JPS6033089B2 - Method for separating precipitated proteins from albumin-containing suspensions - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating precipitated proteins from an albumin-containing suspension obtained by recovering albumin from dish acid.

The object of the present invention is to improve the invention described in Hakama Application No. 50-37713. When recovering albumin from blood, blood products, albumin-containing body fluids, etc., an albumin-containing suspension is obtained which, in addition to dissolved albumin, contains precipitated proteins, especially globulins.

Regarding this, the specification of Tokugan Sho 50-37713 (West German Published Patent Application No. 241507y) discloses a method for isolating albumin from human blood or the like. This method consists of the following steps. 'a - Separate the blood count from the solid components of blood (blood cells and platelets) and, if necessary, recover dissolved non-albumin components from this blood acid, 'b}
The separated liquid is heated in the presence of an alcohol having a composition of CH3-n(CH2)-OH (where n is 0.1 or 2) and an albumin stabilizer such as sodium caprate at a temperature of about 60 to 70%.
The globulins are precipitated by heating to a temperature of 500 °C, and this secretion (nujd) is then separated to enrich the albumin contained in the remaining secretion.

The process disclosed therein involves the separation of precipitated globulins and precipitable proteins by thermal precipitation at a pH of about 4.4 during centrifugation in continuous flow.

The separated proteins are collected in the rotor, but the albumin remains in the supernatant. In conventional separation methods, relatively large amounts of albumin remain in the separated concentrate, and we believe that it is unsatisfactory that multiple washing and centrifugation operations are required to recover this useful residual albumin. It is being

Moreover, the centrifugation process is very time consuming and involves a very expensive operation. Furthermore, centrifuges are very noisy. Despite these shortcomings, we are familiar with the long-standing Cohn method for separating suspensions containing precipitated and lysed globulins by a centrifugation step, and the reservoir operation required Nothing was taken into account. Tests have shown that suspensions obtained by the Cohn process cannot be heated under normal conditions. These materials have been recovered by the method described in the above specification, and due to the fact that the conventional Kohn process yields materials of approximately the same viscosity in the intermediate and final product form from the same starting materials, It was never thought that the materials obtained by these methods could be filtered.

Various furnace methods have been tried to remove proteins precipitated by cold ethanol fractionation. Experience has shown that these precipitates cannot be economically removed by furnace processes, probably because of their inherent consistency. Although other filtration methods have been investigated, it has been found that the consistency of globulin precipitated with hot ethanol is quite different from that of globulin precipitated with cold ethanol. Contrary to the above experience and expectations, surprisingly, according to the present invention, suspension
al) Separation by filtration achieves the objective of separating thermally precipitated proteins from suspensions at substantially lower costs.

The fan furnace itself is known.

Usually, fan filtration is carried out in a centrifugal purification filter mainly consisting of a closed pressure vessel having annular filter elements arranged horizontally or vertically in parallel with each other on a central rotating hollow shaft. These filter elements are usually made of metal strands (wire type) or knitted woven fabric. The material to be filtered is added to and mixed with a furnace filter aid, usually diatomaceous earth.

These auxiliaries are added in a predetermined manner depending on the type, turbidity and properties of the residual liquid. The furnace liquor collected in the filter element is separated by rotation of the filter element and countercurrent flushing.

The residual liquid is discharged as so-called slurry. While other types of filters and filtration methods require long filtration times that provide no advantage over the time-consuming centrifugation method, the present fan filtration provides surprising advantages.

Here, we will show the results of tests on refining furnace filtration using carbon and asbestos plate filters, cellulose-based furnace filtration, glass fiber filters, and refining furnace filtration methods using smelt glass. All of these filtration methods required very long furnace times due to clogging of the filter element or turbidity of the furnace liquor. In contrast, the use of a fan filter alone allows obtaining a clear filtrate, without requiring a separate additional cleaning or purification process, and thus can be the optimal recovery operation for obtaining albumin. Specifically, it is proposed that this fan filtration be carried out in a centrifugal purification filter having a closed container and a filter element having a mesh of 20 to 200 microns.

More specifically, the liquid in question is treated with a 70-90 micron mesh.

This mesh size corresponds to the obtained throughflow (throughH
ow) can be set to the optimum condition and complete filtration can be achieved. In this case, only one furnace step needs to be carried out in each operation. To fully prepare the filter surface for the next filtration, the filter element may optionally be precoated with a neutral liquid primer to a thickness of approx.
．． 5 It is a good idea to pre-coat the skin with a layer of furnace additive.

As the furnace auxiliary agent, Keibushi (trade name: High Flow Super Cell, Celite S45) or Perlite, which are generally available as commercial products, are suitable. Furthermore, cellulose filtering aids are also useful, although the filtering effect is slightly inferior. These furnace aids likewise make it possible to obtain satisfactory products for many applications. It is preferable to swell or impregnate the furnace auxiliary agent in advance. 8 to obtain advantageous filtering effect
Horizontal filter elements of 0 micron mesh metal wire or knitted woven fabric are preferred.

A preferred composition is a suspension of about 4-6% hemiplast protein to which is added 20-70 g of diatom + furnace additive per batch.

This suspension is then filtered. Under certain circumstances, when the protein concentration is low, the amount of furnace aid may be reduced. Albumin present in the slurry or turbidity can also be recovered by cleaning by rotation and countercurrent flushing, or by cleaning the residue at the residue filter with a recirculation system.
It is proposed to isolate this albumin as well by means of a residue filtration device which occupies a small portion of the total filter area.

4.0 to increase yield without reducing furnace overcapacity.
This process can be carried out at ~2.5 gallons (atmospheric pressure).

Hereinafter, the present invention will be explained in detail with reference to Examples.

The following two examples are based on two different suspensions. Example 1 uses a suspension prepared by the method described in Tokugan Sho 50-137713. Example Solid matter (blood cells and platelets) is separated from human blood to remove clotting factors.

This starting solution contains approximately 5-6% blood* protein. According to the method disclosed in said specification, clotting factors and fibrinogen are removed by cold ethanol precipitation. The raw acid has negative Hepatitis activity, normal transaminase values, and no visible hemoglobin. Sodium caprylate is added as an albumin stabilizer to this raw blood mackerel until the concentration becomes 0.004 mol. This mixture, which contains approximately 9% (by volume) ethanol, is heated to a pH of 6.5. PH value is 0. State-Adjusted by HCI. The temperature is then raised to porridge C over about 3 hours, and then the liquid is cooled to 10 qo. This suspension is further cooled and processed at a temperature of 10q○. The processed solution has an albumin content of approximately 2 to 2.5
%contains. The processing of the starting solution is shown diagrammatically in the accompanying drawings. In this method, a modified rotary discharge filter (type ZHF-S, Schenck vertical type) was used to perform 600 blood count separations within a maximum furnace time of 5 hours. The central device of the complete system is a double-walled filter vessel 20 capable of both heating and cooling, with a capacity of approximately 220 mm and a furnace surface area of approximately 3 mm. Annular filter disk 2
2, once the furnace liquid has passed through the filter media covering each disk, to the central column 21, then to line 23 and valve 8.
It is attached to the central rotating tower 21 so that it flows through the outlet to the outlet. Residue from the filtration cycle (furnace aid and precipitated globulin) is removed by centrifugal washing and 1% NaCI reflux washing. As a result, the filter can be easily used for the next cycle. The filter elements are covered with metal strands (wires) or knitted woven fabrics. The raw starting solution is maintained in suspension in a slurry tank 24 which is uniformly sealed.

The starting material enters two filter vessels through a slurry valve 10 and a pair of inlet valves 11,12. This container is pressurized with compressed air from compressed air valve 5. First, add 500ml of filter elemesoto to distilled water on a desk and add about 15% of Celite (trade name of diatomaceous earth furnace super-aid).
000 lbs. in suspension with distilled water and furnace aid to form a primer precoat approximately 0.2 mm thick at atmospheric pressure.

The mixture is then transferred from the slurry tank into a filter vessel and passed through the furnace surface of element 22 at a positive pressure of 2 bar. Before actually dispensing this liquid in a clean tank, the substance is passed through the filter element repeatedly in a so-called recirculation process (recirculation valve 6, pump 2) until the furnace liquid is sufficiently transparent through the see-through glass 25. . A wash solution of 1% NaCI is pumped through the filter as soon as the filter is free of clear albumin-containing solution (the amount of wash solution used is half the volume of the original batch size).

Furnace overspeed is 150 evening/hr. The protein concentration in the furnace fluid at the beginning of the process is about 2.5%, while the final concentration of non-alhumic components is only about 0.05%.
The resulting solution was clear, with an albumin protein concentration of about 1.5% and an osmolarity of about 100 hosm.
ity). The transparent solution was then filtered through a dialysis furnace using a Millipore Vericon cassette system (manufactured by Millipore, Neuesenburg, West Germany, furnace surface area: 9.0).

Dialysis refers to filtering a solution through a dialysis membrane. The initial flow rate in this process is about 5-6 mol/min.

The entire separation process takes three days when operating in eight-hour shifts per day.

Day 1: Precipitation with hot ethanol, followed by cooling and pH
Adjusted to 4.4 (5 hours).

Day 2: Fan oven week (4 hours) and dialysis oven week (flow rate adjusted so that the process ends at the beginning of the third working day).

In both steps, the raw material is maintained at a temperature of 160-18° C. by cooling in double-walled containers. 3rd day: pH (7.0
), protein concentration (5%, 20%), horse mackerel transparency (30℃)
final adjustment of sm); transparent oven filtration; sterilization oven filtration (membrane 0.2 μm)
; Bottling; Heat sterilization (1 field hour, 60°C).

The yield of albumin when using the fan filtration/dialysis filtration process is the sickle percent of albumin present in the original starting material (average of 6 lugches), and the quality of the final product is the same as that of the previous method. The biggest advantage of this method is that one person can do all the steps except for bottling.

The filter is cleaned by rotating the filter element with the drive motor 1 and then discharging the slurry through the discharge valve 9.

The material discharged through valve 4 can be resuspended and filtered. The liquid after passing through the filter no longer shows the Tyndall effect and is clear like water.

This liquid does not contain accompanying substances such as other proteins and lipids. This liquid can be used directly to saturate albumin and perform the following Furashimanpo method (see Tokuka Sho 50-137713). This method basically involves the following steps. Blood acid 600k9: 3.6k9 protein 18.48k9 albumin (:55%) 19% ethanol 10.004 mol sodium cabrylate 10 days CI p
H6.5 2 Thermal precipitation: 3 minutes, 68q○ 3 10HC1 pH 4.4 4 Fan furnace filter solution 5 Dialysis furnace filter 5.1 Concentration 5.2 Dialysis 6 NaOH pH 7.0 7 Transparent furnace weekly albumin solution 17. 2k9 albumin (=93% yield)

[Brief explanation of the drawing]

The figure shows an example of a fan filtration device used in the method of the present invention. 1... Motor, 2... Pump, 3... Hinazusa machine, 4,5
, 6, 8, 11, 12... Valve, 20...
... Filter container, 21 ... Central tower or rotating tower, 22 ... Filter disk, 23 ... Line, 24
...Slurry tank, 25...Transparent glass.

Claims (1)

[Claims] 1. Plasma from which coagulation factors have been separated is heated to about 9% at approximately neutral pH.
% by volume of alcohol and sodium caprylate as an albumin stabilizer at a temperature of about 60-75°C and then, after cooling, lowering the pH to about 4 to precipitate non-albumin components consisting primarily of globulins;
The albumin-containing suspension thus obtained is then fan-filtered through a woven filter element to collect non-albumin components on the filter element.
A method for the separation of precipitated proteins from albumin-containing suspensions, characterized in that a clear albumin solution is obtained as a filtered product. 2 The mesh having a sealed pressure vessel for the filtration is 20
The method according to claim 1, which is carried out in a centrifugal purification filter of ~200 microns. 3. The method of claim 2, wherein one of said filter elements has a mesh of 70 to 90 microns. 4 The filtration mesh is made of 80 micron metal strands (
3. The method as claimed in claim 2, wherein the method is carried out with horizontally arranged filter elements having a wire or a knitted fabric. 5 Approximately 4-6% plasma protein suspension at approximately 30% per liter
5. The method of claims 1 and 4, wherein the method is mixed with ~70g of diatomaceous earth filter aid. 6. The method according to claims 1 and 5, wherein the cellulose filter aid is used in a previously expanded or impregnated state. 7 Discharging the slurry or turbidity contained in the precoat composition through the residue filtration device after cleaning by rotation and countercurrent flushing or after cleaning the contents of the residue filtration device in a recirculation process The method described in Items 1, 2 and 6. 8 The above method was 4.0±2
．． A method according to claims 2 and 6, which is carried out at a positive filtration pressure of 5 par (atmospheres). 9. The method according to claim 1, wherein the transparent albumin solution is diafiltered (through a dialysis membrane).