from each other by applying pressure.

Next, the purified plasma is obtained by circulating the closed circuit formed by the container 4 and the dialyzer 5 to remove sodium chloride from the supernatant blood 1 (L).The purified plasma is collected from the plasma outlet 2, and the plasma preparation It is used in the production of blood cells and plasma exchange therapy.

FIG. 3 is a block diagram of an apparatus for removing proteins from plasma according to another embodiment of the present invention.

In FIG. 3, the device of the present invention has Jhl 'I
F (a flexible container 4 having an E capacity of 7% and containing sodium chloride 3 therein; a second container 9 communicating with the container 4 through the plasma outflow port 2; dialyzer 5
and a plasma separator 7 that communicates with the plasma population 1 and supplies plasma to the container 4 through a sterile joint 6, and the sodium chloride is contained in the plasma contained in the container 4. This amount is more than the amount required to precipitate the protein.

To remove proteins from plasma using this device, plasma layer g! The plasma is supplied from the container 7 to the container 4, mixed with the sodium chloride 3 in the container, and left to stand for a while to precipitate the proteins in the plasma.

Then, by pressing the lower part of the container 4, the supernatant plasma is discharged into the second container 9 from the plasma outlet 2 in the upper part of the container 4. Alternatively, a pump is installed between the plasma outflow port 2 and the second container 9, and the plasma is introduced into the second container 9 by the pump. At this time, it is desirable to clamp the boundary between the protein precipitate layer and the supernatant plasma layer with a clip from the outside of the container 4 to prevent the precipitate from mixing with the supernatant plasma. Next, the supernatant plasma is circulated in a closed circuit formed by the second container 9 and the dialyzer 5 to remove thorium chloride from the supernatant plasma to obtain purified plasma.

FIG. 4 is a block diagram of an apparatus for removing proteins from plasma according to still another embodiment of the present invention.

At the fourth point, the device of the present invention has plasma in the upper part.
, a container 4 having a plasma outflow [J2 in the lower part and containing sodium chloride 3 therein, a second container 9 communicating with the container 4 through a plasma outflow port 2 via a sterile joint 6 and a filter 10, It consists of a closed circuit formed in two containers 9 via a dialyzer 5, and a plasma separator 7 that communicates with the plasma inlet l and supplies plasma to the container 4 via a sterile joint I.6. 1. is necessary to precipitate proteins in plasma contained in container 4. -This is the above dragon.

To remove proteins in plasma using this device, plasma is supplied from plasma separator 7 to container 4, mixed with sodium chloride 3 in the container, and left to stand for a while to precipitate proteins in plasma. let The plasma is then passed through the filter 1 from the outlet 2 of the container 4.
0 into the second container 9. At this time, the precipitate in the plasma is filtered by the filter 10, and the supernatant plasma separated from the precipitate V is stored in the second container. Thorium chloride is removed from the supernatant plasma by circulating it in a closed circuit formed by the second container 9 and the dialyzer 5 to obtain purified plasma.

The sterile joint used in the above device is one that aseptically connects the communication part between the container 4 and the plasma separator 7 or the second container 5, and is #! jfj-)4IJ? 357-2
1i of a tapered sleeve made of ceramic and a tapered hole into which it is fitted, as disclosed in No. 11353.
It is desirable to use a combination with li members.

IV. Effects of the Invention According to the present invention, a method for removing proteins from body fluids is provided, which allows proteins in body fluids to be removed by simple operations.

In the method of the present invention, since an alkali metal chloride such as sodium chloride or 1) potassium silicate is used as a protein precipitant, it is highly safe and does not denature proteins in plasma.

Furthermore, the detergent used in the present invention does not change its precipitation effect even when added in large quantities, and immunoglobulin, 7"-)'
+) / -)y'' 7. Only the proteins of Oita J'cell are selectively precipitated, and the proteins of small molecules J11 such as albumin are hardly precipitated. Therefore, the amount of precipitant added is I'f1. There are no planets to control in the cloth.

Further, according to the present invention, there are provided four apparatuses for use in the negative removal of proteins from body fluids.

The wooden container has at least one body fluid outflow inlet, and separates a container containing an alkaline chemical compound therein, salted protein sediment, and clear body fluid. and a dialyzer that circulates the isolated 1-10 body fluid and removes the alkali metal chloride, and can easily and safely wash and remove proteins in the body fluid. According to the apparatus of the present invention, all operations can be performed in a closed system, so there is no risk of contaminating body fluids or the environment, and conversely, there is no risk of contamination of body fluids with bacteria or the like.

[Brief explanation of drawings]

FIG. 1, FIG. 3, and FIG. 4 are explanatory diagrams showing the configuration of the apparatus of the present invention, respectively, and FIG. 2 is an explanatory diagram showing an example of use of the apparatus of the present invention. 1...Plasma inlet 2...Jm 12 Outflow 113.
... Sodium chloride] Noum 4 ... Container 5 ... Dialyzer
6... Sterile joint 7... Plasma separator 8... Clip 9... Second container lO... Filter Figure 1 Figure 2

Claims (7)

[Claims] (1) Salt out the proteins in the body fluid by adding the necessary amount of alkali metal chloride to the body fluid to precipitate the protein in the body fluid, and separate the white matter precipitate and the supernatant body fluid, and dialysis treatment 1. A method for removing proteins from body fluids, which comprises removing alkali metal 1'4 oxides from supernatant body fluids. (2) The protein removal method according to claim 1, wherein the body fluid is plasma. (3) The protein removal method according to claim 1 or 2, wherein the alkali metal chloride, sodium chloride, or potassium chloride is used. (4) a container having at least one body fluid inlet and an alkali metal chloride therein; a means for separating the salted-out protein precipitate from the clear body fluid; and a means for separating the isolated supernatant body fluid. a dialyzer that circulates and removes the alkali metal chloride;
or f+7 required to precipitate the protein in the body fluid contained in the container. (5) The protein removal apparatus according to claim 4, wherein the isolation means is a member that presses the container from the outside. (6) The protein removal apparatus according to claim 4, wherein the means for isolating is a second container communicating with the upper part of the container. (7) Protein removal method according to claim 4, wherein the isolation means is a filter.