JP2000319294A - Purification of solution of polymer originating from organism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To purify the subject organism-originating polymer that is useful in the fields of medicines and medical treatment in high filtration efficiency, as the contaminants in the polymer solution, for example, viruses or the like are effectively removed by reducing the concentration of DNA or the like in the organism-originating polymer solution to be filtered before the membrane filtration. SOLUTION: After the concentration of DNA and/or RNA in the organism- originating polymer solution to be filtered is reduced, preferably to <=5 ppb, the polymer solution is subjected to the membrane filtration with an ultrafiltration membrane made of cellulose or the like to purify the objective solution. In a preferred embodiment, viruses are removed by this filtration. The concentration of the DNA and/or the RNA is reduced by the membrane filtration. As an organism-originating polymer solution, are cited preferably a γ-globulin solution of 3-8% concentration.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for purifying a biologically derived polymer solution used in the fields of medicine and medical treatment.

[0002]

2. Description of the Related Art In the field of medicine and medical treatment, when impurities are mixed into biological polymers such as proteins, there is a risk of illness such as viral hepatitis (K. Yamaguchi e).
t al. J. Electron. Microsc. 40, 337 (1991)). Accordingly, there is a need for a highly safe biological polymer from which impurities, particularly infectious viruses, are inactivated or removed. As a method of obtaining a biologically-derived polymer substantially containing no impurities, a solution containing the biologically-derived polymer is heated, irradiated with radiation, chromatographed, treated with a solvent and a surfactant (so-called SD) at the production stage. Method), a method using membrane separation, etc. (S. Harada et al. J. Clin. Microb
iol., 22, 908 (1985)).

[0003] However, the above-mentioned method except membrane separation degrades and deactivates the target biological polymer, thereby lowering the recovery rate.
There are problems such as an increase in danger due to denatured products, the difficulty in removing or detoxifying chemicals such as surfactants added for inactivation, and the inability to sufficiently remove impurities. Have.

[0004] On the other hand, the membrane separation method physically relies on the relative size difference between the pores present in the membrane structure and the particles to pass (for example, useful proteins) or the particles to be blocked (for example, viruses). It is based on the so-called size exclusion principle that separates these particles and consequently removes impurities.There is no fear of denaturation or deactivation, and no chemicals are added for inactivation, so removal and harmlessness. Is also unnecessary. For this reason, membrane separation is spreading as a use for purification of a biologically derived polymer solution in the fields of medicine and medical treatment.

However, according to the size exclusion principle, effective removal of impurities as the particles to be removed and efficient membrane permeation and recovery of the biological polymer as the particles to be recovered tend to be a trade-off phenomenon. That is, if the size of the pores of the membrane is set small in order to effectively remove the particles to be removed, the permeation of the particles to be recovered is suppressed, and the filtration efficiency, that is, the filtration rate and the transmittance (sieving coefficient) are reduced. descend. On the other hand, if the size of the pores of the membrane is set large in order to make the permeation and collection of the particles to be collected more efficient, the effect of removing the particles to be removed is sacrificed. In general, these parameters indicating the filtration efficiency deteriorate as the filtration time elapses. The reason is that the pores are blocked by the solute as the filtration is continued. In this respect, the relative relationship between the particle size and the pore size also has an effect. If it is desired to filter the biological polymer as the particles to be recovered in a short time and with high transmittance while maintaining a high effect of removing impurities, the membrane area needs to be increased and the purification cost increases.

As a means for solving these problems, there has been proposed a method for reducing the size of particles to be passed before separation by a membrane, that is, filtration. For example, Japanese Patent Application Laid-Open No. H10-502074 discloses a method of virus-filtering a solution containing at least one kind of polymer, wherein the total salt content of the solution is about 0.2 M to the relevant salt. A method wherein said method is characterized by being in the range of saturation. It is considered that the filtration efficiency is improved by increasing the salt concentration in this manner because the size of the protein particles themselves shrinks and the interaction between the protein particles and / or the protein and the membrane decreases. However, Tokiohei 10-50
In JP 2074, a salt concentration that exhibits the above-mentioned effect is 0.2 M or more, and a range of 0.8 to 1.5 M is particularly preferable, so that a salt concentration that is significantly higher than a physiologically isotonic salt concentration is required. . Therefore, in the case of a drug administered to the human body by intravenous injection or intramuscular injection, an operation of reducing the salt concentration to a physiological isotonic concentration by dialysis or the like after filtration is essential. In addition, when the salt concentration is increased, the solubility of the protein is reduced by salting out, which may have an adverse effect. Therefore, impurities from various biological polymer solutions,
There is a need for a method of separating and removing the membrane safely and with high efficiency, that is, with a high filtration rate and transmittance.

[0007]

In the field of medicine and medical treatment, the membrane filtration method is used as a means for removing impurities such as viruses from a biologically derived polymer solution by filtration. Since there is no activity, the recovery rate is high, and this method is superior to other purification methods. However, it is difficult to achieve both effective removal of impurities and filtration efficiency of the target biological polymer, that is, high filtration rate and transmittance. An object of the present invention is to reduce the content of DNA and / or RNA in a biological polymer solution, while maintaining effective removal of viruses and other impurities mixed in the biological polymer, and And a method for obtaining high filtration efficiency.

[0008]

SUMMARY OF THE INVENTION The present invention provides a biologically-derived polymer obtained by membrane filtration, comprising reducing the concentration of DNA and / or RNA in a biologically-derived polymer solution to be filtered and then performing membrane filtration. This is a solution purification method. The present invention is also a method for purifying a biologically-derived polymer solution in which a target to be removed by membrane filtration is a virus. In the present invention, the concentration of DNA and / or RNA in the biologically-derived polymer solution to be filtered is preferably reduced to 5 ppb or less, and this is preferably subjected to membrane filtration.

[0009] The origin of the biological polymer used in the present invention is irrelevant for the use in the present invention. Thus, any biological macromolecule produced in human, animal, plant, cultured cells using genetic engineering or cell fusion techniques. The biological macromolecules referred to in the present invention are proteins and polypeptides. As an example of the protein referred to in the present invention, blood coagulation
Factor VIII, Factor IX, hemoglobin, fibrinogen, antithrombin III, immunogamma globulin, albumin, interferon, apolipoprotein, and growth hormone. The impurities to be removed by membrane filtration according to the present invention are infectious viruses, bacteria, fungi,
Protozoa is a causative agent of transmissible spongiform encephalopathy.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION A filtration membrane used in the present invention has a mean pore size of 1 μm or less, preferably 100 nm or less, a micro-filtration membrane, an ultra-filtration membrane, a virus removal membrane. And so on. The material of the filtration membrane used in the present invention may be any material as long as it can filter an aqueous solution. Examples include, but are not limited to, cellulose, cellulose acetate, polyvinylidene fluoride, polysulfone, polyvinyl chloride, polyester, polymethyl methacrylate, polyacrylonitrile, nylon, and the like. Among them, those made of cellulose are hydrophilic, and therefore have a high permeation rate of an aqueous solution, little adsorption of proteins, and are suitable and suitable for permeation. As an example of a filtration membrane used for separating impurities such as viruses from a biological polymer solution, Planova manufactured by Asahi Chemical Industry Co., Ltd.
35N and Planova 15N (both are made of regenerated cellulose), Millipore Viresolve 70, Vipresolve 18
0 (all materials are polyvinylidene fluoride), Fluoropore film (material is ethylene tetrafluoride) manufactured by Sumitomo Electric Industries, Ltd., Nuclepore film (material is polycarbonate) manufactured by Corning Costar, and the like.

[0011] The form of the filtration membrane used in the present invention may be any form such as a flat membrane, a hollow fiber membrane, a spiral membrane, and a pleated membrane. In addition, the dead end method and the tangential flow method can be used for the filtration.

The concentration of the biologically-derived polymer solution suitable for the present invention varies depending on the type, and is, for example, 0.05 to 10% in the case of a gamma globulin solution. Preferably it is 3 to 8%. The reason why the concentration is 10% or less is that if the concentration is higher than that, the association between gamma globulin molecules is increased, and it becomes very difficult to perform filtration using the present invention. In general, the higher the concentration of the globulin solution, the higher the possibility of DNA- and / or RNA-mediated association and the more likely the effect of removing DNA and / or RNA to improve permeation efficiency is preferable.

It is desirable that the temperature and pH of the biological polymer solution at the time of carrying out the present invention are within a range where the biological polymer is not denatured. For example, in the case of a gamma globulin solution, it is usually desirable that the temperature of the solution is 2 ° C to 25 ° C and the pH is about 7 to 8.

For the measurement of DNA and / or RNA in a solution, PCR, ultraviolet-visible spectrophotometry, and fluorescence have conventionally been used. In the PCR method, the detection limit is, for example, λ
In the case of phage DNA, it is about 10 ppm (Bio-General Catalog 1997/1998 vol.1 Genetic Engineering, D-17, Takara Shuzo Co., Ltd.). In addition, it is determined to be about 1.0 ppm in an ultraviolet-visible spectrophotometer (A. Higuchi et al. J. Membrane Sci., 1
16,191 (1996)). Further, in a fluorescence measurement method using ethylene bromide (an intercalating agent), it is about 0.1 ppm. However, in the fluorescence measurement method using a newly developed fluorescent probe (BEACON V2165, DNA QUANTITATION KIT, manufactured by Takara Shuzo Co., Ltd.), the detection limit can be reduced to 0.5 ppb or less (R. Bolger et al. Biotech
niques, 23,532 (1997)).

Since the production of a biological polymer is performed by a living organism or a cell, usually, the biological polymer solution before purification contains DNA and / or RNA inevitably. For example, freeze-dried gamma globulin (G-
5009, from cattle) was dissolved in a buffer solution to prepare a 5000 ppm (0.5%) aqueous solution, and the DNA concentration at this time was measured.
It was 8 ± 2 ppb. The DNA concentration in a 5% solution obtained by dissolving frozen gamma globulin (197-7000, bovine) manufactured by Life Technologies was 37 ± 4 ppb.

Known methods for reducing the concentration of DNA and / or RNA in a solution include a restriction enzyme method, affinity chromatography, ion exchange, and removal by membrane filtration. Also, by adding a flocculant,
A method has been reported in which DNA and / or RNA is precipitated and separated by centrifugation. For example, there is a substance such as chitosan that aggregates by electrostatic interaction (Japanese Patent Application Laid-Open No. 63-56300). According to the present invention, if the concentration of DNA and / or RNA in the biological polymer solution is reduced, and then membrane filtration is performed using an appropriately selected membrane, effective impurity removal can be performed at the same time as compared with the conventional method. And a very efficient filtration is realized.

When a biologically-derived polymer solution containing DNA and / or RNA is subjected to membrane filtration, the biologically-derived polymer associates with the DNA and / or RNA to increase the particle size and pass through the membrane pores. Therefore, the filtration efficiency, that is, the filtration speed and the transmittance are reduced. In addition, if filtration is continued, pores on the membrane surface and inside the membrane are closed with the passage of time, and the filtration efficiency is usually further reduced, but this tendency is caused by the enlargement of biological polymer particles due to association. Is accelerated. These phenomena can be eliminated by reducing the concentration of DNA and / or RNA that promotes association and then performing membrane filtration.

The level of removal of DNA and / or RNA from a biologically-derived polymer solution that exhibits the effect of improving filtration efficiency depends on the type of biologically-derived polymer substance, temperature, pH, etc., the properties of the solution, and the type of filtration membrane. Although it varies depending on the concentration, the removal rate with respect to the stock solution concentration is preferably in the range of 10% or more, and more preferably in the range of 20% or more. More preferably 40%
This is the above range. For example, in the case of gamma globulin,
After removing DNA in the range of DNA removal rate of 15 to 50% (DNA concentration after removal is in the range of 20 ppb to 4 ppb), membrane filtration was performed. Improvements were seen. When the DNA concentration was reduced to 5 ppb or less, the filtration efficiency was significantly improved, and the improvement effect was maintained with respect to the lapse of time from DNA removal to impurity removal. Furthermore, the cloudiness phenomenon due to the re-generation of impurities in the solution after the impurities were removed could be suppressed.

[0019]

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

Example 1 Gamma globulin (manufactured by Sigma, G-5009,
(Derived from cattle) was dissolved in a buffer solution of trisaminomethane hydrochloride (5 mM / l) / sodium ethylenediamine-4 acetate (0.5 mM / l), and acetic acid was added to prepare a 5000 ppm gamma globulin aqueous solution at pH 8.0. . This gamma globulin aqueous solution
In 3 ml, add Beacon DNA Quantitation Kit (V2165, PanVer
a Corporation, purchased from Takara Shuzo Co., Ltd.). Excitation wavelength 485nm, fluorescence wavelength
Under the condition of 530 nm (using Spectrofluorometer FP-777 manufactured by JASCO Corporation), the DNA concentration in the aqueous gamma globulin solution was determined by DN
Quantification was performed based on the fluorescence intensity of the fluorescent probe intercalated into A. The DNA concentration at this time was 8 ± 2 ppb.

Next, a fluoropore membrane (FP-100, manufactured by Sumitomo Electric Industries, Ltd.) having a pore size of 1.0 μm and a membrane area of 17.3 cm ² was ultrafiltrated (RP
-1, UHP-43K, manufactured by Advantech Co., Ltd.).
Filtration was performed at 5 ° C. to perform a DNA removal operation. Collect 3 ml of the permeated liquid that has passed through the membrane, and use the same
The NA concentration was quantified. The value at this time was 4.5 ppb.
That is, the DNA removal rate in this case was 43.8%.

After attaching a Nuclepore membrane (111105, manufactured by Corning Costar, a microorganism removing filter) having a pore diameter of 0.1 μm and a membrane area of 17.3 cm ² to an ultrafiltration apparatus (RP-1, UHP-43K, manufactured by Advantech), Above DNA concentration 4.5ppb
Was filtered under the conditions of a pressure of 0.3 atm and 25 ° C. The gamma globulin transmittance after collecting 40 g of the permeate was 84.9 ± 2%. In addition, 40 g of permeate
The permeation rate after collection was 16.3 l / m ² / hr. Here, the transmittance of gamma globulin was defined as follows. Permeability (%) = ((gamma globulin concentration in permeate) /
(Gamma globulin concentration in stock solution)) × 100

[0022]

Comparative Example 1 A 5000 ppm gamma globulin aqueous solution (pH 8.0) was prepared in the same manner as in Example 1. A Nuclepore membrane (111105, Corning Costar, Microbial Removal Filter) having a pore size of 0.1 μm and a membrane area of 17.3 cm ² containing 8 ± 2 ppb DNA was prepared without performing the DNA removal operation performed in Example 1. And filtered in the same manner as in Example 1. The gamma globulin transmittance after collecting 40 g of the permeate was 60.0 ± 5%. This value was significantly lower than the transmittance of gamma globulin after collecting 40 g of the permeate in Example 1. The permeation rate is
After collecting 40 g, the flow rate was 11.6 liter / m ² / hr. This value is
This is about 70% of the transmission speed of the first embodiment.

[0023]

Examples 2 to 5 A 5000 ppm gamma globulin aqueous solution (pH 8.0) was prepared in the same manner as in Example 1. The operation of DNA removal was performed under the following conditions. In Example 2, the hole diameter
0.1 μm, 17.3 cm ² film area
5, Corning Costar, microbial removal filter)
Was attached to an ultrafiltration device (RP-1, UHP-43K, manufactured by Advantech), and the aqueous gamma globulin solution prepared above was filtered at a pressure of 0.3 atm and 25 ° C to obtain DNA.
A removal operation was performed. The DNA value at this time was 0.5 ppb. That is, the DNA removal rate in this case was 93.8%.

In Example 3, the pore size was 0.22 μm, and the membrane area was 17.3.
The DNA was removed by filtration twice using a cm ² fluoropore membrane (FP-022, manufactured by Sumitomo Electric Industries, Ltd.) under the ultrafiltration conditions of Example 2. The DNA value at this time was 0.75 ppb.
That is, the DNA removal rate in this case was 90.6%.

In Example 4, the pore diameter was 1.0 μm and the membrane area was 17.3.
Filtration was performed twice under the ultrafiltration conditions of Example 2 using a cm ² fluoropore membrane (FP-100, manufactured by Sumitomo Electric Industries, Ltd.). Furthermore, the hole diameter
The DNA was removed once using a Fluoropore membrane (FP-022, manufactured by Sumitomo Electric Industries, Ltd.) having a membrane area of 17.3 cm ² and a membrane area of 17.3 cm ² under the ultrafiltration conditions of Example 2. The DNA value at this time is
It was 1.00 ppb. In other words, the DNA removal rate in this case is
87.5%.

In Example 5, the pore diameter was 1.0 μm and the membrane area was 17.3.
The DNA was removed by filtration twice using a fluoropore membrane (FP-100, manufactured by Sumitomo Electric Industries, Ltd.) of ² cm ² under the ultrafiltration conditions of Example 2. The DNA value at this time was 2.50 ppb. That is, the DNA removal rate in this case was 68.8%.

Using the gamma globulin aqueous solutions of Examples 2 to 5 as stock solutions, a Nuclepore membrane (111105, manufactured by Corning Costar Co., Ltd.) having a pore size of 0.1 μm and a membrane area of 17.3 cm ² was prepared.
Ultrafiltration was performed using a microorganism removing filter) in the same manner as in Example 1 under the conditions of a pressure of 0.3 atm and 25 ° C. Table 1 shows the results of measuring the transmittance and the transmission rate of gamma globulin after collecting 40 g of the permeate. In the second embodiment,
The gamma globulin concentrations of the permeate and the stock solution were consistent within the margin of error. The permeation rate in Example 2 was as high as 45% of the permeation rate of 279 l / m ² / hr when a pH 8.0 buffer solution was subjected to ultrafiltration using a membrane having a pore size of 0.1 μm. Was.

[0028]

[Table 1]

[0029]

Comparative Example 2 5 containing 0.5 ppb DNA prepared in Example 2
DNA (Sigma, Cal.
f Thymus, Type I) to a final DNA concentration of 8 ppb
Was prepared. Using this 8 ppb DNA-containing aqueous gamma globulin solution as a stock solution, using a Nuclepore membrane (111105, manufactured by Corning Costar Co., Ltd., a microorganism removing filter) having a pore size of 0.1 μm and a membrane area of 17.3 cm ² , using the same method as in Example 1, and applying a pressure of 0.3 Ultrafiltration was performed under the conditions of atmospheric pressure and 25 ° C. The permeation rate of gamma globulin after collecting 40 g of the permeate was 12.3 l / m ² / hr. This value was obtained in Comparative Example 1.
The transmission speed was almost equivalent to Further, the value was 1/10 of the transmission speed of Example 2. That is, it is clear that the permeation speed is significantly reduced when DNA is contained in the stock solution.

[0030]

Example 6 In the same manner as in Example 5, a 5000 ppm gamma globulin aqueous solution (pH 8.0, DNA concentration 2.5 ppb) from which DNA was partially removed was prepared. Using this gamma globulin aqueous solution as a stock solution, a fluoropore membrane (FP-010, manufactured by Sumitomo Electric Industries, Ltd.) having a pore diameter of 0.1 μm and a membrane area of 17.3 cm ² was prepared in the same manner as in Example 1 at a pressure of 0.3 atm and 25 ° C. Ultrafiltered under. The permeate was collected in 10 g increments, and the permeate No. was numbered sequentially from 1. Gamma globulin permeability and permeation rate of the permeate were measured. Table 2 shows the results.
Shown in The permeation rate when a pH 8.0 buffer was filtered using the above fluoropore membrane was 608 liters / m ² / hr, so the permeation rate shown in Table 2 was the same as when the buffer was used.
High transmittance of 65% or more.

[0031]

[Table 2]

[0032]

Comparative Example 3 In the same manner as in Comparative Example 1, a 5000 ppm gamma globulin aqueous solution (pH 8.0, 8
± 2 ppb DNA). Using this gamma globulin aqueous solution as a stock solution, a fluoropore membrane with a pore diameter of 0.1 μm
Ultrafiltration was performed using FP-010 (manufactured by Sumitomo Electric Industries, Ltd.) in the same manner as in Example 1 under the conditions of a pressure of 0.3 atm and 25 ° C. The permeate was collected in 10 g increments, and the permeate No. was numbered sequentially from 1. Gamma globulin permeability and permeation rate of the permeate were measured. Table 3 shows the results. The permeation rate after collecting 130 g of the permeate shown in Table 3 was measured in Example 6.
Was 1/5 of the permeation rate after collecting 130 g of permeate. Further, the transmittance of gamma globulin was significantly reduced as compared with the transmittance of Example 6.

[0033]

[Table 3]

[0034]

Example 7 5% gamma globulin solution (Life Technol
ogies, 197-7000, from cows)
Thawed. The DNA concentration in this gamma globulin solution was quantified as in Example 1. The DNA concentration at this time was 37
It was ± 4 ppb. Next, a Nuclepore membrane with a pore size of 0.22 μm and a membrane area of 17.3 cm ² (111106, manufactured by Corning Costar)
For ultrafiltration (RP-1, UHP-43K, Advantech)
, And the aqueous gamma globulin solution prepared above was filtered under the conditions of a pressure of 0.3 atm and 25 ° C. to perform a DNA removal operation. Thereafter, the permeate was diluted with physiological saline to a 3% gamma globulin solution. When 3 ml of this solution was collected and the DNA concentration was determined in the same manner as in Example 1,
It was 18.5 ppb. That is, the DNA removal rate in this case was 15.9%.

A fluoropore membrane (FP-010, manufactured by Sumitomo Electric Industries, Ltd.) having a pore diameter of 0.1 μm and a membrane area of 17.3 cm ^{2 was} subjected to an ultrafiltration apparatus (RP-1, UHP).
-43K, manufactured by Advantech Co.)
A gamma globulin solution containing 18.5 ppb A
Filtration was performed at 25 ° C. under atmospheric pressure. The permeate was collected in 10 g increments, and the permeate No. was numbered sequentially from 1. Gamma globulin permeability and permeation rate of the permeate were measured. Table 4 shows the results.

[0036]

[Table 4]

[0037]

Example 8 In the same manner as in Example 7, thawing was carried out in a 5% aqueous solution of gamma globulin. The operation of DNA removal performed in Example 7 was performed several times, and then this gamma globulin solution was diluted with physiological saline to a 3% gamma globulin solution. At this time, the DNA quantification method performed in Example 1 revealed that 4.8 ppb of DNA was contained in the 3% gamma globulin solution. That is, the DNA removal rate in this case was 78.2%. Using this gamma globulin aqueous solution as a stock solution, a fluoropore membrane (FP-010, manufactured by Sumitomo Electric Industries, Ltd.) having a pore size of 0.1 μm and a membrane area of 17.3 cm ² was prepared in the same manner as in Example 1 at a pressure of 0.3 atm and a pressure of 25 ° C. Ultrafiltered under. The permeate was collected in 10 g increments, and the permeate No. was numbered sequentially from 1. Gamma globulin permeability and permeation rate of the permeate were measured. Table 5 shows the results.
Shown in

[0038]

[Table 5]

[0039]

Comparative Example 4 Thawed in a 5% gamma globulin aqueous solution in the same manner as in Example 7. Without performing the DNA removal operation performed in Example 7, the 5% gamma globulin solution was directly diluted to 3% gamma globulin solution with physiological saline. At this time, according to the DNA quantification method performed in Example 1,
It became clear that 22 ± 2 ppb of DNA was contained in the 3% gamma globulin solution. Using this gamma globulin solution as a stock solution, a Fluoropore membrane (FP-010, manufactured by Sumitomo Electric Industries, Ltd.) having a pore size of 0.1 μm and a membrane area of 17.3 cm ² was used to prepare Example 7.
Filtration was performed in the same manner as described above. At this time, the permeate was sampled in 10 g increments, and the permeate No. was numbered sequentially from 1. Gamma globulin permeability and permeation rate of the permeate were measured. Table 6 shows the results. The permeation rate after collecting 40 g of the permeate was 1/6 and 1/10 of the permeation rate after collecting 40 g of the permeate in Examples 7 and 8, respectively. Also,
The transmittance of gamma globulin in Table 6 was reduced as compared to the transmittance of Examples 7 and 8.

[0040]

[Table 6]

[0041]

Example 9 A 3% gamma globulin solution from which DNA was removed (a DNA concentration of 4.96 ppb was prepared. That is, the DNA removal rate in this case was 86.6%. Was used as a stock solution, using a virus removal filter, PLANOVA 35N (manufactured by Asahi Kasei Kogyo Co., Ltd., effective membrane area 0.001 m ² ) in the same manner as in Example 7 at a pressure of 0.3 atm and a dead end method at 25 ° C. 5 g of the permeate was collected, and the permeate No. was numbered sequentially from 1. The transmittance and permeation rate of gamma globulin with respect to this permeate were measured. Shown in

[0042]

[Table 7]

[0043]

Example 10 In the same manner as in Example 7, thawing was carried out in a 5% aqueous gamma globulin solution. Using this gamma globulin aqueous solution as a stock solution, a virus removal filter, PLANOVA 75
Using N (manufactured by Asahi Kasei Kogyo Co., Ltd., effective membrane area 0.001 m ² ), filtration was performed by the dead end method under the conditions of 0.3 atm and 25 ° C. in the same manner as in Example 9. This gamma globulin solution was then diluted with physiological saline to a 3% gamma globulin solution. At this time, D
According to NA assay, DN in 3% gamma globulin solution
A was found to be contained at 19.0 ppb. That is, the DNA removal rate in this case was 13.6%. Using this gamma globulin aqueous solution as a stock solution, using a virus removal filter, PLANOVA 35N (manufactured by Asahi Kasei Kogyo Co., Ltd., effective membrane area 0.001 m ² ) in the same manner as in Example 9 under a pressure of 0.3 atm and 25 ° C. And filtration by the dead end method. The permeate was sampled in 5 g increments, and the permeate No. was numbered sequentially from 1. The transmittance and the transmission rate of gamma globulin to this permeate were measured. Table 8 shows the results.

[0044]

[Table 8]

[0045]

Comparative Example 5 In the same manner as in Comparative Example 3, a 3% gamma globulin solution (22 ± 2 ppb DNA
Was prepared. Using this gamma globulin aqueous solution as a stock solution, using a virus removal filter, PLANOVA 35N (manufactured by Asahi Kasei Kogyo Co., Ltd., effective membrane area 0.001 m ² ), at a pressure of 0.3 atm and 25 ° C. in the same manner as in Example 9 Then, ultrafiltration was performed by a dead end method. The permeate was sampled in 5 g increments, and the permeate No. was numbered sequentially from 1. Gamma globulin permeability and permeation rate of the permeate were measured. Table 9 shows the results.

[0046]

[Table 9]

The permeation rate after collecting 35 g of the permeate was very slow and could not be measured. The permeation rate after collecting 30 g of the permeate was one of the permeation rate after collecting 30 g of the permeate in Example 9.
/ 24, which was 1/22 of the permeation rate after collecting 30 g of the permeate in Example 10. Further, the transmittance of gamma globulin in Table 9 was lower than the transmittances of Examples 9 and 10.

[0048]

Example 11 Porcine Japanese encephalitis virus was added to the aqueous gamma globulin solution in Example 10, and according to Example 10, 10 g of the solution (stock solution) was filtered, and the permeate was collected. This is referred to as Experiment A. Porcine Japanese encephalitis virus was added to the aqueous gamma globulin solution in Comparative Example 6, and 10 g of the solution (stock solution) was filtered according to Comparative Example 6 to collect a permeate. This is referred to as Experiment B. Aqueous solution before filtration (stock solution)
In each of Experiment A and Experiment B, the infectious titer of the virus in each was 8.2 logs. The infectious titer of the virus in the aqueous solution (undiluted solution) before filtration and in the permeate was analyzed, and the following results were obtained.

[0049]

[Table 10]

However, the virus removal rate (LRV) in Table 10 was defined as follows. LRV = −log ((virus infection titer in permeate) / (virus infection titer in stock solution))
The above results sufficiently satisfy the guidelines of the regulatory authorities on virus safety, and the present invention also enables virus removal as efficiently as in the conventional method.

[0051]

Example 12 As in Comparative Example 1, 5000 ppm of pH5
A 200 ml aqueous gamma globulin solution was prepared. This aqueous solution was added to a 1-liter flask together with 75 ml of an anion exchange resin (SP Sepharose HP, manufactured by Amersham Pharmacia Biotech). The mixture was stirred in a thermostat at 15 ° C. for 24 hours.
Thereafter, the stirring was stopped and the anion exchange resin was removed by decantation. After the above DNA removal operation, 5000pp
m The DNA concentration in the gamma globulin aqueous solution was determined in Example 1.
It was confirmed to be 1 ppb or less by the DNA quantification method measured in the above. After adjusting the pH of the above gamma globulin aqueous solution to pH 8, a fluoropore membrane having a pore size of 0.1 μm (FP-010, manufactured by Sumitomo Electric)
And filtered in the same manner as in Example 6. The permeation rate after collecting 40 g of the permeate was 300 l / m ² / hr, and a permeation flow rate increase 1.7 times that of Comparative Example 2 was observed. The transmittance of gamma globulin was 93.5 ± 1%, which was higher than that of Comparative Example 2.

[0052]

Example 13 Gamma globulin (manufactured by Sigma, G-500
9, bovine) was dissolved in ISOGEN (311-02501, manufactured by Nippon Gene), a reagent for isolating RNA, DNA and protein. Thereafter, chloroform was added and centrifuged. The aqueous phase containing RNA was removed, and ethanol was added to the remaining intermediate phase and the organic phase to homogenize.
After centrifugation again, the supernatant was extracted (DN was contained in the precipitate).
A remains). Isopropanol was added to the supernatant, followed by centrifugation again. The precipitate was washed with isopropanol and dried in vacuum. By the above operation, the recovery of gamma globulin was 83%. A 5000 ppm gamma globulin aqueous solution (pH 8.0) was used in the same manner as in Example 1 using gamma globulin from which DNA had been removed by the above method.
Was prepared.

The DNA concentration in this gamma globulin aqueous solution was confirmed to be 1 ppb or less by the DNA quantification method measured in Example 1. Using this gamma globulin aqueous solution as a stock solution, using a fluoropore membrane (FP-010, manufactured by Sumitomo Electric Industries, Ltd.) having a pore size of 0.1 μm and a membrane area of 17.3 cm ² , a pressure of 0.3 atm and a temperature of 25 ° C. Under the conditions described above. Permeation rate after collecting 40 g of permeate is 529 liters
/ m ² / hr, and an increase in the transmission speed 3.1 times that of Comparative Example 2 was observed. Gamma globulin transmittance is 96.5 ± 1%
Which was higher than that of Comparative Example 2.

[0054]

Example 14 A 5000 ppm gamma globulin aqueous solution (pH 8.0) was prepared in the same manner as in Example 1. Bovine pancrea
tic DNase I (Sigma) was added to the above aqueous gamma globulin solution to a concentration of 100 ng / ml. Then 37
The mixture was stirred in a thermostat at 72 ° C. for 72 hours to digest the DNA. After the above operation, DNA in 5000ppm gamma globulin aqueous solution
The concentration was confirmed to be 1 ppb or less by the DNA quantification method measured in Example 1. Thereafter, filtration was carried out in the same manner as in Example 6 using a fluoropore membrane (FP-010, manufactured by Sumitomo Electric Industries) having a pore size of 0.1 μm. Permeation rate after collecting 40 g of permeate is 4
It was 05 l / m ² / hr, and an increase in the permeation rate 2.3 times that of Comparative Example 2 was observed. The transmittance of gamma globulin was 95.5 ± 1%, which was higher than that of Comparative Example 2.

As is clear from the above, the decrease in DNA in the protein (gamma globulin) solution reduces
When the protein solution was subjected to membrane filtration, the permeation rate was increased, and the protein permeability was significantly increased.

[0056]

Example 15 A 5000 ppb gamma globulin aqueous solution (pH 8.0) was prepared in the same manner as in Example 1. In addition, RNA tra
nscript (luc gene) was added to prepare a 12 ± 2 ppb RNA-containing gamma globulin aqueous solution. Beacon DNA Quantit
RNA transcript (luc genogen) using the ation Kit (V2165, manufactured by Pan Vera Corporation, purchased from Takara Shuzo Co., Ltd.).
Using e) as a test amount, a calibration curve of the fluorescence intensity with respect to the RNA concentration was prepared. Using this calibration curve, the concentration of RNA in the aqueous gamma globulin solution prepared above was quantified, and it was determined that the aqueous gamma globulin solution contained 12 ± 2 ppb of RNA.

Next, a fluoropore membrane (FP-022, manufactured by Sumitomo Electric Industries, Ltd.) having a pore diameter of 0.22 μm and a membrane area of 17.3 cm ² was passed through an ultrafiltration apparatus (RP-1,
UHP-43K, manufactured by Advantech Co., Ltd.)
Filtration was performed under the condition of ° C. to perform an RNA removal operation. Collect 3 ml of the permeate that has passed through the membrane, and use the same method as above.
RNA concentration was quantified. The value at this time was 5 ± 1 ppb.

This gamma globulin aqueous solution was used as a stock solution.
And pore size 0.1 μm, membrane area 17.3cm ^TwoThe Nuclipor
Membrane (111105, Corning Costar Co., Ltd.
Pressure) using the same method as in Example 1
Ultrafiltration was performed under conditions of 0.3 atm and 25 ° C. 40 g of permeate
The gamma globulin permeation rate after collection is 14.8 l
Torr / m^Two/ hr and gamma globulin transmittance is 90.5%
Met.

[0059]

Comparative Example 5 In the same manner as in Example 15, a 5000 ppm gamma globulin aqueous solution (pH 8.0) containing 12 ± 2 ppb was prepared. Without performing the operation of removing RNA performed in Example 15, in the state containing 12 ± 2 ppb RNA, the pore size was 0.1 μm,
Filtration was carried out in the same manner as in Example 1 using a Nuclepore membrane (111105, manufactured by Corning Costar) having a membrane area of 17.3 cm ² . The gamma globulin permeability after collecting 40 g of the permeate was 74.3 ± 5%.
This value was significantly lower than the gamma globulin transmittance after collection of 40 g of the permeate in Example 15.
The permeation rate was 12.5 liters / m after collecting 40 g of permeate.
² / hr. This value is the transmission speed of 84% of Example 15.

[0060]

According to the method of the present invention, when removing impurities such as viruses by filtration from a biological polymer solution, the filtration efficiency, that is, the filtration rate and the transmittance are improved as compared with the conventional method. Thus, the production efficiency can be improved and the production cost can be reduced.

Claims (3)

[Claims] Claims: 1. DN in a biological polymer solution to be filtered
A method for purifying a biologically-derived polymer solution, comprising reducing the concentration of A and / or RNA, followed by membrane filtration. 2. The DN in a biological polymer solution to be filtered.
The concentration of A and / or RNA is 5 ppb or less.
The method for purifying a biological polymer solution according to the above. 3. The method according to claim 1, wherein the target to be removed by filtration is a virus.