JP2002085056A - Virus inactivation process - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a virus inactivation process that can surely inactivate and eradicate the viruses in the liquid to be treated such as blood preparation without deactivation and deterioration of the liquid to be treated such as a blood preparation in low costs with high safety. SOLUTION: In the virus inactivation process according to this invention, the treatment temperature is set to one at which the liquid to be treated is in the supercooled state, the liquid component in the liquid to be treated permeates the head shell of the virus for a minimum treatment time and the conditions are set for the time until the hydrogen bonds formed with the inner living body elements reaches the thermodynamic equilibrium so that the liquid is permeated into the virus by retaining the above-stated treatment temperature and the above-stated pressure for more than the minimum treatment time and the hydrogen bonds formed in the head shell is kept in the thermodynamically equilibrium. Then, the pressure is gradually removed to allow the liquid permeating the virus to phase-transfer to the ice crystals whereby the head shell or the living body element of the virus is broken by the volume expansion of the formed ice crystals.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for inactivating a virus contained in a liquid to be treated, such as blood or blood products, at a temperature below freezing within a relatively short time. It is.

[0002]

2. Description of the Related Art Conventionally, when producing blood products such as albumin preparations and immunoglobulin preparations, it has been concerned that hepatitis virus, human immunodeficiency virus (hereinafter referred to as HIV) and the like may be mixed into the blood preparations. It is well known that hepatitis and AIDS infection actually caused this and became a major social problem. In light of these circumstances, research is still ongoing to eradicate hepatitis and AIDS.

[0003] By the way, the heat inactivation method which has been conventionally adopted to prevent contamination of hepatitis virus and HIV is used. This method is known to be a method for effectively inactivating viruses.

[0004] However, heat treatment has essentially the following serious problems. In other words, there is a problem that the active ingredient of a blood product lacks thermal stability because it is a protein. When the virus is inactivated by heat treatment, the protein is inevitably denatured, the activity of the blood product itself is lost, and an insoluble substance is produced. Then, in order to avoid such a decrease in the activity and the generation of insoluble substances, a large amount of an additive for a metamorphic inhibitor is required when performing the heat treatment. Additives of anti-metastatics to such drugs for use in the body are not desirable. If an attempt is made to use a non-heated preparation to avoid this, virus inactivation is not thorough and social problems occur as described above.

[0005] As described above, inactivation of a virus by heat treatment causes protein denaturation, and the liquid to be treated such as a blood product is easily denatured. Therefore, development of a virus killing method that does not have such a problem is urgently required.

Incidentally, in addition to the heat treatment, there has been proposed a method of inactivating a virus by subjecting an object to be treated to a pressure treatment (Japanese Patent Laid-Open No. 6-142197). In order to inactivate a virus in an object that lacks heat stability, such as a blood product, the object to be treated that may be contaminated with the virus is pressurized under a condition of 1,000 to 6,000 atm. The pressure to be applied is 1000 atm
6000 atm, preferably about 3000 to 5000 atm. The reason is that the processing pressure is 10
If the pressure is lower than 00 atm, it is difficult to inactivate the virus.
There is no problem if the pressure exceeds the atmospheric pressure, but a sufficient effect can be obtained at this pressure. In this high-pressure treatment, the treatment temperature is not particularly limited, but is usually from −15 ° C. to room temperature (2 ° C.).
The treatment is preferably performed at a temperature of about 10 ° C. to room temperature, and the time of the high-pressure treatment is appropriately adjusted depending on the treatment pressure, the treatment temperature, the type of virus, and the like.
About 30 minutes, and preferably 10 minutes to 20 minutes.

Unfortunately, however, according to the description, it is unclear why the virus is inactivated by the pressure treatment, and the mechanism of inactivation is not clear. However, since the envelope of the virus having the envelope is destroyed by pressurization, it is speculated that inactivation due to the disruption of the envelope is a major factor. Therefore, this conventional technology simply inferred that pressurization is useful for inactivating the virus having an envelope, and did not focus on the processing temperature or processing time, but was one step further for application development. Met.

According to the study by the present inventor, when a high pressure of 4,000 atm or more, which is preferable here, is applied, the processing temperature varies, but when the pressure medium is water, pressure ice is formed, Since pressure may not be available, use of an antifreeze-based pressure medium such as a mixed solution of ethanol and ethylene glycol is inevitable as the pressure medium. However, this method does not use water that is most easily used and has high safety, and is not practical.

By the way, the above-mentioned prior art is an inactivation technique targeting only a virus. However, prior to the present application, the present inventors reduced microorganisms such as bacteria contained in foods and pharmaceuticals to minus temperature. A method of autoclaving or autoclaving in a region or under slow heating has been proposed (JP-A-9-285).
No. 526). Hereinafter, this conventional pressure sterilization method will be described.

In the conventional pressure sterilization method proposed by the present inventors, water is penetrated into the microbial cells under pressure, and the water is adiabatically expanded from the inside to the outside of the cells, and then the cell membrane or membrane tissue is formed. (Hereinafter referred to as membrane tissue) physically destroys the cell membrane of microorganisms. When destroying membrane tissue,
It utilizes the impact of water, which is the most energy efficient. That is, foods and materials contaminated by microorganisms are dispersed in a liquid, the liquid is sufficiently pressurized, and the liquid is sufficiently penetrated into the inside of the microorganism. Then, the pressure is increased until the pressure difference between the inside and the outside of the membrane tissue disappears or the pressure difference between the two becomes infinitely zero. That is, by pressurizing the liquid at 200 to 800 MPa for 60 minutes or more, the above-described environment is sufficiently produced. Thereafter, the pressure of the pressurized liquid is released instantaneously. At this time,
The liquid inside the microorganisms follows its own bulk modulus,
Adiabatic expansion is forced instantaneously, and a very large impact force is generated in the membrane of the bacterial body. This impact forces the membrane tissue from the inside out, while destroying the microorganisms. At this time, the impact force applied to the membrane of the cells is several times or several tens times the pressure applied to the liquid. Therefore, any microorganism can be sterilized at less than 80 ° C.

In this conventional sterilization method, since water is used as the pressure medium, sterilization can be performed even in a negative temperature range. The reason for this is that water at 250 MPa is a liquid that does not freeze even at -25 ° C, and can be instantaneously sterilized against microorganisms such as vegetative cells at 250 MPa and -25 ° C by adiabatic expansion. And Bacillus stearothermophilus (Baci
llus stearothermophilus IFO 12550 spores), the adiabatic expansion start temperature is at least -25 ° C or more and less than 80 ° C, preferably 70 ° C, and the applied pressure is 200 to 800 MPa.
The pressurization time is at least 60 minutes so that sterilization is possible.

[0012]

As described above,
The conventional method of inactivating a virus by pressurization is based on only a few experiments that the pressure is useful for inactivating an enveloped virus. The mechanism of inactivation is unknown. However, from some experiments, it was found that
00 to 6000 atm, preferably 3000 to 5 atm
Inactivation is realized by pressurizing under the condition of 000 atm. At this time, since the envelope is destroyed, it is possible that inactivation based on the destruction of the envelope may be a major factor. Points out. And there is no sufficient disclosure about the influence of processing temperature and processing time. Therefore, the disclosure is generally poor enough to be applied.

[0013] Further, this inactivation method is not so precise that an inactivation treatment can be performed specifically for each virus, does not take into account the depressurization at all, and simply applies pressure. It is. Moreover, it is presumed that the protein is slightly denatured. And as an effect,
It describes the obvious matter, for example, that a virus that can easily die will die at about 3000 to 5000 atmospheres. It is rather a matter of ensuring that the most indelible viruses can be killed. In this sense,
This conventional virus inactivation method lacked applicability and true practicality.

In the conventional autoclaving method proposed by the present inventors, the temperature is from -25 ° C. to less than 80 ° C., preferably 70 ° C.
The pressure is 200MPa ~ 800MPa, the processing time is that at least 60 minutes sterilization is possible,
It was a very good sterilization method. However, this proposal
Since the entire framework that enables the sterilization of various microorganisms has been disclosed as a whole, the most effective and specific conditions for killing viruses have not been disclosed.

Further, practicality is required for a virus inactivating device. The conventional technique proposed by the inventor is still a little high in pressure, and is a step in terms of cost, weight, and size. It is desired to develop a deactivation device in which these are improved to a practical level. If the critical pressure for virus inactivation decreases, the lower the cost, the greater the weight and the size of the device, and the higher the performance of the device, both in terms of safety and control. I can have.

Therefore, what kind of problems must be newly solved in order to lower the pressure of this conventional technique to a practical level with respect to the virus at this time will be further described below.

As disclosed in Japanese Patent Application Laid-Open No. 9-285526, in the case of bacteria, especially gram-negative bacteria, the cell membrane is easily destroyed by the adiabatic expansion force of water and has the property of being sterilized. It has also been found that the head shell of the virus is composed of a lipid bilayer similar to the cell membrane of Gram-negative bacteria. From this fact, it is expected that the virus can be inactivated by almost the same method if the conventional technique is applied as it is. For example, the strain of water generated at a pressure of 400 MPa, which varies depending on the temperature, is generally about 10%. Therefore, when this strain is released in about 0.1 second, Gram-negative bacteria such as Escherichia coli and Salmonella are destroyed and sterilized. . From this, it is considered that a virus having a lipid bilayer similar to the cell membrane of Gram-negative bacteria can be inactivated under similar conditions as Gram-negative bacteria.

However, the size of the skull of the virus is extremely small (1/1) compared to the size of the gram-negative bacteria.
0 to 1/100 or less). For this reason, the virus has a relatively large surface area on a volume basis as compared with a gram-negative bacterium, and has a higher destruction limit per unit area. Further microscopically, the inside of the virus's head and shell is very small, and the ratio of DNA is very high.
NA forms strong hydrogen bonds, and a decrease in processing pressure makes it difficult to destroy viruses, leading to a decrease in inactivation rate. The present inventors have confirmed this by experiments and have found that when the treatment pressure is reduced from 300 MPa to 200 MPa, the inactivation rate of the virus is sharply reduced.

That is, since the size of the head shell of the virus is extremely small, it cannot be destroyed under the same conditions as Gram-negative bacteria. In order to inactivate the virus at a relatively low pressure that is practical, it is necessary to exceed the destruction limit at a low pressure.
A device other than the conventional pressure treatment is required. Simply converting the conventional method to a virus as it is, killing the virus,
Deactivation is difficult. Therefore, first, it is necessary to examine how the size difference relates to sterilization or inactivation.

As described above, the virus head shell and the cell membrane of Gram-negative bacteria have a lipid bilayer structurally similar to each other. It makes the following critical differences in the mechanisms for achieving virus inactivation. That is, since the size of Gram-negative bacteria is relatively large, the destruction of the cells by the conventional method is macroscopic, and can be sterilized by destroying the cell membrane by utilizing the adiabatic expansion rate of water.

However, in the case of a minute virus, a more microscopic viewpoint at the molecular and atomic levels is required, and it is thought that the only mechanism for protecting the skull and the living body is to destroy it. In other words, enzymes essential for survival form hydrogen bonds between N atoms and O atoms and surrounding water molecules, and stabilize the hydrogen bonds themselves. However, it is necessary to separate or recompose these hydrogen bonds. It is thought that this leads to inactivation and destruction of the virus, thereby destroying the mechanism of survival. By breaking this mechanism,
It should be possible to weaken the head shell and biological elements from the inside of the virus, the biological elements themselves are inactivated, and the skull should be able to be destroyed very easily, and there is a great prospect for virus inactivation. Be expected. Thus, a new issue for virus inactivation can be said to be the creation of a new means capable of separating and recombining hydrogen bonds, which are mechanisms for maintaining survival in the virus.

[0022] In addition to the above-described problems, there is still another urgent and greatest problem in the conventional virus inactivation technique. That is, there is no effective way to protect the liquid to be treated such as blood products from HIV.
It can be said that the inactivation of the virus was solved only after HIV measures were established.

Therefore, in order to solve the problems described above, the present invention provides a low-cost, safe, and reliable method for reducing the activity of a liquid to be treated, such as a blood product, without causing denaturation. An object of the present invention is to provide a virus inactivation method capable of inactivating and killing a virus.

[0024]

In order to solve the above-mentioned problems, the virus inactivating method of the present invention sets the processing temperature to a temperature at which the liquid to be processed is in a supercooled state under a freezing point above a predetermined pressure. Along with the setting, the minimum processing time is maintained at the processing temperature and pressurized at the pressure, so that the liquid component of the liquid to be processed permeates through the head and shell of the virus and between the internal biological elements. Set the time until the hydrogen bonds formed are thermodynamically equilibrated, penetrate the liquid into the virus while maintaining the processing temperature and pressure for a minimum processing time,
After the hydrogen bonds formed in the skull are thermodynamically equilibrated, the pressure is then reduced to change the phase of the liquid component that has penetrated into the virus into ice crystals. And destruction or deactivation of the virus's head and shell and its biological components by volume expansion force.

Thus, the activity of the liquid to be treated such as a blood product is lost, and the virus in the liquid to be treated can be inactivated and killed safely and reliably at low cost without causing denaturation. .

[0026]

DESCRIPTION OF THE PREFERRED EMBODIMENTS According to the first aspect of the present invention, a flexible hermetically sealed container filled with a liquid to be treated is accommodated in a pressure medium, and the whole is kept at a predetermined treatment temperature below freezing for a minimum treatment time or longer. A method for inactivating a virus in which a pressure is applied and then depressurized to inactivate a virus contained in a liquid to be treated, wherein the treatment temperature is a temperature at which the liquid to be treated is in a supercooled state at a freezing point above a predetermined pressure. In addition, the minimum processing time is kept at the processing temperature and pressurized at the pressure, so that the liquid component of the liquid to be processed permeates through the virus's skull, and between the living body elements. The time required for the hydrogen bonds formed to equilibrate thermodynamically is set, the liquid is allowed to penetrate the virus into the virus while maintaining the processing temperature and the pressure for a minimum processing time, and the hydrogen bonds formed in the skull To thermodynamic equilibrium After that, by removing pressure, the liquid component that has penetrated into the virus undergoes a phase change to ice crystals, and the structural change of the ice crystals and the volume expansion force destroy or inactivate the virus's head and shell. Initially, for example, normal pressure, 4 ° C.
Biological elements in the virus (enzymes and DN
A, nucleic acid, etc.) and water molecules are recombined into new hydrogen bonds that are imposed in a new environment by the processing temperature and pressure, and when the newly formed hydrogen bonds are in thermodynamic equilibrium. By decompressing the virus component in the supercooled state, the phase changes to ice crystals, and the volume inside the virus increases due to the adiabatic expansion of the supercooled water based on the decompression and the volume expansion force caused by freezing ( About 20 at 400MPa
%), Whereby the skull is physically destroyed, and the hydrogen bonds of the biological elements are forced to dissociate and associate due to structural changes in the ice crystals, so that they can be inactivated or damaged.

Oxygen and nitrogen atoms of the liquid to be treated, for example, water, which is the main component of blood, and viruses, blood proteins and virus biological elements (enzymes, DNAs, nucleic acids, etc.) are separated from surrounding water molecules. They form hydrogen bonds between them and protect their proteins and biological elements with surrounding water molecules, contributing to the stabilization of blood and viruses.However, after cooling from room temperature to below freezing and simultaneously pressurizing When depressurized, pressure compresses water and bioelements, causing strain, and these hydrogen bonds reform to form the most thermodynamically stable and optimal conformation in the new environment. I do. This hydrogen bond has a different structure from the hydrogen bond initially formed under normal pressure at 4 ° C., so that the hydrogen bond dissociates and re-associates to form a reasonable shape in a new environment.

This phenomenon is similar to the phenomenon when urea or alcohol, which is a hydrogen bond-cleaving agent, is applied to proteins or carbohydrates, but the amount of liquid compression is extremely small.
The dissociation and association of this hydrogen bond cannot be forcibly promoted. Therefore, it is necessary to additionally provide heat energy. This is the reason why a predetermined processing temperature must be maintained in the present invention. However, instead of heating as in a heat treatment, cooling below the freezing point obtains auxiliary energy for inducing hydrogen bond dissociation and association. Therefore, it is different from the cleavage of a hydrogen bond by heating.

The minimum processing time depends on the level of the pressure to be applied, and is about 20 minutes at 400 MPa, and 4 minutes at 300 MPa.
The 0 th quantile is appropriate. Pressure from 400MPa to 300M
When the pressure is reduced to Pa, the driving energy that can be taken out to cleave the hydrogen bond decreases, so that a new hydrogen bond is formed at 300 MPa and the time required for thermodynamic equilibrium is longer than in the case of 400 MPa. . That is, as the pressure decreases, the minimum processing time increases.

The invention according to claim 2 is the method for inactivating a virus according to claim 1, wherein the liquid to be treated is blood or a blood product. Even if the liquid to be treated undergoes heat denaturation, the activity of the blood or blood product itself is lost,
No insoluble material is produced.

The energy transmitted to the protein contained by pressurization is extremely small (0.08 Kcal (0.3328 Jo).
ul) / mol), so that it is less susceptible to denaturation such as heating. Moreover, protein damage due to volume changes due to ice crystal formation is extremely low. The reason is that there is no membrane-like tissue structure such as a cell membrane in the protein. Therefore,
It is very unlikely that the solution to be treated loses its activity and is denatured. Since the phase change of the liquid (mainly water) is used, the safety is extremely high and the virus in the liquid to be treated can be surely inactivated. Also, almost no additives are required for stabilization.

In the invention according to claim 3, the processing temperature is -2.
The method for inactivating a virus according to claim 1 or 2, wherein the method is at 5 ° C to -5 ° C, the minimum treatment time is 20 minutes to 120 minutes, and pressure is applied at 200 MPa to 400 MPa. The conditions for the specific treatment for inactivating the liquid are clear, and since the energy transmitted to the liquid to be treated is extremely small because the treatment is performed at a temperature of 400 MPa or less below freezing, it is difficult for the liquid to be treated to be denatured by heat. No, very high safety. Further, the virus in the liquid to be treated can be reliably inactivated in a short time. Furthermore, additives for stabilizing the protein are almost unnecessary and safe.

The invention according to claim 4 is to pressurize at a processing temperature at which the indicator virus can be deactivated and at a minimum processing time to inactivate all viruses contained in the liquid to be processed.
3. The method for inactivating a virus according to any one of 3.
In addition to specifically killing the indicator virus having the highest pressure resistance (a virus having a very small head shell size), it is possible to inactivate any virus having a smaller resistance. (Embodiment) Hereinafter, a virus inactivating device capable of inactivating a virus according to an embodiment of the present invention will be described. FIG. 1 is a configuration diagram of a virus inactivation device that can carry out a virus inactivation method according to an embodiment of the present invention.

In FIG. 1, reference numeral 1 denotes a high-pressure container made of a corrosion-resistant metal such as stainless steel and which withstands a pressure of 1000 MPa at the maximum, 2 denotes a cylindrical upper lid screwed to the high-pressure container 1, and 3 denotes a high-pressure container formed in the high-pressure container 1. The high-pressure chamber 4 is a stainless steel pressure-resistant cylinder which is inserted into the high-pressure container 1 to form the high-pressure chamber 3. Reference numeral 5 denotes a male screw formed on the outer periphery and a female screw formed on the inner periphery. After the pressure-resistant cylinder 4 is accommodated in the space of the high-pressure container 1 to form the high-pressure chamber 3, The first holding member 6 is a second holding member provided with a communication passage communicating with the high-pressure chamber 3 at the center, and is screwed to a female screw formed on the inner periphery of the first holding member 5. The high-pressure container 1 is provided with a heat exchange coil through which a refrigerant passes, and the high-pressure chamber 3 is a constant-temperature water tank. The high-pressure chamber 3 contains a flexible sealed container made of polypropylene or the like filled with a liquid to be treated such as blood or a blood product. A flexible bag made of a sealed flexible sheet may be used. After this accommodation, the pressure medium is accommodated in the high-pressure chamber 3, and the whole is kept at a predetermined processing temperature (-25 ° C to -5 ° C) below the freezing point.

7 is a tank for storing the pressure medium, 8 is a pressure pump for sending the pressure medium to the high-pressure chamber 3, and 9 is a pressure pump for increasing the pressure of the pressure medium pressurized by the pressure pump 8 and sending it to the high-pressure chamber 3. It is a booster. 10 is an area ratio of 1:20 accommodated in the pressure intensifier 9
, A primary pressure line connecting two cylinder chambers having an area ratio of 1:20, and a primary pressure line 1
1 is a diaphragm pump having a discharge pressure of about 60 MPa.

Reference numeral 13 denotes a secondary pressure line which is connected to the pressure intensifier 9 and is connected to a communication passage provided in the second holding member 6 to pressurize the pressure medium accommodated in the high pressure chamber 3; A check valve 16 capable of sending the high-pressure medium increased in pressure by the pressure unit 9 to the secondary pressure line 13 without backflow, and a pressure-reducing valve 16 (operating speed （0.1 second). Reference numeral 17 denotes a pressure sensor for detecting the pressure of the pressure medium in the high-pressure chamber 3, and reference numeral 18 denotes a temperature sensor for detecting the temperature of the pressure medium in the high-pressure chamber 3. It is provided in the next pressure line 13. 19 is a heat source unit, 20 is a timer for measuring the time for pressurizing or keeping the temperature of the pressure medium, 21 is composed of a microcomputer or the like, and controls the pressure of the diaphragm pump 12 based on the pressure detected by the pressure sensor 17, The control unit controls the heat source unit 19 based on the water temperature detected by the temperature sensor 18. The control unit 21 performs pressurization and heating according to the time counted by the timer 20.

Next, a procedure for killing a virus in a liquid to be treated packed in a flexible container using the virus inactivating apparatus of the present embodiment will be described. In addition, as the liquid to be treated of the present invention, for example, an albumin preparation, a blood preparation such as an immunoglobulin preparation, and blood as its raw material,
Examples include liquids such as plasma and foods such as meat. In addition, extracorporeal circulation of serum or blood (for example, artificial dialysis, cardiopulmonary bypass, etc.)
In this case, extracorporeally circulating blood and the like are also included.

When the flexible container is accommodated in the high-pressure container 1 and a start button (not shown) of the control unit 21 is pressed, the operation of the pressure pump 8 and the diaphragm pump 12 is started. The pressurized pressure medium is sent into the high-pressure chamber 3 through the check valves 14 and 15 while bleeding air.
In the present embodiment, the pressure medium is water. Phase change (freeze)
Considering that, water is the best pressure medium. At the same time, the control unit 21 starts heating the heat source unit 19 and starts heat exchange. The heat source 19 and the temperature sensor 18 maintain the water inside at a processing temperature selected below the freezing point (−20 ° C. to −5 ° C.). When the pressure sensor 17 detects that the set pressure selected for each virus has been reached, the minimum processing time specific to each virus is set to the timer 20.
To count. In addition, in this specification, the head shell of a virus means the outer membrane (Envelope) of a virus, or a head shell (Capsid). During this processing time, the control unit 21
Controls the processing temperature and the set pressure. By maintaining the processing temperature and the set pressure, it is possible to destroy the virus crust even if the pressure and the temperature are not relatively high, and the 200M
The virus can be inactivated at a relatively low pressure of Pa to 400 MPa.

When the timer 20 counts the processing time from 20 minutes to 120 minutes, the control unit 21 receives the processing time, and
Release 6 at once. At this time, the virus in the flexible container in the high-pressure chamber 3 is destroyed at once by the volume expansion force accompanying the phase change and is killed. After that, the upper lid 2 is removed and the flexible container is taken out of the high-pressure container 1. Virus inactivation is achieved by taking the above steps.

Next, according to the virus inactivation method of the present embodiment, why the virus can be inactivated or killed, and why the protein components such as blood are not denatured, will be described in detail below. .

Enzymes, nucleic acids, and the like, which are biological elements of a virus, contain protein molecules and contain N atoms and O atoms. DN
It is well known that A contains N atoms and O atoms and is bonded by hydrogen bonds, but protein molecules such as enzymes and nucleic acids also contain N atoms and O atoms, which interact with surrounding water molecules. They form hydrogen bonds between them, protecting enzymes and nucleic acids with water molecules. A major cause of denaturation of protein molecules is that the hydrogen bond formed between the N atom or O atom of these protein molecules and the O atom of the water molecule is cleaved to remove the protection mechanism. So, first, we will explain how the hydrogen bonding of protein molecules is affected by pressure.

The pressure denaturation of the protein is performed at a pressure of 250 MPa.
Occurs when the temperature reaches the degree, and changes from the undenatured state to the denatured state. However, this pressure band is called a “fluctuation” region, and when pressure is released, the pressure is released from denaturation and returns to the original undenatured state. The “fluctuation” region and the pressure at which pressure denaturation differs depending on the structure of the protein molecule.

For example, bovine serum albumin having four SS bonds (disulfide bonds) in a protein molecule is 25 bovine serum albumin.
It is not denatured by pressurization at 400 ° C. for 9 minutes, and pressurization at 25 ° C., 600 MPa for 9 minutes or more is required to impart denaturation. A region around 450 MPa is a “fluctuation” region.

By the way, under a pressure of 300 MPa, not only the water molecules as the dispersion medium but also the protein molecules themselves are compressed, causing a strain of about 8% or more (however, the processing temperature is slightly different). However, this distortion is caused by the water molecules around which N atoms and O atoms contained in protein molecules exist.
Denaturation does not occur because hydrogen molecules form hydrogen bonds with the atoms and the water molecules themselves protect the protein molecules. However, when the pressure is increased to 300 MPa or more, the strain generated in water molecules and protein molecules increases, and it is no longer thermodynamically possible to maintain the hydrogen bond formed at normal temperature and normal pressure. Cleaves. At this point, denaturation of the protein begins. Then, under this pressure, the 8% distorted water molecules and the distorted protein molecules adapt to each other's environment and form new hydrogen bonds necessary to create the most thermodynamically efficient conformation. Over time, this formation proceeds and denaturation of the protein molecule proceeds.

However, the denaturation under pressure does not proceed to the final stage unlike the denaturation by heating. The reason is that the energy required to completely destroy the three-dimensional structure of the protein molecule is not present in the strain of water, that is, in pressurized water.

From this, it seems that it is impossible to completely destroy the protein molecule by applying the pressure treatment, and it seems that the biological element of the virus cannot be inactivated by the application of pressure. Non-freezing area by pressurizing water shown in
That is, the use of supercooled water (hereinafter, supercooled water) solves this problem. Therefore, first, the influence of the phase change on the denaturation and how the structure of the protein molecule relates to the denaturation will be described below. FIG. 2 (A)
FIG. 2B is a phase diagram of water, and FIG. 2B is a diagram showing the relationship between temperature and pressure on the rate of volume increase accompanying depressurization.

As shown in FIG.
Water at Pa and −25 ° C. is supercooled water. Because it is compressed by about 7%, it is not water having the same structure as water having a regular tetrahedral structure under normal pressure. However, the supercooled water undergoes a phase transition to hexagonal Ih-type ice via a water structure close to a tetrahedral structure in a very short time when the pressure is released. Therefore, when normal water is pressurized to −20 ° C. and 300 MPa and then depressurized, the structure of water at 4 ° C. and normal pressure is −20 ° C. and 300 M
Dissociation and association of hydrogen bonds are forcibly performed to a Pa structure and further to an ice crystal structure at −20 ° C. and normal pressure, and recombination of hydrogen bonds is performed.

The volume change that occurs during this process is 300MP
a, about 19% increase compared to the volume at -20 ° C. This volume increase is sufficient to destroy the virus's head shell with volume expansion force. The dissociation and association of hydrogen bonds that occur during this phase transition inevitably denature proteins.
However, the denaturation of various protein molecules is not the same,
Denaturation depends on the intrinsic structure of the protein molecule.

For example, bovine serum albumin having four SS bonds (disulfide bonds) in the molecule is not denatured by such treatment. And human serum albumin has 17 SS bonds and has a stronger structure. However, some proteins contained in Gram-negative bacteria and a small amount of proteins constituting a biological element contained in Escherichia coli T4 phage are denatured because they include those having a small number of SS bonds. . In other words, it can be seen that a protein having a large number of SS bonds is less likely to be denatured even when pressurized for a relatively long time, and a protein having a small number of SS bonds is denatured when pressurized for a long time. The energy itself transmitted from the pressure to the water molecules is about 0.06 Kc at 300 MPa.
Since it is extremely small at al / mol (0.2496 Joule / mol), it does not give fatal denaturation to proteins unlike heat treatment. For example, for egg albumin which has no SS bond and easily undergoes pressure denaturation, when compared to a protein denatured under pressure of 400 MPa for 9 minutes and a protein denatured by heating at 80 ° C. for 9 minutes, the former has a surface hydrophobicity of the latter. It is 1/30 to 1/50 times the value. The reason is that the energy transmitted from the pressure to the protein is extremely small, and the pressure cannot transform the protein fatally.

As described above, proteins such as human blood and blood products are relatively strong even when subjected to pressure treatment, and include proteins having high pressure sensitivity to protein molecules of biological elements which are present in very few viruses. For this reason, viruses are relatively easily inactivated by sub-zero freezing. In addition, this inactivation method utilizes the phase change from water to ice crystals,
A large volume expansion can be elicited and, for viruses, this volume expansion force acts in addition to the above-mentioned denaturation, so that the skull can be specifically and completely destroyed. And then,
Even if proteins of human blood and blood products are mixed, these proteins are not destroyed because they do not have a membrane-like tissue structure. As described above, this inactivation method has an excellent effect that the cleavage of hydrogen bonds and the destruction of the skull due to phase transition act synergistically on viruses, and do not denature human blood or blood products. It can be seen that the virus inactivation method was used.

Incidentally, there are various types of viruses, and their sizes are also various. The basic explanation of the destruction of the skull of the virus has been given above, but it is explained that the skull size is closely related to the destruction of the skull. That is, as the head shell size decreases, the volume change accompanying the phase change of the liquid decreases in proportion to the cube of the size, so that the breaking force due to the volume change relatively decreases. The reason is,
This is because, contrary to the volume change, the surface area relatively increases, and the breaking force per unit surface area decreases opposite to the volume.
However, the above explanation is a general explanation of the tendency of skull destruction because it is unique to each virus. Therefore, a virus with a very small head shell size is inactivated,
It is difficult to die. This is why viruses that are extremely small in size are considered indicator viruses. As described above, in the case of a virus having a small head shell size, the processing temperature, pressure, and minimum processing time for increasing the destructive power should be further examined.

Next, as having a relationship with skull destruction,
Have a fever. Heat increases the electron radius of a substance, increases the radius of the nucleus, and weakens the bonding of molecules. In the process of heating, the hydrogen bonds that form them naturally break. The minimum expression temperature is about 60 ° C. for a biopolymer. For example, for egg protein, it is about 62 ° C., which corresponds to the lowest temperature at which boiled eggs can be formed. Because heat is transferred directly, it acts equally on all biological materials, including viruses.
Thus, increasing the processing temperature will cleave the hydrogen bonds of the virus, promoting the rate of virus inactivation. And even if the virus has a small head shell size, if the treatment temperature is raised, the DNA of the virus and the enzymes essential for maintaining the life of the virus can be damaged or inactivated. This is an application of a so-called heat treatment.

However, the processing temperature of the present invention is not heated for such a purpose. Rather, since the amount of liquid compression is extremely small, this alone cannot promote the dissociation and association of hydrogen bonds, It is used as a heat energy source. Therefore, the temperature does not need to be high enough to inactivate the virus by itself, but is used to weaken the molecules and to produce the destruction due to the phase change under pressure and below freezing.

Next, the specific conditions under which the head shell of the virus is destroyed by the volume expansion force will be described. As shown in FIG. 2 (B), the volume of water increases by about 9% from 0 ° C. water to 0 ° C. ice due to the phase change. At this time, if the pressure is 100 MPa,
Further, about 4.2%, 200 MPa, about 7.6%, and 300 MPa, about 10% increase. Accordingly, depressurization from a pressurized state below the freezing point involves an extremely large volume increase.
This increase in volume directly destroys the skull of the virus.

For example, when the pressure is increased from −5 ° C. at a pressure of 200 MPa to normal pressure, the volume expands by 15.5% due to the volume expansion during non-freezing and the volume expansion accompanying freezing. Therefore, a processing temperature T and a pressure p are used, and a change ΔT
With respect to the change Δp of p (hereinafter, ΔT and Δp are omitted in the notation), the compression rate in the same phase is C (T, p), the volume expansion rate accompanying the phase change is Ci, and the phase change is The average value of the bulk modulus before the occurrence of the phase change is βav1 (T, p), and the average value of the bulk modulus after the occurrence of the phase change is βav2 (T, p).
p), when the bulk modulus at the time of the phase change is βav, the average instantaneous volume expansion force per unit volume after the phase change is caused and βav1 (T, p) · C (T, p) +
βav2 (T, p) · C (T, p), to which βav · Ci at the time of freezing is added, so that the phase change that occurs when the pressure is reduced from the processing temperature T and the pressure p to normal temperature and normal pressure. Is the volume expansion force F with F = βav1 (T, p) · C
(T, p) + βav2 (T, p) · C (T, p) + βa
v · Ci, which causes destruction when it exceeds the destruction limit value R (T, p) of the skull specific to each virus. That is, F> R (T, p) is a condition for specifically killing the virus.

As described above, the method of inactivating the virus according to the present embodiment comprises, as the first step, 200 MPa to 40 MPa.
A pressure of 0 MPa was applied, and the processing temperature was -2.
By keeping the temperature at 5 ° C. to −5 ° C. and maintaining it for at least 20 minutes to 120 minutes, the thermodynamically dissociated and associated hydrogen bonds are brought into an equilibrium state under pressure. Then, as a second stage, the pressure is released, the hydrogen bond in the supercooled state is broken again, and the enzymes and the like that play an important role in maintaining the life of the virus are inactivated. At the same time, the supercooled water undergoes rearrangement of hydrogen bonds and shifts to an Ih ice crystal type structure. This structural change causes volume expansion, and the virus's head and shell is destroyed and completely killed.

As described above, according to the present embodiment, the biological element of the virus, which is the most important in maintaining the life, and the destruction of the skull is precisely controlled by the dissociation and association of hydrogen bonds centering on water. Deactivation and destruction can be easily controlled by pressure and sub-zero temperatures.

Further, since the treatment is performed at a temperature below the freezing point, it can be applied to a liquid to be treated which is frozen such as a blood product, and the energy transmitted to the blood is small even if the treatment is performed at 400 MPa. .08Kcal (0.3328
Joule) / mol), although the virus is inactivated,
Protein components such as blood do not agglomerate or insolubilize.

Further, the power cost is extremely small due to the pressurization of the liquid of the incompressible fluid. Moreover, since the treatment is performed at a relatively low pressure, the safety is extremely high, and the virus in the liquid to be treated can be surely killed. Also, since the energy added to blood is small, the amount of stabilizers for suppressing protein denaturation can be reduced as much as possible.

The other conditions are the same as those of the present embodiment, and if the pressure is increased to 400 MPa or more, the virus can be reliably inactivated, but the denaturation of blood proteins proceeds.
Therefore, when the pressure exceeds 400 MPa, it should be noted that there is a pressurized region which shows a uniform effect in terms of virus inactivation, although the practicality is slightly inferior.
Similarly, in the non-freezing region, even at −25 ° C. or less,
It should also be noted that, although less practical, there is a region that shows a uniform effect in terms of deactivation.

[0061]

FIG. 3 is a graph showing the relationship between the pressurizing time and the pressure on the number of surviving viruses in the decompression treatment to induce freezing, and FIG. 4 is a graph showing the survival of the virus in the decompression treatment to induce freezing. 9 is a graph showing the relationship between pressure and temperature on the number. The virus used for the experiment was Escherichia coli phage T4, which was used for a predetermined time.
The cells were kept warm and pressurized to measure the number of surviving bacteria (cfu / ml). In the experiment, one 56MPa diaphragm pump was used.
1:20 booster installed on the next line and up to 1000M
The test was performed in a high-pressure vessel of Pa. The pressure medium of the secondary line is water, and the liquid to be treated has an inner diameter of 10 mm and a height of 16 mm (with an inner volume of 1 mm).
ml) of polypropylene containers. Pressure is 0
５００500 MPa and the temperature was in the range of −20 ° C. to 50 ° C.

The preparation of the T4 phage suspension was carried out in the following procedure. One plaque was punctured with a platinum wire, and peptone top agar (Peptone Top Agar: peptone 10)
g, NaCl 5g, agar 5g, total volume 1000ml, pH
7.2) to which the host cells (Escherichia coli I
FO 13168) is overlaid with 0.1 ml of an overnight culture,
And cultured. After culturing for 3-4 hours, 3 ml of T2 buffer (3.0 g of Na2HPO4, 1.5 g of KH2PH4, NaCl
4.0g, K2SO4 5.0g, MgSO4 0.1M10m
1, 0.1 ml of CaCl 2, 1 ml of gelatin 1%, total volume 1000 ml), 1 drop of chloroform was added dropwise, and the mixture was cultured for 1 hour. After the culture, the upper layer liquid was collected, and adjusted by low-speed centrifugation (3000 rpm, 10 minutes).

The surviving cell count (cfu / ml) was determined as shown in FIGS.
Were measured at the experimental points shown in FIG. An overnight culture of the host bacterium, a phage suspension of T4 phage, and a peptone top agar were overlaid on a peptone agar medium, and cultured at 37 ° C. for 1 day.
The number of surviving bacteria (cfu /
ml).

As shown in FIG. 3, according to this experiment, the number of surviving bacteria decreases almost in proportion to the treatment time. As can be seen from the measurement results at −5 ° C., the number of bacteria decreased by about one digit at a treatment time of 30 minutes at 200 MPa, but the virus was almost completely inactivated at 30 minutes at 300 MPa,
In a, the virus was completely inactivated by the treatment for 20 minutes.

Next, the relationship between the pressure and the processing temperature becomes clear from FIG. This processing time is 120 minutes. As can be seen from the experimental results, at a processing temperature of room temperature or higher, 300
The virus does not decrease unless the pressure is higher than MPa. 500 MPa is required for complete killing. 300M
The number of bacteria decreased under the condition of Pa or more and 25 ° C. or 50 ° C., because the DNA of the virus or the hydrogen bond of the important enzyme was cleaved by high pressure and heat.

However, at 400 MPa, the strain amount has not yet reached the level at which hydrogen bonds can be completely broken. So 400MP
In order to break the hydrogen bond below a, the help of thermal energy is required separately. In other words, cooling to -5 ° C or less even at 400 MPa or less, depressurizing, and forming ice crystals from supercooled water easily causes re-destruction of hydrogen bonds and formation of new hydrogen bonds accompanying ice crystal formation. Can be. This inactivates the enzyme and, at the same time, the rapid volume expansion accompanying the phase change completely destroys the crucial skull of the virus, which is vital for life, and causes death. And
At −20 ° C., the virus is completely killed at 300 MPa.

As described above, 200 MPa to 400 MPa
a, desirably, at a pressure of 300 MPa to 400 MPa, and at a processing temperature of −25 ° C. to −5 ° C.,
By maintaining this for at least the minimum processing time of 20 minutes to 120 minutes, the virus can be killed and inactivated.

[0068]

As described above, according to the first aspect of the present invention,
In the method for inactivating viruses described in (1), the treatment is performed at a temperature below freezing point. Less. Pressure is 200MPa ~ 400MPa
Is sufficient, the equipment cost is easy and the safety is high.
Viruses in the liquid to be treated, including HIV, can be reliably killed and inactivated. In addition, the use of additives for stabilization can be reduced to zero or less.

The method for inactivating a virus according to claim 2 of the present invention relates to a method for inactivating a virus such as blood or blood products, even if the liquid to be processed undergoes denaturation of proteins when subjected to heat treatment. It is very unlikely that the activity of itself will be lost and an insoluble substance will be produced.

In the method for inactivating a virus according to claim 3 of the present invention, the treatment temperature is from -25 ° C to -5 ° C, the minimum treatment time is from 20 minutes to 120 minutes, and
Since the pressure is increased at 400 MPa, the liquid to be treated does not deteriorate, and the pressure is low.
Viruses in the liquid to be treated can be reliably killed and inactivated in a short time.

The virus inactivation method according to claim 4 of the present invention is capable of specifically killing the most resistant indicator virus and inactivating all viruses having a lower resistance. it can.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of a virus killing apparatus capable of performing virus inactivation according to an embodiment of the present invention.

FIG. 2A is a phase diagram of water. FIG. 2B is a graph showing the relationship between temperature and pressure on the rate of volume increase due to depressurization.

FIG. Graph showing the relationship between pressurization time and pressure on the number of surviving viruses in the decompression treatment that induces freezing

FIG. 4 is a graph showing the relationship between pressure and temperature on the number of surviving viruses in a virus in a decompression process that induces freezing.

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 high-pressure container 2 top lid 3 high-pressure chamber 4 pressure-resistant cylinder 5 first holder 6 second holder 7 tank 8 pressure pump 9 intensifier 10 pressure-increasing piston 11 primary pressure line 12 diaphragm pump 13 secondary pressure line 14, 15 Check valve 16 Depressurizing valve 17 Pressure sensor 18 Temperature sensor 19 Heat source unit 20 Timer 21 Control unit

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) C12R 1:94) C12R 1:94)

Claims (4)

[Claims] 1. A flexible sealed container filled with a liquid to be treated is accommodated in a pressure medium, and the whole is kept warm and pressurized at a predetermined processing temperature below freezing for a minimum processing time, and then depressurized to remove the liquid to be treated. A method for inactivating a virus that inactivates a virus contained therein, wherein the processing temperature is set to a temperature indicating a supercooled state of the liquid to be processed under a pressurized freezing point equal to or higher than a predetermined pressure, and the minimum processing time is reduced. By performing the heat retention at the treatment temperature and the pressurization at the pressure, the liquid component of the liquid to be treated permeates through the virus crust and the hydrogen bond formed with the internal biological element is formed. Set to the time until thermodynamic equilibrium, penetrate the liquid into the virus while maintaining the processing temperature and the pressure for the minimum processing time or more, thermodynamically the hydrogen bonds formed in the skull To equilibrium From,
Thereafter, the liquid component permeated into the virus is phase-changed into ice crystals by depressurization, and the head and shell of the virus and biological elements are destroyed or inactivated by the structural change and volume expansion force of the ice crystals generated at that time. A method for inactivating a virus, characterized in that: 2. The method according to claim 1, wherein the liquid to be treated is blood or a blood product. 3. The method according to claim 1, wherein the processing temperature is -25.degree. C. to -5.degree. C. and the minimum processing time is 20 minutes to 120 minutes.
3. The method for inactivating a virus according to claim 1, wherein pressurization is performed at Pa to 400 MPa. 4. The inactivation of the virus according to any one of claims 1 to 3, wherein pressure is applied at a treatment temperature for inactivating the indicator virus and a minimum treatment time to inactivate all viruses contained in the liquid to be treated. Method.