JP2000005569A - Series multistage filtration method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means for improving the rejection rate of a material such as virus to be removed without lowering the permeability of the material such as useful protein to be recovered. SOLUTION: A material to be removed is filtered off from a soln. contg. a material to be removed and a material to be recovered. In this case, a filter having >=2 rejection rate of the material to be removed per stage and >=90% permeability of the material to be recovered is plurally used in series to conduct multistage filtration. The filter is a porous hollow fiber made of the regenerated fiber of the cuprammonium process, and the material to be removed is virus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for efficiently removing a substance to be removed from a solution, and more specifically to a method in which a virus such as plasma, a plasma fraction preparation, a cell culture medium, or a biopharmaceutical is contaminated. The present invention relates to a filtration method for efficiently removing a virus from a potential solution.

[0002]

BACKGROUND OF THE INVENTION Multistage filtration methods have long been known in the field of filtration filters as an efficient means of filtration. For example, Japanese Patent Application Laid-Open No. 2-167232 discloses a multistage filtration method in which a blood coagulation factor VIII preparation is produced using a porous membrane hollow fiber made of cellulose regenerated by a copper ammonia method. In this multi-stage filtration method, a method for removing a virus from a blood coagulation factor VIII product is disclosed, and the arrangement is such that the average pore size of the hollow fiber membrane used in the first stage filter is not smaller than that of the filter used in the second stage. It is characterized by doing. In this method, large substances clogged by the main filter used in the subsequent stage are removed by the pre-filter used in the previous stage, so that the original performance of the main filter used in the subsequent stage (high transmittance of useful proteins and virus High removal rate).

Further, in general, for the purpose of improving performance, means for arranging main filters themselves in multiple stages has been adopted. However, there has not been established any means for simultaneously satisfying the trade-off performance at a high level of suppressing the decrease in the transmittance of useful proteins while enjoying the effect of improving the virus removal performance by multi-stage filtration.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide means for improving the removal rate of a substance to be removed such as a virus without reducing the transmittance of a useful protein.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems of the prior art, and as a result, the removal rate of the virus to be removed by one-stage filtration is 2 or more, and Means in which multi-stage filtration is performed in series using a plurality of filters having a transmittance of a useful protein of a substance of 90% or more as a substance, whereby a virus removal rate can be additionally increased and a decrease in the transmittance of a useful protein can be suppressed. And completed the present invention.

That is, according to the present invention, when filtering and separating a substance to be removed from a solution containing the substance to be removed and the substance to be recovered, the removal rate of the substance to be removed by one-stage filtration is 2 or more, and A filtration / separation method in which a plurality of filters having a substance transmittance of 90% or more are filtered in series using a plurality of filters. The filter is a porous hollow fiber made of regenerated cellulose using a copper-ammonia method. Is a virus / filtration / separation method.

[0007] During the production process of a plasma fraction preparation in which one-stage filtration is carried out using a filter having a certain degree of effectiveness with respect to the removal of the target virus, a similar or even higher removal performance is attempted to increase the removal rate. If a multi-stage filtration is carried out by adding a filter having a high molecular weight, generally the overall removal rate can be improved, but the permeability of useful proteins to be permeated and recovered is reduced. On the other hand, if a membrane having a larger average pore size is used to maintain the permeability of the useful protein, the permeability of the useful protein is improved, but the removal rate of the target virus is additionally improved even in multi-stage filtration. Without
In some cases, a sufficient effect cannot be obtained. The present invention selects a filter having a specific removal rate for a substance to be removed and a specific transmittance for a substance to be removed between the two trade-off characteristics as described above, and using the filter in multiple stages, thereby making it additive. It has been found that it is possible to improve the virus removal performance and to suppress a decrease in useful protein permeation performance.

The removal rate referred to in the present invention is represented by -log (concentration of the substance to be removed in the filtrate / concentration of the substance to be removed in the original solution). In the present invention, the solution to be filtered includes plasma, serum, a solution of an intermediate purified product in the production process of a plasma fraction preparation, a solution of an intermediate purified product in the production process of a biopharmaceutical, etc., in which case, the substance to be removed is HIV. , HCV, Polio, Parvo
And the target substances to be collected include blood coagulation factor VIII, factor IX, thrombin, a thrombin / antithrombin complex, fibrinogen, immunoglobulin, albumin and the like.

As a filtration method, there are a dead-end method in which the original liquid is injected into a closed primary space, and a tangential method in which a part of the original liquid is filtered while circulating the original liquid. The properties of the original liquid, such as the concentration, are determined as appropriate from both aspects of maintaining the characteristics of the object to be recovered and exhibiting certain filtration characteristics. The filter use conditions such as filtration pressure, filtration volume (filtration amount per unit membrane area), and original liquid circulation rate (in the case of tangential filtration) are defined as the removal level of the substance to be removed, the purity of the substance to be removed, and the time required for filtration. It is determined comprehensively from the viewpoints of the objectives such as and the like, the performance of the filter, and the effects of those conditions on maintaining the characteristics of the target substance.

In the present invention, since a plurality of filters are arranged in series, it is preferable to employ a dead-end system. According to the present invention, a filtration method for efficiently removing a substance to be removed from a solution is provided. When the substance to be removed is, for example, a virus, the removal rate is usually 4 or more, preferably 5 or more. In the case of the two-stage filtration method, the removal rate of the substance to be removed by the one-stage filtration method is desirably 2.5 or more in order to realize this. Similarly, in order to maintain the total transmittance of the substance to be recovered at 90% or more, the transmittance of the substance to be recovered by the single-stage filtration method is desirably 95% or more.

The filter in the present invention is preferably a hollow fiber membrane made of cellulose regenerated by a copper-ammonia method, a flat membrane and a hollow fiber membrane of polyvinylidene fluoride, a flat membrane and a hollow fiber membrane of polyacrylonitrile, etc. for the purpose of removing viruses. Is raised. In particular, the hollow fiber membrane made of regenerated cellulose using the cuprammonium method is easy to control the pore size of the membrane for efficiently sifting relatively similar viruses and useful proteins, and adsorbing proteins due to the chemical characteristics of the material. Is preferred.

Prior to describing an embodiment according to the present invention,
The methods for measuring various physical properties used in the present specification are shown below. [Measurement of Average Pore Diameter] The outflow amount V of water when water was filtered with a filter was measured, and the average pore diameter (D) was obtained from the equation (1). D = 2.0 (VTμ / PAρr) ^1/2 (1) where, V: water outflow T: film thickness of hollow fiber P: pressure difference A: membrane area ρr: porosity μ: viscosity of water rate

[Measurement of Virus Removal Rate] The effect on virus removal in the present invention was determined by measuring a virus removal rate expressed as a logarithmic reduction rate (LRV) of the virus. The LRV of the virus was determined from the formula (2) by filtering a solution containing the virus (original solution) and measuring the virus concentration in the filtrate. The measurement of the virus concentration is generally performed by an infectivity test using animal cells, a PCR method, or the like. In Examples and Comparative Examples in the specification of the present application, all were measured by an animal cell infectivity test. LRV = -log (virus concentration in filtrate / virus concentration in original solution) (2)

[Measurement of transmittance of protein to be recovered] The transmittance of the protein to be recovered in the present invention is calculated by the formula (3). Transmittance [%] = (protein concentration in filtrate / protein concentration in original solution) × 100 (3) In addition, protein concentration is generally measured by reacting with various coloring reagents and measuring absorbance using a spectrophotometer. The method is performed by a liquid chromatography method or the like. In the examples and comparative examples in the specification of the present application, all were measured by an absorbance measurement method.

[0015]

Next, the present invention will be described more specifically with reference to examples and reference examples.

[0016]

Example 1 An effective membrane area of 0.001 m ² and an average pore diameter of 35
nm filter ("PLANOVA 35N": manufactured by Asahi Kasei Corporation) was used. BVDV with a titer of 10 ⁹ or more
(Bovine Viral Diarrhea Virus) spiked 1%
The bovine gamma globulin solution is used for dead-end
The solution was filtered at a constant pressure of 0.25 kg / cm ² . As shown in FIGS. 1, 2, and 3, the hollow fiber membrane was filtered by connecting a first stage, a second stage, a third stage, and a filter in series. Table 1 shows the results.

[0017]

[Table 1]

As shown in Table 1, using a PLANOVA 35N filter having a BVDV removal rate of 2 or more and a bovine gamma globulin transmittance of 90% or more in one-stage filtration, two stages in series,
By increasing the number of stages to three, the BVDV removal rate becomes 4.
96, 6.36 greatly improved. On the other hand, the bovine gamma globulin transmittance was 88 88 respectively in the two-stage filtration and the three-stage filtration.
% And 86%, both could be maintained at high levels.

[0019]

[Comparative Example 1] Effective pore area of 0.001 m ² and average pore size of 15
nm filter (“PLANOVA 15N”: manufactured by Asahi Kasei Corporation) was used. BVDV with a titer of 10 ⁹ or more
(Bovine Viral Diarrhea Virus) spiked 1%
The bovine gamma globulin solution is used for dead-end
The solution was filtered at a constant pressure of 0.25 kg / cm ² . As shown in FIG. 1, FIG. 2, and FIG.
The filter was connected to the second and third stages and filtered. Table 2 shows the results. W

[0020]

[Table 2]

As shown in Table 2, the BVDV removal rate by one-stage filtration was 2 or more, but the bovine gamma globulin transmittance was 90%.
Even if the number of stages is increased to two or three in series using the following filters, a high BVD exceeding 6.68 in any case is obtained.
V removal rate could be maintained, while bovine gamma globulin permeability was 54% and 51% in two-stage filtration and three-stage filtration, respectively.
%.

[0022]

Comparative Example 2 An average pore diameter of 35 with an effective membrane area of 0.001 m ^2.
nm filter ("PLANOVA 35N": manufactured by Asahi Kasei Corporation) was used. Poli with a titer of 10 ^7.4 or more
o Using a 1% bovine gamma globulin solution spiked with the virus, 0.25 kg /
Filtered at a constant pressure of cm ² . 1, 2 and 3
As shown in Table 1, the filters were connected in series with one, two, and three stages and filtered. Table 3 shows the results.

[0023]

[Table 3]

As shown in Table 3, the filtration rate of bovine gamma globulin is 90% or more by one-stage filtration, but the removal rate of Polio virus is less than 2, using a PLANOVA 35N filter having a filtration rate of 2 or less, and two-stage filtration is performed. Even if the number of steps and the number of steps are increased, Po
The removal rate of lio virus was as low as 0.25 and 0.42.

[0025]

According to the present invention, the removal rate of the substance to be removed by one-stage filtration is 2
As described above, by performing multiple-stage filtration in series using a plurality of filters having a transmittance of the target substance of 90% or more, for example, it is possible to suppress a decrease in the protein transmittance as the target substance and to remove the target substance. According to the present invention, a filtration method for efficiently removing a substance to be removed from a solution can be provided.

[Brief description of the drawings]

FIG. 1 is a conceptual diagram showing a configuration of an apparatus of a one-stage filtration method.

FIG. 2 is a conceptual diagram showing a configuration of an apparatus of a two-stage filtration method.

FIG. 3 is a conceptual diagram showing a configuration of an apparatus of a three-stage filtration method.

Continued on the front page F term (reference) 4C077 AA11 BB02 CC06 KK11 LL05 MM05 MM09 NN03 NN15 PP03 4D006 GA02 KA53 KA55 MA01 MA03 MA22 MB06 MC13 MC29 MC39 PB52 PB55 PC41

Claims (3)

[Claims] When filtering and separating a substance to be removed from a solution containing the substance to be removed and the substance to be recovered, the removal rate of the substance to be removed by one-stage filtration is 2 or more, and the permeation of the substance to be collected is permeated. A method for filtering / separating a substance to be removed, characterized in that a plurality of filters having a ratio of 90% or more are used to perform multistage filtration in series. 2. The method for filtration and separation according to claim 1, wherein the filter is a porous membrane hollow fiber made of cellulose regenerated by a copper ammonia method. 3. The method according to claim 1, wherein the substance to be removed is a virus.