JPH06279296A - Removal of associated protein from plasma fraction preparation - Google Patents

Abstract

PURPOSE:To remove associated proteins from plasma fraction preparation where as it was impossible until now, this method enables secured and rapid removal while keeping a high recovery of proteins by a relatively simple method. CONSTITUTION:Associated proteins contained in a preparation are removed by filtering an immunoglobulin preparation for intravenous injection and a fractionated albumin preparation through a porous high polymer membrane having 10 to 30nm average pore diameter.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for removing protein aggregates from a plasma fractionation preparation. More specifically, it relates to a method for separating an aggregate of proteins mainly from intravenous immunoglobulin preparations and fractionated albumin preparations separately from useful proteins.

[0002]

2. Description of the Related Art In a protein solution, there are non-covalently associated associations of a plurality of protein molecules. This aggregate is generated by irreversible denaturation of the protein and generally cannot be easily dissociated. Further, the aggregates tend to aggregate with each other to form larger aggregates, which may result in cloudiness or precipitation in the solution.

Immunoglobulins, which are responsible for the immunity of the living body, have proteins, bacteria, viruses, etc. as their cores and bind to their periphery to form huge protein aggregates called antigen-antibody complexes.

[0004] Plasma or plasma fraction preparations containing protein as a main component, or protein-associated products present in cell culture medicines are considered to be one of the causes of side effects that appear in the human body when the preparations are intravenously injected into the human body. .

For example, in the case of immunoglobulin for intravenous injection, it is said that shock-like reactions such as cyanosis and hypotension, respiratory tract symptoms such as dyspnea, and skin rash are caused by immunoglobulin aggregates.

Therefore, many methods for removing these aggregates from the solution have been devised. For example,
Chromatographic methods using porous carrier gels, chemical treatment methods, addition of adsorbents, and the like.

However, in the case of the chromatography method, although a considerable effect of removing aggregates can be obtained, it is impossible to process a large amount of solution, and the time required for the work becomes long.

[0008] Further, although the chemical treatment method allows large-scale treatment, it is necessary to remove the chemicals used for the treatment from the solution, and further, the treatment is likely to cause inactivation or denaturation of the useful protein. Will reduce the recovery rate of.

The method of adding the adsorbent cannot be said to have a high efficiency of removing the associated substances, and further, the work of removing the adsorbent is required as in the chemical treatment.

Furthermore, a method for removing aggregates of globulin by filtering with an ultrafiltration membrane formed of polysulfone resin (Japanese Patent Publication No. 62-3815) has been devised.
The recovery rate of the protein after carrying out this method is extremely low at around 40%, and the filtration rate and the filtration capacity are both low, so it cannot be said that the industrial effectiveness is high.

It is also said that the globulin-associated product can be removed by using a membrane for separating plasma from blood (Japanese Patent Laid-Open No. 61-69732). It is considered that it is not due to the size relationship of the particles, but rather due to the adsorption of the association product on the surface of the film by the interaction force between the film material and the association product. PH and ionic strength)
In many cases, the expected removal effect cannot be obtained with a slight change in conditions.

In addition, in these conventional methods, there is a great risk that microorganisms such as bacteria and viruses are mixed into the solution from the outside during the work. This is particularly important in the case of pharmaceuticals, so for protein solutions that have been treated to remove aggregates,
Work is required to inactivate or remove these microorganisms.

[0013]

DISCLOSURE OF THE INVENTION The present invention is a method for removing protein aggregates from a plasma fractionated preparation, which cannot be achieved by conventional techniques, reliably, quickly and relatively easily while maintaining a high protein recovery rate. Provide a way to do it by operation. The high protein recovery rate referred to here is 70% or more, preferably 80%.
It means that it is above.

[0014]

According to the present invention, an aqueous solution of immunoglobulin for intravenous injection or serum albumin, which is a plasma fractionation preparation, is filtered through a proteinaceous polymer membrane and recovered as a solution containing no protein aggregates. Is the way.

The present invention is carried out using a porous polymer membrane having an average pore size of 10 to 30 nm.

The average pore size of the membrane is of particular importance. Since the exact size of the aggregates to be removed is still unknown, it is not clear whether there is a suitable average pore size for removal.
However, as a result of examining the removal performance of aggregates by a series of porous polymer membranes having different average pore diameters, the present inventors have found that aggregates are distributed within a substantially constant region of 20 to 30 nm. The present invention has been accomplished by discovering what is being done. Therefore, when the average pore size is too large, exceeding 30 nm, the protein-associated product permeates the membrane and the purpose cannot be achieved. On the other hand, if the average pore size is too small, such as 10 nm or less, even a single molecule of the protein cannot pass through the membrane, so a membrane having such an average pore size cannot be used.

The porosity of the film means a state in which innumerable holes penetrating from one surface of the film to the other surface are opened on the film surface. In this case, the hole does not necessarily have to penetrate the membrane as a straight tube, but may be bent inside the membrane. Also, some holes may be fused inside the membrane, or conversely, one hole may be branched and these may be mixed.

Further, the average pore diameter is expressed as a diameter obtained by approximating the average tube diameter of the pores penetrating the membrane as the entire membrane as a straight section of the pore as a circle. Such average pore size can be calculated indirectly by measuring the amount of liquid permeation through the membrane.

In the present invention, various shapes such as a hollow fiber shape, a flat membrane shape, and a tube shape can be used as the polymer membrane, but the hollow fiber shape having a large filtration effective membrane area in comparison with the volume is effective. Is.

Further, in the present invention, the material of the polymer film used is not particularly limited and may be either an organic polymer or an inorganic polymer. However,
In order to increase the recovery rate of useful proteins, it is desirable to exclude materials that tend to adsorb proteins and materials that denature proteins by contact. In that sense, the copper-ammonia method regenerated cellulose excellent in protein permeability and non-denaturing property is a preferable material.

Among the membranes made of the regenerated cellulose,
Particularly, a virus-removable copper-ammonia method regenerated cellulose membrane is more preferable. By filtering with this membrane, not only protein-associated substances but also microorganisms such as viruses can be efficiently removed.

When a protein solution is filtered using such a polymer membrane, the proteins in the solution that associate into large particles cannot pass through the pores of the polymer membrane, and The solution of the membrane is trapped on the surface on the inflow side or in the pores in the polymer membrane. On the other hand, proteins that are not associated with each other can pass through the pores of the polymer membrane and are thus recovered in the filtered solution. Thus, the protein aggregate is removed from the protein solution.

[0023]

【Example】

(Example 1) A hollow fiber membrane having an effective membrane area of 3.5 cm ^{2 made} of regenerated cellulose having a copper-ammonia method and having an average pore diameter of 15 nm obtained from the amount of permeated water passing through the membrane was prepared. The film is formed by JP-A 61-254202 and JP-A 61-254202.
-274707.

On the other hand, 0.1% of γ-globulin dry powder derived from bovine plasma was dissolved in 20 mM sodium phosphate buffer (pH 7.4) containing 0.9% sodium chloride to prepare a protein solution. .

4 ml of this protein solution was filtered through the above hollow fiber membrane by applying an air pressure of 0.27 kgf / m ² to the protein solution. The time required for filtration is 170
It was a minute.

The protein solution filtered in this way was analyzed by high performance liquid chromatography to determine the recovery rate of the associated product at each association number. As shown in Table 1, it is composed of 3 or more molecules of γ-globulin. Aggregates had been removed. There was no change in the amount of protein single molecule.

[0027]

[Table 1] (Example 2 and Comparative Example 1) Each having an average pore diameter of 15 n
A hollow fiber membrane having an effective membrane area of 0.7 cm ^{2 and made} of cuprammonium regenerated cellulose having m, 35 nm, 40 nm and 75 nm was prepared in the same manner as in Example 1.

Next, a solution of human-derived serum albumin dissolved in physiological saline at 1% was prepared, and 0.15 ml of each was filtered through the above membrane. The air pressure during filtration was 0.27 kgf / cm ² .

The protein contained in the obtained filtrate was analyzed by polyacrylamide gel electrophoresis and the results are shown in FIG.
a is an albumin solution before filtration, and b to e are analyzes of solutions after filtration through membranes having pore sizes of 75 nm, 40 nm, 35 nm, and 15 nm in order.

Of the three bands shown in FIG. 1, 3 are at least 3 molecules, and 2 are associated molecules consisting of 2 molecules of albumin.
Corresponds to one molecule of albumin. Average pore size is 15nm
It is clear that the association product of 3 or more molecules can be completely removed by performing filtration through the membrane. At this time, there is no change in the amount of albumin that is not associated. On the other hand, even if filtration was carried out using a membrane having pores having an average pore diameter of 35 nm, the association product of 3 molecules or more could not be removed at all. (Comparative Example 2) The same operation as in Example 1 was performed using a membrane having an average pore size of 8 nm.

At this time, as shown in Table 2, the recovery rate of the γ-globulin single molecule was about 55%.

[0032]

[Table 2]

[0033]

EFFECTS OF THE INVENTION By carrying out the present invention, it becomes possible to remove the protein-associated product from the plasma fractionated preparation.

Since the present invention only filters the protein solution through the polymer membrane, it can be carried out as an extremely simple operation as compared with the conventional method. Further, in the present invention, since the treatment can be performed without changing the composition of the solution except that the protein-associated product is removed, it is not necessary to additionally perform an operation such as removal of an additive chemical. In addition, since no physical or chemical action is applied to the protein, the useful protein is not deactivated.

Furthermore, according to the present invention, if the polymer membrane and its accompanying equipment (membrane supporting structure, piping for transferring the solution, etc.) and the container for collecting the solution are kept sterilized, The fine pores of the applied polymer membrane are removed by the polymer membrane, not only fungi but also smaller viruses (the smallest virus currently known has a particle size of about 20 nm). Therefore, it is not necessary to perform operations such as inactivation and removal of these microorganisms on the recovered solution.

[Brief description of drawings]

FIG. 1 is a chart showing the results of analyzing the proteins contained in the filtrates obtained in Example 2 and Comparative Example 1 by polyacrylamide gel electrophoresis.

Claims (6)

[Claims] 1. A plasma fractionation product having an average pore size of 10 to 30 n.
A method for removing a protein-associated product from a plasma fractionation preparation, which comprises filtering with a porous polymer membrane of m. 2. The plasma fraction preparation is an immunoglobulin preparation for intravenous injection, and the porous polymer membrane has an average pore diameter of 10 to 10.
The method for removing an immunoglobulin-associated product according to claim 1, which has a thickness of 20 nm. 3. The method for removing albumin aggregates according to claim 1, wherein the plasma fractionation preparation is a fractionated albumin preparation, and the porous polymer membrane has an average pore diameter of 10 to 20 nm. 4. The method for removing a protein-associated product according to claim 1, wherein the porous polymer film is a virus-removable polymer film made of a copper-ammonia method regenerated cellulose. 5. A protein preparation obtained by cell culture, which is filtered through a porous polymer membrane having an average pore size of 10 to 30 nm to obtain a protein-associated product from the protein preparation obtained by cell culture. Removal method. 6. The method for removing protein-associated product according to claim 5, wherein the porous polymer film is a virus-removable polymer film made of regenerated cellulose by the cuprammonium method.