JP3372638B2 - Filtration aid comprising substance that blocks virus passage and filtration method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a filter aid comprising a substance that blocks the passage of a virus and a filtration method using the same. More specifically, the present invention relates to a filter aid comprising a substance which is safe for a living body and which itself passes through a membrane filter but prevents virus from passing through the membrane filter when it comes into contact with a virus, and a filtration method using the same. About.

[0002]

2. Description of the Related Art The removal and inactivation of viruses has become a serious problem in the production process of pharmaceutical products produced using biologic-based preparations and genetic recombination techniques, and various studies and developments have been carried out. ing. Conventional virus inactivation methods include 1) disruption of the envelope lipid membrane by an organic solvent (ether, chloroform), 2) denaturation of virus proteins by phenol, formalin, a surfactant, etc., 3) physical conditions (heat, Drying, osmotic pressure change, etc.), and 4) damage of viral nucleic acids by radiation or ultraviolet rays. In many cases, these virus inactivation methods involve protein denaturation and nucleic acid damage of not only the virus but also other components, and the biological activity of the active component is not sufficiently maintained.

[0003] In order to capture microorganisms, there is a method of separating microorganisms according to the size of the microorganisms using a membrane filter. For capturing microorganisms such as bacteria and yeast, for example, those having a pore size of 0.22 μm or 0.45 μm are usually used. Separation of microorganisms by such a filter has advantages such as easy use, short-time operation, no chemical change, no chemical addition to the treatment liquid, no change in composition, and no thermal denaturation. However,
Smaller viruses and mycoplasmas often permeate the membrane and cannot be captured.

For this reason, recently, a porous membrane capable of separating small-sized viruses by reducing the pore diameter or devising the pore structure has been proposed (for example, Japanese Unexamined Patent Publication No. Sho.
0-142860, JP-A-63-236501, JP-A-1-22305). These techniques attempt to capture a small virus according to its size, and basically reduce the pore size of the membrane.

[0005] In the above method, in order to separate extremely small viruses, the pore size of the filter is set to 20 nm (0.02 µm).
The holes need to be extremely small. In such a method for removing virus particles, since the pore size is further reduced to 1/10 to 1/20 of that of a normal membrane filter, it may be necessary to increase the filtration pressure or increase the filtration time. As a result, the filtration speed is reduced, and clogging is likely to occur. Further, if a large hole diameter pinhole or the like is present in the filter, the virus cannot pass through and cannot be removed, and the method for removing the virus has not been sufficiently satisfactory.

[0006]

SUMMARY OF THE INVENTION Accordingly, the present invention provides a method for removing or concentrating a virus very simply and quickly while retaining all the advantages of a known method for separating microorganisms using a filter. The purpose is to do. When applied to the production of pharmaceutical preparations, only the virus is removed or inactivated to achieve the purpose of eliminating infectious particles from the preparation,
It is desirable to establish conditions under which the active ingredient in the preparation is not damaged or lost as much as possible. Therefore, the present invention
It is an object of the present invention to provide a method capable of easily and efficiently removing or capturing a virus and easily recovering an active ingredient.

[0007]

DISCLOSURE OF THE INVENTION The present invention provides a method for removing or concentrating a virus more rapidly and more efficiently than in a preparation containing a virus or a sample containing a virus, and without losing an active ingredient. The purpose is to be able to fully collect. The purpose of this is to make a sample that is contaminated with a virus a substance that is safe for the living body and that passes through the membrane filter by itself, but prevents the virus from passing through the membrane filter when it comes into contact with the virus, that is, a safe and water-soluble substance. It is achieved by filtration through a membrane filter in the presence of a substance that is a viscous polymer compound, for example, hyaluronic acid or alginic acid.

Accordingly, the present invention provides a filter aid comprising a substance which is safe for living organisms and which itself passes through a membrane filter but, when in contact with a virus, prevents the virus from passing through the membrane filter, and a filter aid therefor. The present invention provides a filtration method in which a liquid to be treated is treated with a membrane filter in the presence of.

(1) Starting Material (Liquid to be Treated) The starting material used in the present invention is a solution containing water or protein or other useful components contaminated with the virus. For example, a biological solution containing human plasma protein, urine-derived protein and the like is exemplified. A solution containing a protein expressed by using a virus as a vector by genetic engineering is also exemplified in that the virus is contained in the protein recovery solution. Further, a virus research sample and the like are also included. The processing target of the present invention is not limited to those described above, but is a sample in which the virus is likely to be mixed or a sample containing the virus. In the treatment of the present invention, it is essential that the raw materials be in a solution state.

(2) Target virus The virus to be removed or concentrated in the present invention includes herpes virus, baculovirus, vaccinia virus, Sendai virus, Newcastle disease virus (NDV) and the like.

(3) Membrane Filter The pore size (pore diameter) and membrane material of the membrane filter used in the present invention are safe for the target virus and living organisms, and pass through the membrane filter by themselves, but not with the virus. The optimal combination is selected according to the type of substance that blocks virus passage through the membrane filter upon contact. That is, the pore size of the membrane filter and the material of the membrane are determined by an additional experiment whether or not the filter aid used prevents the passage of the virus when it comes into contact with the virus. For example, when the purpose is virus removal, the pore size is preferably 0.45 μm or less in order to obtain effective virus removal efficiency.

For example, when the protein to be treated does not coexist and the target virus is a herpes virus such as herpes simplex virus 1 (herpes simplex type-1), and the pore size of the membrane is 0.45 to 0.22 μm. If
As the material of the membrane, a cellulose mixed ester gives a good result. When a protein coexists in the liquid to be treated, the pore size is 0.
Good virus removal can be achieved with a cellulose mixed ester or polyvinylidene fluoride of 22 μm or less.

(4) Filtration Aid Substances that are safe for living organisms and pass through the membrane filter by themselves, but prevent virus from passing through the membrane filter when they come into contact with viruses are substances having such properties. Anything is fine. Such water-soluble mucilage polymer compounds known to be safe to the living body is like we are as material, the present invention includes a living body safety and is passed through a membrane filter is itself virus A filter aid comprising a water-soluble viscous high molecular compound which prevents virus from passing through the membrane filter when contacted, and a liquid to be treated, in the presence of a filter aid comprising the above water-soluble viscous high molecular compound, in a membrane filter And a filtration method.

Examples of the water-soluble viscous high molecular compound include water-soluble viscous polysaccharides.
And, a filter aid consisting of a water-soluble mucous polysaccharide that passes through the membrane filter by itself but prevents virus from passing through the membrane filter when it comes into contact with the virus,
Further, the present invention provides a filtration method for treating a liquid to be treated with a membrane filter in the presence of a filter aid comprising the water-soluble mucous polysaccharide. Examples of the water-soluble mucous polysaccharides include methylcellulose, dextran, pectin, starch, gum arabic, guar gum, and the like, in addition to chrycosaminoglycan and glycuronane (polyuronic acid) described in detail below.

Examples of the above water-soluble mucous polysaccharides include water-soluble mucous acidic polysaccharides. The present invention is directed to a bio-safety product which is safe for a living body and passes through a membrane filter by itself, but when it comes in contact with a virus, the virus becomes viscous. Filtration aid consisting of a water-soluble viscous acidic polysaccharide that blocks passage through a membrane filter, and filtration in which a liquid to be treated is treated with a membrane filter in the presence of the filter aid consisting of the water-soluble viscous acidic polysaccharide. Provide a way.

As the above-mentioned water-soluble viscous acidic polysaccharide, glycosaminoglycan (mucopolysaccharide) is particularly preferred because it has high safety to living organisms and a high effect of capturing viruses.
Therefore, the present invention provides a filter aid comprising glycosaminoglycan which is safe for living organisms and which itself passes through a membrane filter but prevents virus from passing through the membrane filter when it comes into contact with a virus, and a liquid to be treated. And a filtration method for treating with a membrane filter in the presence of a filter aid comprising the glycosaminoglycan.

The glycosaminoglycans used in the present invention include chondroitin sulfate, chondroitin, hyaluronic acid, dermatan sulfate, heparin, heparan sulfate, keratan sulfate, keratopolysulfate and derivatives thereof (acylated, sulfated, deacylated). And desulfated products). These are preferably physiologically acceptable salts thereof, and water-soluble salts are used. These are obtained by extraction from biological components such as cartilage, cockscomb, umbilical cord, etc., those produced by fermentation, those obtained by known methods such as those enzymatically or chemically synthesized or modified, etc. Its origin and production method are not limited. The molecular weight is not particularly limited.

Further, when the water-soluble viscous acidic polysaccharide is, for example, glycuronan (polyuronic acid) such as alginic acid,
The present invention provides a filter aid consisting of glycuron, such as alginic acid, which is safe for a living body and which itself passes through a membrane filter but prevents virus from passing through the membrane filter when it comes into contact with a virus, and a liquid to be treated. Provided is a filtration method for treating with a membrane filter in the presence of a filter aid comprising glycuronane such as alginic acid.

In the present invention, hyaluronic acid or a salt thereof is most preferably used as the water-soluble viscous high molecular compound, and alkali metal (sodium, potassium, etc.) salts of hyaluronic acid, amines (tri-n-butylamine, triethylamine, etc.) Pyridine and the like) salts and the like. The molecular weight of the hyaluronic acid is not particularly limited, but usually, the average molecular weight is about 500,000 to 3,500,000, more preferably 800,000 to 1.2 million, and purified one is used. If the degree of purification is low, impurities may be mixed into the filtrate.

Accordingly, the present invention provides a filter aid comprising hyaluronic acid which is safe for living organisms and which itself passes through a membrane filter but prevents virus from passing through the membrane filter when it comes into contact with a virus, and a liquid to be treated. Is provided with a membrane filter in the presence of a filter aid comprising hyaluronic acid.

The case where sodium hyaluronate (hereinafter sometimes simply referred to as "hyaluronic acid") or sodium alginate (hereinafter sometimes simply referred to as "alginic acid") is used will be described below. (5) Treatment Conditions For the purpose of virus removal, hyaluronic acid is added to the target sample in advance, the final concentration of hyaluronic acid is 5 mg / ml or more, and the viscosity is defined as the shear rate (S ^-1 ) 1.
At ~ 40, apparent viscosity 1500-150 mPa · s
So that the condition This hyaluronic acid-containing sample is filtered with a membrane filter. The degree of pressurization depends on the molecular weight, concentration, and viscosity of hyaluronic acid.
For example, at 0.45 μm, hyaluronic acid: 10 mg
/ Ml can be filtered at low pressure down to 0.22 μm
Hyaluronic acid: 10 mg / ml requires high pressure.

In particular, the present invention is directed to a method of passing a virus through a membrane filter which is safe for a living body and is itself composed of a cellulose mixed ester membrane or a polyvinylidene fluoride membrane having a size of 0.45 μm or less, but comes into contact with a virus. A filter aid made of hyaluronic acid or alginic acid that prevents passage through a filter; and a liquid to be treated which is safe for a living body and is itself a cellulose mixed ester membrane or a polyvinylidene fluoride membrane of 0.45 μm or less. Provided is a filtration method wherein the membrane filter is treated in the presence of a filter aid consisting of hyaluronic acid or alginic acid, which passes through the membrane filter but contacts virus when the virus stops passing through the membrane filter.

When the liquid to be treated contains protein, it is preferable to use a cellulose mixed ester film or a polyvinylidene fluoride film of 0.22 μm or less as a membrane filter. In that case, the final concentration of hyaluronic acid is added to 5 mg / ml or more of sodium hyaluronate. Alternatively, it is carried out by adding alginic acid as a final concentration to sodium alginate at 10 mg / ml or more. After the treatment with the membrane filter, the filter aid that has passed through the membrane filter can be separated or decomposed and removed.

Accordingly, the present invention relates to a filter aid for a liquid to be treated in which a protein is present, which comprises a cellulose mixed ester membrane or a polyvinylidene fluoride membrane which is safe for a living body and has a thickness of 0.22 μm or less. A filter aid consisting of alginic acid that passes through the membrane filter but blocks the passage of the virus when it comes in contact with the virus; Sodium alginate is passed through a membrane filter made of a cellulose mixed ester membrane or polyvinylidene fluoride membrane of 22 μm or less, but when it comes in contact with virus, it blocks virus from passing through the membrane filter. Is added to the membrane filter by 10 mg / ml or more. A filtration method for treating with a luter is provided.

The present invention also relates to a filter aid for a liquid to be treated in which a protein is present, which comprises a cellulose mixed ester membrane or a polyvinylidene fluoride membrane which is safe for a living body and has a thickness of 0.22 μm or less. A filter aid for a liquid to be treated containing a protein consisting of hyaluronic acid that passes through the membrane filter but prevents virus from passing through the membrane filter when it comes into contact with the virus; A hyaluronic acid which passes through a membrane filter consisting of a cellulose mixed ester membrane or polyvinylidene fluoride membrane having a thickness of 0.22 μm or less by itself but, when in contact with a virus, prevents the virus from passing through the membrane filter; As a final concentration of hyaluronic acid, sodium hyaluronate 5 mg / ml or more A filtration method for adding and treating with the membrane filter is provided.

The filtration conditions can be appropriately set depending on the amount of virus contained and the properties of the protein to be recovered. In other words, regarding the final concentration of hyaluronic acid and the pore size of the membrane, increasing the concentration or molecular weight of hyaluronic acid increases the virus removal efficiency but increases the filtration time, and decreasing the pore size increases the virus removal efficiency but increases the filtration time. Determine the optimal conditions while referring to the relationship of extension. It goes without saying that it differs depending on the type of virus.

(6) The water-soluble viscous high molecular compound that has passed through the purification membrane filter is fractionated and removed depending on the difference in molecular weight and charge from the target substance, or means for specifically decomposing only the filter aid (for example, And enzymatic degradation using an immobilized enzyme). In particular, hyaluronic acid is a high molecular weight polysaccharide having a very large molecular weight, and ordinary proteins can be easily fractionated by a gel filtration method or the like, except for special proteins (a very small number of proteins such as hyaluronectin). In order to hydrolyze hyaluronic acid, hyaluronidase, chondroitinase ABC, chondroitinase AC and the like can be used.

Accordingly, the present invention relates to a filter aid comprising hyaluronic acid, which is used for treating a liquid to be treated, which is safe for a living body and which itself passes through a membrane filter but, when in contact with a virus, blocks the virus from passing through the membrane filter. The present invention provides a filtration method in which a filter aid is treated with a membrane filter in the presence, and then the filter aid that has passed through the membrane filter is separated or decomposed and removed from the filtrate.

[0029]

The present invention will be described below with reference to examples. The present invention is not limited by the examples.

Example 1 A medium containing herpes simplex virus type 1 (HSV-1) [Eagle MEM medium containing 2% bovine serum] and an aqueous sodium hyaluronate solution (5 mg / ml,
Phosphate buffered saline, pH 7.4) mixed at a ratio of 1: 9 (in the liquid to be treated, bovine serum in the virus solution is present at a final concentration of 0.2%). The solution was filtered through a membrane filter (0.45 μm: Myrex HA, Millipore). The amount of the virus in the filtrate was measured for its infectious titer by the Braak formation method using Vero cells as host cells. The results are shown in Table 1 (fluctuation of virus amount before and after filtration of virus-containing sample). As shown in the table, a remarkable difference was observed in the amount of virus permeated between the case where hyaluronic acid was not added to the virus-containing medium and the case where hyaluronic acid was added. No inactivation of the virus by hyaluronic acid itself was observed.

[0031]

[Table 1]

Example 2 The hyaluronic acid concentration and the membrane filter pore size and material with respect to the virus membrane permeation amount were examined as shown in Table 2 (Effect of membrane pore size and material and hyaluronic acid addition concentration on virus removal efficiency). . The experimental operation was performed in the same manner as in Example 1. as a result,
Pore size 0.22μm, 0.45μm, 0.88μ
When compared by m, 0.22 μm had the smallest amount of virus permeated through the membrane. It was considered that the higher the final concentration of hyaluronic acid was, the more effective it was. As a result of comparing the cellulose mixed ester and the polyvinylidene fluoride with the same pore size (0.45 μm) for the material of the membrane, the membrane mixed amount of the cellulose mixed ester was significantly smaller than that of the membrane mixed virus.

[0033]

[Table 2] UN: uncountable: The number of plaques was too large to be measured as a result of measuring the filtrate with a stock solution. Membrane filter GV used: Mirex GV (Material: polyvinylidene fluoride, manufactured Millipore) HA: Mirex HA (Material: mixed cellulose ester, manufactured Millipore) PF: Mirex PF (material: mixed cellulose ester, manufactured Millipore) Company)

From the above results, the following conditions can be considered as preferable conditions for removing the virus from the liquid to be treated in which no protein is present. 1) Hyaluronic acid addition concentration: 5 mg / ml or more in final concentration 2) Pore size: 0.22 to 0.45 μm 3) Membrane material: mixed cellulose ester 4) pH: Near neutral, temperature: room temperature, 5) pressure A: No special high pressure is required. (High pressure is required only when the hyaluronic acid is 10 mg / ml or more at 0.22 μm)

Example 3 The effect of hyaluronic acid concentration on the amount of virus permeated through the membrane was examined in the presence of bovine serum (hereinafter abbreviated as "serum") in the presence of cellulose (final concentrations 1.0 and 5.0%). Ester film (0.22 μm: Mailex GS, Millipore) and polyvinylidene fluoride film (0.22 μm: Mailex G)
V, Millipore) and FIG. 2 (Effect of hyaluronic acid concentration on viral membrane permeation in the presence of 1% serum) and FIG. 2 (hyaluronic acid on viral permeation in the presence of 5% serum) Influence of concentration). In FIG. 1, GS (serum 1.0%) is 0.1%.
22 μm cellulose mixed ester membrane (Mirex G
S), GV (serum 1.0%) represent the results when a 0.22 μm polyvinylidene fluoride membrane (Mirex GV) was used. In FIG. 2, GS (5.0% serum) was 0.22.
μm cellulose mixed ester membrane (Mirex G
S) and GV (5.0% serum) represent the results when a 0.22 μm polyvinylidene fluoride membrane (Mirex GV) was used. The virus concentration of the virus-containing sample used in these experiments before filtration was 4 × 10 ⁴ PFU / ml. The experimental operation was performed in the same manner as in Example 1.

As a result, in both the cellulose mixed ester membrane and the polyvinylidene fluoride membrane, as the concentration of added hyaluronic acid increased, the permeation amount of the virus decreased. Regarding the effect in the presence of serum protein, an increase in the amount of viral membrane permeation was observed with an increase in the amount of serum protein in the cellulose mixed ester membrane, but even in 5% of bovine serum, 2.5 mg of hyaluronic acid / Above ml, the virus removal effect was maintained. In the polyvinylidene fluoride membrane, no change in the amount of virus permeated by the serum protein was observed.

Example 4 The effect of serum protein concentration on the amount of virus permeated by the membrane was examined in the absence and presence of hyaluronic acid in a cellulose mixed ester membrane (0.22 μm: Milex GS,
Millipore) and polyvinylidene fluoride film (0.22 μm:
(Milex GV, Millipore), and the study was performed as shown in FIG. 3 (Effect of serum concentration on virus membrane permeation). In FIG. 3, GS (hyaluronic acid 5 mg)
/ Ml) is 0.22 μm cellulose mixed ester membrane (Mirex GS), GV (hyaluronic acid 5 mg /
) (in the presence of hyaluronic acid) represents the result of membrane filtration of a 0.22 μm polyvinylidene fluoride membrane (Mirex GV) in the presence of 5 mg / ml of hyaluronic acid. 22 μm cellulose mixed ester membrane (Mirex GS), GV (without hyaluronic acid)
Represents the result of membrane filtration of a 0.22 μm polyvinylidene fluoride membrane (Mirex GV) in the absence of hyaluronic acid. The experimental operation was performed in the same manner as in Example 1.

As a result, when the pore size of 0.22 μm of the cellulose mixed ester membrane and the polyvinylidene fluoride membrane was used, the hyaluronic acid (final concentration: 5 mg / ml) in both membranes was higher than that in the absence of hyaluronic acid. In the presence, even in the presence of high concentrations of serum proteins, the amount of virus permeated significantly decreased.

Example 5 Alginic acid (sodium alginate: 500-600 cp, Wako Pure Chemical Industries) was used as another mucopolysaccharide, and a comparative study with hyaluronic acid was performed using a polyvinylidene fluoride membrane of 0.22 μm. 4 (the effect of alginic acid on the amount of virus permeated by membrane compared with hyaluronic acid). In FIG. 4, GV (alginate) is alginate (0, 1.0, 2.5, 5.0,
GV (hyaluronic acid) indicates the result of membrane filtration performed in the presence of hyaluronic acid at the same concentration. As the filtration membrane, a polyvinylidene fluoride membrane of 0.22 μm (Mirex GV) was used for both. The experimental operation was performed in the same manner as in Example 1.

As a result, it was confirmed that alginic acid also had the same virus removing effect as hyaluronic acid. It is considered that the reason why the virus permeation amount is higher in alginic acid than in hyaluronic acid is that alginic acid has lower viscosity than hyaluronic acid at the same concentration.
It is considered that alginic acid can also be a filter aid in good virus removal at a concentration that gives sufficient viscosity.

[0041]

According to the present invention, the virus can be removed or concentrated very easily and quickly while retaining all the advantages of the known method for separating microorganisms using a filter. A water-soluble, viscous polymer compound that is safe for a living body, such as hyaluronic acid, to be added to the treatment solution has no toxicity to the living body and does not adsorb to proteins. The components can be easily recovered.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing the effect of hyaluronic acid concentration on the amount of virus permeated in the presence of 1% serum.

FIG. 2 is an explanatory diagram showing the effect of hyaluronic acid concentration on the amount of virus permeated in the presence of 5% serum.

FIG. 3 is an explanatory diagram showing the effect of serum concentration on the amount of virus permeated through the membrane.

FIG. 4 is an explanatory diagram showing the effect of alginic acid on the amount of virus permeated through the membrane compared with hyaluronic acid.

Claims (9)

(57) [Claims] 1. A filter aid comprising a substance which is safe for a living body and which itself passes through a membrane filter but prevents virus from passing through the membrane filter when it comes into contact with a virus. 2. The filter aid according to claim 1, wherein the substance is a water-soluble viscous polymer compound. 3. The filter aid according to claim 2, wherein the water-soluble viscous high molecular compound is a water-soluble viscous polysaccharide. 4. The filter aid according to claim 3, wherein the water-soluble viscous polysaccharide is a water-soluble viscous acidic polysaccharide. 5. The filter aid according to claim 4, wherein the water-soluble viscous acidic polysaccharide is glycosaminoglycan or glycuronane. 6. The filter aid according to claim 5, wherein the glycosaminoglycan is hyaluronic acid. 7. The filter aid according to claim 5, wherein the glycuronane is alginic acid. 8. A membrane to be treated in the presence of a filter aid consisting of a substance which is safe for living organisms and which itself passes through the membrane filter but, when in contact with a virus, blocks the virus from passing through the membrane filter. A filtration method of treating with a filter. 9. After the treatment with a membrane filter, face
The filter aid that has passed through the run filter may be separated from the filtrate.
9. The filtration method according to claim 8, wherein the filtration is carried out.