HU181926B - Process for the isolation of precipitated proteins from suspensions containing albumin - Google Patents

Abstract

The production of albumin for non-therapeutic applications from a suspension obtained from blood plasma is carried out by flotation filtration of the suspension on a fabric filter element. The non-albumin-containing constituents of the emulsion are collected on the fabric filter element and a clear albumin solution is obtained as the filtrate. The process enables the separation of heat-precipitated proteins from the suspension with a substantially smaller outlay compared with known filtration processes, within a shorter filtration time and avoiding a blockage of the filter inserts.

Description

The present invention relates to an improved process for the separation of precipitated proteins in the recovery of albumin from plasma albumin-containing suspensions.

; It is known that in 1946, Cohn et al. Developed a method for fractionating human blood plasma. In the process, five major and more subfractions are obtained by varying the five variables, ethyl alcohol concentration, pH, temperature, ionic strength and protein concentration, respectively.

The procedure was of great importance because the therapeutic use of the plasma constituents isolated in this way is more economical and allows them to undergo specific therapy without the unnecessary use of whole blood plasma. This also means better use of plasma, since it is not available in unlimited quantities.

However, Cohn's alcohol fractionation is a very labor-intensive and energy-intensive process that takes 8 days to process a portion. Most operations require refrigeration and / or refrigeration equipment, large numbers of manpower, and working conditions are very uncomfortable due to high temperature differences and noisy centrifuges.

The main fractions obtained in the Cohn process were fibrinogen (fraction I), gamma globulin (fractions II and III), alpha and beta globulins (fraction IV), and finally albumin precipitated from the supernatant (V. fraction), the latter of which is obtained by enrichment (lyophilization, dissolution, filtration, stabilization, pH and concentration adjustment) of 5 to 15 and 5 to 15%, respectively, for pharmaceutical purposes. 20% albumin.

In practice, of the above products of the Cohn process, only albumin, fibrinogen and gamma-globulin have been used in medicine.

In practice, it has later been found that fibrinogen produced from large plasma pools has a very high risk of hepatitis 5 transmission, so that small amounts of blood cryoprecipitate are used instead.

Practice has also shown that, apart from specific hyperimmune globulins, it can be found in Part II. The demand for gamma-globulin in the fraction A is also low.

Therefore, in practice, out of the 5 fractions, only albumin is in fact the most valuable product, which, in turn, becomes even more expensive.

Despite all these disadvantages, the 15 Cohn method for plasma fractionation is still widely used throughout the world.

In addition, however, there has been a long history of research into lowering the cost of the process, since blood supply and blood products are largely non-profit services, and on the other hand, only a limited amount of plasma base serves growing needs.

The first proposal to reduce costs and improve working conditions was made in 1962 by Kistlerés Nitschmann, who proposed that the I-III program should be implemented. fractions are combined and automatically separated and discarded. However, the resulting savings have been very limited, as most of the costs are borne by cooling systems and centrifuges, which, however, cannot be eliminated.

Also recommended is column separation, this method

-1181926, on the one hand, resulted in pyrogen products and, on the other hand, did not improve profitability and was also unsuitable for processing larger portions.

An important step in 1964 was the suggestion of Polsen et al., Who determined that polyethylene glycol (PEG) is a good, non-toxic and non-pyrogenic precipitant. Therefore, it is nowadays widely used in the production of gamma globulin and coagulation factor concentrates. Further details of the Cohn procedure remained unchanged.

Also significant was the invention of Gabriel et al., Who found that the addition of caprylates protects albumin from denaturation during heat treatment.

2415 079 proved to be an important step in improving economy. The procedure described in the disclosure document of the Federal Republic of Germany, which consists of four main stages:

1. Isolating plasma from solid and coagulable soluble constituents of the blood and, if desired, recovering the soluble non-albumin constituents;

2. Fractionation of plasma by heating, whereby globulins are precipitated while albumin remains in solution. The operation is carried out by treating the globulins at a temperature between 60 and 75 ° C in the presence of albumin stabilizers, for example sodium caprylate, with 7-12% by volume of alcohol (s) CH ₃ - (CH ₃ - OH), wherein n = 0, 1 or 2 is treated;

3. Separation of the heat-precipitated globulins by centrifugation as described above;

4. Enrichment of albumin from the albumin-containing solution.

The process succeeded in eliminating the use of expensive refrigeration equipment, reducing the processing time to 4 days to 4 days, reducing equipment demand and improving working conditions by eliminating cold chambers, while maintaining product quality.

However, laborious, expensive equipment and very noisy centrifugation have still not been eliminated.

Several attempts have been made to replace centrifugation and several suggestions have been made.

Various methods have been investigated, such as clarification filtration on carbon and asbestos disk filters, filtration on cellulose-based filter layers, fiberglass filters, and sintered glass filters. However, because either these filtration methods were either too long in filtration, or the filter cartridges were clogged too quickly, or the filtered fluid was too cloudy, the only effective method for separating globulin albumin remained the expensive and uncomfortably noisy centrifugation, the view that globulins from the Cohn process cannot be filtered.

Recently, ultrafiltration on membrane filters has been proposed for the purposes of the Cohn process, similar to the so-called reverse osmosis process successfully introduced in seawater desalination, for smaller power plants.

Such a procedure is recommended in U.S. Patent No. 2,005,260. Also in the disclosure document in the Federal Republic of Germany. The object of this method is to concentrate a solution of a macromolecular substance or to separate the macromolecular substance by removing the solvent and any salts dissolved therein through a membrane. For example, according to Example 1 of said disclosure, bovine serum albumin is recovered from aqueous bovine serum albumin solution by removing water and dissolved salts.

However, this method cannot separate the precipitated proteins from the albumin-containing solution. If the suspension of the present invention were to be processed by this method, a mixture of the desired albumin and unwanted proteins (e.g., gamma globulins) would be obtained, which would be contrary both to the purpose of the process of the invention and to the . purposes. However, ultrafiltration would in principle be well suited for concentrating or enriching the albumin solution already separated by the process of the present invention, although this task is still too expensive on an industrial scale today. In principle, ultrafiltration could also be used to solve the problem of the present invention, provided that pore size cheap membranes could be developed which would allow both the solvent and albumin molecules to pass through, but not the proteins to be separated. However, such membranes have not yet been developed and are not described in this specification. In addition, the process would be expensive for this purpose and would only be applicable on a smaller scale.

Finally, separation of the serum proteins with pairs of solvents, carried out in many stages and in countercurrent, stepped, distributed centrifuges, has also been recommended. Such a procedure is described by Tavel and Bolliger in Helvetica Chimica Acta Volume 51, pp. 278-293, 1968.

The process involves treating a material with a lower and higher specific gravity liquid phase, where the two liquid phases are composed of two solvents of different solubilities while accelerating the separation of the layers by centrifugation.

By this method attempts have been made to separate proteins of different molecular sizes.

However, according to the inventors themselves, there are serious difficulties in applying the procedure. First, it is difficult to find solvent pairs that dissolve protein compounds satisfactorily without denaturing, and secondly, the process can only be applied when large differences in molecular weight or size are present, since molecules of similar size are in the same phase, the method is very difficult, if at all possible. But albumin is pretty uniform. In addition, the recommended centrifuge is much more expensive and much less efficient than the Cohn process and its improved processes, making the recommended process completely unsuitable for industrial scale.

Therefore, in the development of the present invention, U.S. Patent No. 2,415,079, which represents a significant improvement on the original Cohn process. It is an object of the present invention to further improve the process disclosed in the disclosure document of the Federal Republic of Germany for the isolation of albumin from human blood and the like, wherein the task is to develop a cheaper, more productive and health-safe method of albumin separation.

The isolation of the globulins precipitated by heat treatment at 68 ° C (as used in the literature: heat-precipitated) and any other precipitated proteins are disclosed in U.S. Patent No. 2,415,079. According to the procedure disclosed in the German Federal Publication, continuous flow centrifugation at pH 4.4. The precipitated proteins accumulate in the rotors while the albumin remains in the liquid above them.

This method, which is commonly used in other methods as well, and is known in the art, is not perfect for two reasons. First, not because it is relatively large, approx. 20% of the albumin remains in the separated concentrate, so several valuable washing and centrifugation operations are required to obtain the valuable albumin residue. Second, not because centrifugation, as we have already pointed out, is very time consuming and labor intensive in its own right, as well as making a lot of noise. However, as noted above, despite these drawbacks, the use of a different working method was not considered, since it is customary and necessary for the Cohn method to separate the suspension of precipitated globulins and dissolved albumin by centrifugation, and the experiments performed have also shown that suspensions according to the Cohn method are practically impossible to filter under normal conditions.

The negative results obtained by filtration, namely that the precipitated proteins obtained by alcoholic fractionation cannot be removed or recovered by filtration, are obviously due to the particular consistency of the materials.

Surprisingly, on the one hand, we have found that globulins obtained by alcoholic heat precipitation, i.e. alcohol precipitation at a higher temperature (60-75 ° C), are significantly different from cold precipitates.

On the other hand, contrary to experience and expectation, it has been found that the problem can be solved efficiently and with significantly less expense by separating the higher temperature, if present, precipitated proteins from the slurry according to the invention instead of centrifugation by auxiliary filtration.

The auxiliary filtration is carried out in well known, generally centrifugal purification disk filters. They consist essentially of a closed pressure vessel which is arranged horizontally or vertically on a central hollow shaft capable of being driven by a motor, and in parallel with round filter elements. The filter elements are generally provided with braided metal cloth on one side, in the case of horizontal discs.

The suspension to be filtered is mixed with the filtration auxiliaries, usually silica, which are added depending on the turbidity of the residues.

The wet sludge consisting of the filter aid and the solid to be filtered is located on the filter elements.

The clear filtrate travels radially in the direction of the shaft and passes through the collecting duct without much resistance.

The densely digested sludge that accumulates on the filter elements is sludged by rotation of the filter elements and rinsing in the opposite direction upon completion of filtration. The residue is removed as a "slurry".

In our studies, it has been found that the auxiliary filtration according to the present invention has surprising results, because this method alone produces a clear filtrate without noise and with little effort but with high productivity, which does not require additional clarification filtration and recovery.

It is particularly advantageous to carry out the filtration in a centrifugal-cleaned disk filter apparatus with a closed pressure vessel, the filter elements having a mesh size of between 20 and 200 μ.

In the case of the suspensions to be filtered, it is particularly advantageous to work with filter elements having a mesh size of 70-90 μ. At this aperture, there is an optimum between the available throughput and the total filtration and only one operation is required at a time.

In order to prepare the filter surface for the next filtration step satisfactorily, in some cases it is preferable first to first slurry it with a neutral liquid by applying a 0.5 cm thick filter aid to the filter element. Diatomaceous earth, commercially available as Hyflo-Super-Target, Celite 545 or Perlite, is usually a suitable filter aid. In addition, known cellulose filtration aids are suitable, but their filtration performance is less good. However, for many purposes, they can provide a product of satisfactory quality. It is recommended to pre-swell the filter aids.

An advantageous filtering effect is obtained by using a horizontally arranged 80 µ mesh braided metal cloth filter element.

It is favorable if approx. Plasma protein of 4-6% is suspended and this suspension is ca. Mix 20-70 g of silica with a filter aid and filter the slurry. Smaller concentrations of protein may require less amount of filter aid.

In order to recover the albumin present in the solution bound in the filtration aid, a preferred embodiment of the process of the present invention also separates it from a small portion of the total filter surface, after rotating and reverse washing or recycle cleaning of the filtration apparatus. We cleaned. In order to increase the yield, a pressure of 4 ± 2.5 bar can be used without reducing the filtration capacity.

The process of the present invention will be described in more detail in the following examples. In the two examples, we start from two different suspensions.

In Example 1, we start from a suspension of a

No. 415,079 It was prepared using the disclosure procedure in the Federal Republic of Germany.

Example 1

Solids, blood cells and platelets are isolated from human blood and clotting factors are extracted. The stock solution is ca. Contains 5-6% plasma protein. No. 2415079; According to the procedure described in the disclosure document of the Federal Republic of Germany, Annex VIII. clotting factor and fibrinogen are removed by cold precipitation with ethyl alcohol. The prothrombin complex is isolated by absorption. Original plasma is HBAg negative, has normal transaminase, and does not contain visible hemoglobin. Sodium caprylate is added to the original plasma to a concentration of 0.004. The approx. 9% ethyl alcohol mixture

Heat at pH 6.5. The pH is adjusted to 0.5 with hydrochloric acid. The temperature is approx. Raise to 68 ° C over 3 hours with uniform heat transfer. The liquid was then cooled to 10 ° C. The slurry was treated in an outer jacket with a centrifugal-cleaned disk filter cooled at 10 ° C. The further processed solution contains about 2 to 2.5% albumin.

In the drawing, the processing of the stock solution is illustrated in principle. The central element of the whole apparatus is the double-walled filter tank 20, in which heating and cooling can be performed.

The tank capacity is approx. 220 liters and a filter surface of approx.

m ² . The central rotating cavity 21 of the round filter elements 22 (filter discs)? they are connected to the shaft in such a way that the filter surface and the filter element covering each filter element

The filtrate passing through excipient -3181926 flows into the middle column from where it is aspirated and fed through the conduit 23 and valve 8 into the filtrate container.

The filter elements are coated with a stretch metal fabric. The filter elements essentially form a hollow storage space connected to the hollow shaft 21. The filter aid and the precipitated globulins are removed from the filter elements by centrifugal separation between each filtration cycle; After rinsing with 1% sodium chloride solution, the filters are again prepared for the next cycle.

The unfiltered starting fluid is suspended in the mud tank 24 with constant agitation (agitator 3). The starting material is fed via the pump 2 via the valve 10 via two inlet valves 11 and 12 into the filter container 20. The filter tank may be pressurized with air drawn through valve 5.

The filter elements are pressurized at atmospheric pressure with 500 liters of distilled water and 1 kp of Celite 545 commercially available silica with a first (primary) slurry. Apply a 0.2 cm thick filter aid filter. Then, 500 liters of the albumin-containing suspension was mixed with 25 kp of Celite 545. The mixture is fed from the mud tank 24 to the filter tank and pressed at a pressure of 2 bar over the filter surfaces of the elements 22. Prior to emptying the filtered liquid into the filtrate container for storage, the so-called circular process (6 return valves, 2 pumps) is repeatedly passed through the filter elements until the filtrate through the sight glass 25 appears to be sufficiently pure. The filtrate is then passed through valve 8 into the filtrate tank.

As soon as the purified albumin-containing solution leaves the filter, a 1% aqueous solution of saline is simultaneously pumped through the filter. The volume of this solution is about half the volume of the starting material.

The flow rate is about 150 liters per hour. The protein concentration of the filtrate at the beginning of the process is about. 2.5%, while the final concentration is ca. 0.05%.

The resulting solution is clear with a protein concentration of ca. 1.5% (albumin) with an osmolality of ca. 1000 cpm.

The clear filtrate is then dialyzed on a Millipore Pellicon cassette system (filter surface approx. 0.9 m ² , manufactured by Millipore GmbH, Neu-Isenburg, Germany) until 25% osmolality of protein between 100 and 200 μm is achieved. solution is not obtained. The flow rate at the beginning of the process is approximately 1 minute per minute. 5-6 liters of 45 filtrates.

The filter is cleaned by means of the drive motor 1 by rotation of the filter elements and removal of the slurry through the discharge valve 9. Through the valve 4, the effluent can be re-slurried and filtered if desired

Even after any filtration, the liquid shows no Tyndall effect and is clear. It is virtually free of ancillary substances such as lipids or foreign proteins. The liquid can be transported directly for albumin enrichment and the subsequent filtration step described. Enrichment of albumin is known per se and is disclosed, for example, in German Patent Publication No. 2415 079.

In principle, and thus in total, the following steps are carried out Plasma, 600 kg contains: 33.6 kg protein, of which 18.48 kg (= 55%) albu as

1. + 9% Ethyl Alcohol, + 0.004 M Sodium Caprylate, + Hydrochloric Acid Solution (to adjust pH to 6.5)

2. Heat loss: 30 minutes, 68 ° C,

3. + Hydrochloric acid solution, pH 4.4

Auxiliary Filtration 4 Filtrate

5. dialysis filtration (concentration and dialysis),

6. + Sodium hydroxide solution, pH 7.0

7. Pure filtration

Albumin solution: Contains 17.2 kg of albumin (efficiency: 93%).

Example 2

Placental extract is first used to remove the hemoglobin present with solvents such as trichloroacetic acid, chloroform, diethyl ether. Thereafter, the proteinaceous residue was heated to 68 ° C at pH 6.5. After cooling, the pH is adjusted to 4.4 with 0.5 N hydrochloric acid, and 100 liters of the resulting liquid are mixed with 3 kp of filter aid20 (silica filter aid, marketed as Hyflo-Super-Target).

The liquid is pressed at 18 ° C and 4 bar through the 70 µ mesh auxiliary filter described above. Thus, without first (slurry) slurry, we steam. The liquid is recirculated until it is clear from the viewing window and free from the Tyndall effect. The albumin in the liquid is then concentrated to the desired concentration by a method known per se.

After passing through all the volumes of liquid, the solution in the excipient filter, fixed on the filter aid, is washed with distilled water or 0.9% sodium chloride solution. The resulting liquid is also clear and still contains about 0.5-20% albumin. This can also be implicitly introduced, for example, in U.S. Patent No. 2,415,079. Albumin enrichment procedure described in the disclosure document of the Federal Republic of Germany.

It should be emphasized that the auxiliary filtration can be performed with or without first slurry.

The filter elements are cleaned by rotation and reverse washing and rinsing, whereby the filter elements are rotated and the residue is removed as slurry.

Using a sufficient amount of washing liquid (distilled water or sodium chloride solution) yields up to 96% of the introduced albumin. In other separating processes, such as centrifugation techniques, particularly elution of first-separated proteins, residues resulting from changes in solvent / solid specific gravity and very fine mechanical distribution can be avoided by filtration of the cleaned. Filtration prior to further processing of the diluted albumin solution may be delayed due to the high purity achieved.

The separation of solids is independent of temperature. The filtration rate changes only slightly with decreasing or increasing temperature and is negligible. In a conventional manner of carrying out the process of the present invention, the heat-treated plasma, acidified to pH 4.4, is isolated at room temperature in a centrifugal purification disk-cooled disk filter apparatus. The filtration may also be carried out at other temperatures. Experience has shown that we can operate in a temperature range of 4 to 40 ° C. The filtration pressure may be raised to 4 ± 2.5 bar after the first slurry, which is almost completely free of pressure65. That's it

10 available filtering capacities approx. 150 liters of filtrate / m ² / hour. The diatomaceous earth mentioned in the introduction proved to be suitable as a filter aid.

The amounts mentioned in the examples may vary according to the capacity of the filter. Successful experiments were carried out on small - approx. 10 liters - and large - approx. Also in filter tanks with a capacity of 500 liters.

The quality of the products obtained has been found to be perfect in every respect.

The centrifugal cleaning disk filter used is much cheaper than the centrifuges commonly used.

The process according to the invention reduces the number of filtration operations.

The processing of a 600 liter batch reduces the labor requirement to 1 instead of the previous 4.

Noise stops.

Thus, according to the invention, albumin can be produced in a cheaper, more productive and labor-safe manner.

Claims (4)

Patent claims A process for isolating albumin and separating precipitated proteins from protein-containing suspensions of 25 plasma treated with alcohol at 60-75 ° C, characterized in that the suspension containing 4-6% plasma protein is in a centrifuge with closed pressurized centrifuge, 20 to 5200 μ, preferably 70 to 90 μ, of mesh filter fabric, preferably metal fabric on the filter element (s) using a filter aid at a pressure of 4 ± 2.5 bar, optionally removing the filter aid from the filter element (s) and In this case, the pure albumin solution is dialyzed in a known manner. 2. A process according to claim 1 wherein the suspension is mixed with 30-70 g / l of filter aid. 15 3. A process according to claim 1, characterized in that it is filtered with a pre-expanded filtration aid. 4. A method according to any one of claims 1 to 4, characterized in that the filter aid and the slurry therein are removed by rotation and reverse rinsing or continuous washing of the filter elements, and then the solution fixed on the filter aid is drained through a residual volume filter device. with the main filtrate.