JPH0321342A - Virus and cell adsorbing and separating agent and method for separation of virus and cell - Google Patents

Abstract

PURPOSE:To enhance the adsorbability to a virus or cell by forming a calcium phosphate type porous granule having two kinds of open pore structures one of which is composed of open micropores having a specific mean pore size and the other one of which is composed open small voids having a specific mean void size. CONSTITUTION:A slurry in which a crystal particle raw material of a calcium phosphate compound subjected to wet synthesis is suspended is granulated into secondary particles by direct spray drying and these particles are again suspended in a slurry form and subjected to wet molding to mold a block body. This block body is baked at about 500-1300 deg.C and subsequently ground and further classified to obtain a calcium phosphate type porous granule having two kinds of open pore structures one of which is composed of open micropores having a mean pore size of 20-50nm and the other one of which is composed of open small voids having a mean void size of 1-50mum is obtained. This porous granule is useful as a virus and cell adsorbing and separating agent.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Application The present invention relates to an adsorption/separation agent for viruses and animal and plant cells, and a method for separating viruses and animal and plant cells using the same.

"Prior Art and its Problems" Prevention and treatment of viral diseases such as influenza have long been an important issue in the medical field. In particular, in recent years, acquired immunodeficiency syndrome (AID)
S) The need for this has become even more important as the rapid increase in the number of people infected with the virus has become a hot topic. To date, methods have been proposed for separating and removing hepatitis B virus mainly in blood using polymer membranes, hollow fibers, or ion exchange resins. However, these separation systems are not only complicated but also expensive equipment, and have not been put into practical use.

In recent years, calcium phosphate compounds have been extensively studied as biomaterials for artificial bones, artificial tooth roots, etc. due to their excellent biocompatibility. In addition, it has long been used as a packing material for liquid chromatography to separate and purify biopolymers such as proteins, and with recent advances in technology, high-performance packing materials have been developed and are also used for analysis. It's starting to look like this. Furthermore, as shown in Japanese Patent Application Laid-Open No. 61-235752, specific adsorption ability for cells has recently been discovered, and immunological applications are also being considered. However, this publication does not consider the microstructure that calcium phosphate granules should have as a cell separation agent, and although the operation time is shortened, there is a problem with water retention, and there is still room for improvement in separation performance. There is.

Furthermore, JP-A No. 63-284 describes that fibrous apatite improves water retention and separation performance. It is difficult to fill a separator such as a column uniformly in a granular type, and there is a large variation depending on the loft, which has the disadvantage that the performance is less stable than that of a granular type.

On the other hand, JP-A No. 63-16045 discloses calcium phosphate-based porous granules as a packing material for liquid chromatography, but the fine pore structure that makes them effective as an adsorption/separation agent for viruses or cells is unknown. No consideration has been made. "Objective of the Invention" The object of the present invention is to improve the adsorption capacity of calcium phosphate compounds for cells, and to create fine particles that have higher separation adsorption performance, sufficient water retention, and excellent stability. An object of the present invention is to provide an adsorption/separation agent for viruses and cells, and a separation method using the same.

"Structure of the Invention" The virus and cell adsorption/separation agent of the present invention has an average pore size of 2
Continuous micropores of 0-500 nm and average pore diameter of 1-5
It is characterized by being a calcium phosphate-based porous granule having two types of continuous pore structure of 0 μm continuous small pores.

The adsorption/separation agent of the present invention is a porous granule having two types of continuous pore structures as described above. In order to adsorb viruses to granular adsorption/separation agents, it is necessary to make the granules porous in order to lengthen the passage route. The size of the virus is
Although it varies depending on the type, it generally has a diameter in the range of 20 to 300 nm. Therefore, the pore diameter for the virus to pass through needs to be 20 nm or more. In addition, if the pores are much larger than the virus, it will only reduce the chance of adsorption and become meaningless, so the pore diameter should be 50
It is sufficient if it is 0 nm or less. On the other hand, in order to provide these granules with sufficient water retention, they must have continuous small pores of 1 to 50 μm through which the suspension of viruses and cells can penetrate relatively easily.

In the adsorption/separation agent of the present invention, the granules include the above-mentioned 2
It is preferable to have a different pore structure and a porosity of 10 to 75%. The porosity of the granules is closely related to water retention; if it is less than 10%, the water retention required for adsorption of viruses and cells cannot be obtained, and if it exceeds 75%, the granules themselves cannot maintain their strength. Furthermore, the adsorption/separation agent of the present invention has an average particle size of 10 to 2000.
Preferably they are micron granules. For cell separation, if the diameter is 100 μm or more, cells can pass through the gaps between the granules. However, if it exceeds 2000 μm, the gap becomes too large and cells cannot be adsorbed. Further, for the purpose of separating viruses, a particle size of 1 μm or more is sufficient, but for practical purposes, a particle size of 10 μm or more is preferable.

Calcium phosphate-based porous granules having two types of pore structures as described above can be produced by various methods using crystal particles of a calcium phosphate-based compound wet-coagulated by a known method as a raw material. For example, a slurry in which raw material particles are suspended is directly granulated into secondary particles by spray drying, or additives such as a viscosity modifier, pyrolyzable organic compound particles, or fibers are added to this slurry and then spray dried. It is granulated into secondary particles by methods such as. These secondary particles are suspended again in the form of a slurry and shaped into a block by either a wet process or a dry process using pressure. At this time, an organic compound may be added to form pores of 1 to 50 μm by thermal decomposition by burning. Even without additives, the temperature can be improved by adjusting other conditions such as firing temperature. Pore diameter can also be controlled. The obtained block body is burned at a temperature in the range of 500°C to 1300°C. If the temperature is less than 500°C, the thermal decomposition of the organic compound and the sintering of the block body will not take place sufficiently. Furthermore, if the sintering is performed at a high temperature exceeding 1300° C., there is a risk that the sintered body will become too dense or that the calcium phosphate will decompose.

After pulverizing the thus fired block, it can be classified to obtain granules of the required particle size. 20-5 of this granule
The fine pores of 0.0 nm can be adjusted by appropriately adjusting the size of crystal particles in the raw material slurry for secondary particle granulation, the viscosity of the slurry, additives, etc. Moreover, the small pores of 1 to 50 μm can be adjusted by appropriately selecting the forming method and additives when forming the secondary particles into blocks as described above.

The calcium phosphate used in the adsorption/separation agent of the present invention has a Ca/P ratio of 1. 4-1. Various types of 8 can be used, but considering the sinterability and strength of the granules, 1. It is preferable that it is 5-1.67. More specifically, various abatites such as hydroxyapatite and fluoroabatite, α- and β-tricalcium phosphate, tetracalcium phosphate, and these two
Mixtures of more than one species can be used.

In the adsorption/separation agent of the present invention, multi-tJM [, muco-poly#M and It is also possible to partially adsorb one or more proteins or their derivatives and modulate the physicochemical or immunological properties of the granule surface to skillfully change the adsorption activity for each target cell group. Thereby, the separation characteristics of the column can be effectively changed, and the separation characteristics that provide a sharp elution pattern and the separation spectrum can be controlled. Furthermore, it is also possible to selectively isolate a specific subbobulation by modulating the adsorption effect on each subbobulation of the cell. The adsorption/separation agent of the present invention adsorbs viruses and specific animal and plant cells. Therefore, the present invention provides a method for separating viruses and cells from biological fluids using this adsorption separation agent.

Biological fluids include blood, serum, urine, saliva or suspensions of cells and/or viruses. The separation method of the present invention can be used to remove viruses in biological fluids, collect specific cells, for example, collect T cells from whole blood or lymph cells. When whole blood is used, it is preferable to add a blood coagulation inhibitor such as heparin or citric acid at the time of blood collection, and dilute it 1 to 10 times with a culture medium. The use of a diluent reduces the viscosity of the blood, thereby facilitating separation. However, if it is diluted more than 10 times, the amount to be handled will be large, which is inconvenient. To separate viruses or cells using the adsorption/separation agent of the present invention, fill a separator such as a column with glass wool or the like on the liquid outlet side to prevent the adsorption/separation agent from flowing out.
Fill the adsorption separation agent, wash it, then pour in the biological liquid, allow this biological liquid to fully penetrate, and then pour out the cleaning liquid.
Non-adsorbed cells can be washed away and collected.

The suspension solution, dilution culture solution, or washing solution can be appropriately selected depending on the virus or cells contained, but examples include physiological saline, Hank's medium (HBSS), serum medium (e.g. RPMI-1640), and serum-free medium. A culture medium etc. can be used.

In addition, when separating useful cells such as TII cells, it is necessary to
It is preferable to carry out the separation operation at a temperature of about °C. The T cells thus collected were substantially the same as before the isolation procedure in terms of survival rate, antibody production regulatory function, and other properties.

"Examples of the Invention" Next, the present invention will be described in more detail based on Examples, but the present invention is not limited thereto.

In addition, in the following Examples and Comparative Examples, influenza virus PR8 was used suspended in physiological saline. In addition, the titer was measured using the following method. How to measure titer When the influenza virus attaches to red blood cells, the red blood cells agglutinate with each other. Using this reaction, the virus suspension was serially diluted 2 times, 4 times, 8 times, 16 times, etc. with physiological saline (0.9% sodium chloride aqueous solution), and the same amount 0. The titer of the stock solution (liquid before dilution) is evaluated by mixing it with a 4% chicken red blood cell suspension and determining how many times the dilution causes an agglutination reaction. Example I I20 hydroxyapatite with a Ca/P ratio of 1.67
Calcined at 0℃, porosity 20%, particle size 300-600am
granules were produced. The average micropore diameter of this granule is 20n
m, and the average small pore diameter was 2 μm. Add glass wool to a syringe with an internal volume of 6 d. 0 3 g
Fill it to approximately lm1, and add the above granules 1 on top of it.
Filled with g. A suspension of influenza virus PR8 was passed through the column thus prepared, and the titer of the virus suspension after passing through the column was measured by the following method. Results first
It is shown in the table. Example 2 Example 1 except that the particle size of the granules is 100 to 300 μm
Using the same granules, the same procedure as in Example 1 was carried out, and the titer of the virus suspension after passing through the column was measured. The results are shown in Table 1.

Example 3 The titer of the virus suspension after passing through the column was measured in the same manner as in Example I, except that 3 g of the same granules as in Example 2 were used, and the results are shown in Table 1.

Example 4 Hydroxyapatite with a Ca/P ratio of 1.67 was
Calcined at °C, porosity 45%, particle size 100-300μm
granules were produced. The average micropore diameter of this granule is 50n
m, and the average small pore diameter was 4 μm.

The same operation as in Example 1 was carried out using 1 g of the obtained granules, and the titer of the virus suspension after passing through the column was measured. The results are shown in Table 1.

Example 5 Hydroxyapatite with a Ca/P ratio of 1.67 was
Calcined at °C, porosity 60%, particle size 100-300μm
granules were produced. The average micropore diameter of these granules is 100
nm, and the average small pore diameter was 6 μm.

The same operation as in Example 1 was carried out using 1 g of the obtained granules, and the titer of the virus suspension after passing through the column was measured. The results are shown in Table 1.

Example 6 The same operation as in Example 1 was carried out using 2 g of the same granules as in Example 5, and the titer of the virus suspension after passing through the column was measured. The results are shown in Table 1.

Example 7 Calcium phosphate with a Ca/P ratio of 1.5 was calcined at 1100°C to produce granules with a porosity of 30% and a particle size of 100 to 300 μm. The average micropore diameter of these granules is 300 nm,
The average small pore diameter was 8 μm.

The same operation as in Example 1 was carried out using 1 g of the obtained granules, and the titer of the virus suspension after passing through the column was measured. The results are shown in Table 1.

Example 8 Ca/P ratio 1. Calcium phosphate No. 5 was calcined at 700° C. to produce granules with a porosity of 60% and a particle size of 100 to 300 μm. The average micropore diameter of this granule is loonm,
The average small pore diameter was 10 μm.

The same procedure as in Example I was carried out using 1 g of the obtained granules, and the titer of the virus suspension after passing through the column was measured. The results are shown in Table 1. Example 9 The same operation as in Example 1 was carried out using 2 g of the same granules as in Example 8, and the titer of the virus suspension after passing through the column was measured. The results are shown in Table 1.

Example 10 Ca/P ratio l. Calcium phosphate No. 57 was calcined at 1100° C. to produce granules with a porosity of 25% and a particle size of 100 to 300 μm. The average micropore diameter of the granules was 50 nm, and the average small pore diameter was 3 pm. The same procedure as in Example 1 was carried out using 1 g of the obtained granules, and the titer of the virus suspension after passing through the column was measured. The results are shown in Table 1. Comparative Example I Calcium phosphate with a Ca/P ratio of 1.67 was calcined at 700°C, and had only fine pores with an average pore size of 50 nm, a porosity of 50%, and a particle size of 100 to 300 μm. The same procedure as in Example 1 was carried out using 1 g of granules, and the titer of the virus suspension after passing through the column was measured, and the results are shown in Table 1.

Comparative Example 2 The same procedure as in Comparative Example 1 was used except that 2 g of the same granules as in Comparative Example 1 were used, and the titer of the virus suspension after passing through the column was measured. The results are shown in Table 1.

The titers shown in Table 1 are proportional to the concentration of influenza virus per unit volume in the effluent. Therefore, most rIi. Even in Example 1, which had poor tl, the number of viruses was reduced by half compared to Comparative Examples 1 and 2.
Moreover, in Examples 6 and 9, all the viruses were almost completely adsorbed and did not leak out, demonstrating the high adsorption performance of the calcium phosphate granules of the present invention. Further, from Examples 5, 6, 8, and 9, the adsorption amount of the granules according to the present invention can be increased by increasing the amount thereof. On the other hand, in Comparative Example 2, although twice as many granules as in Comparative Example 1 were used, the amount of adsorption did not improve.

Example 11 Glass wool was added to a syringe with an internal volume of 6 d. 0 3 g
was packed to approximately lIIIi, and 1 g of the granules produced in Example 1 was filled thereon. 0.2 d of Hank's medium in which 5 x 10" human peripheral blood lymphocytes were suspended was poured into the column prepared in this way, and after this suspension had sufficiently permeated the granules, 3III1 Hank's medium was poured to allow non-adsorbed cells to flow out. After labeling the collected cells with fluorescently labeled antibodies, anti-Leu 4 and anti-Leul 2,
F A C S (fluorescence acti
The positive rate of T cells and B cells was examined using a vated cell sorter. The results are shown in Table 2.

Example 12 Hydroxyapacite with a Ca/P ratio of 1.67 was added to 900
Calcined with ``C, porosity 45%, particle size 300-600μm
granules were produced. The average micropore diameter of this granule is 50n
m, and the average small pore diameter was 4 μm. Using 1 g of these granules, the same procedure as in Example 1l was performed to examine the positive rate of T cells and B cells. The results are shown in Table 2. Example 13 Ca/P ratio l. 67 hydroxyapatite to 700
Calcined at °C, porosity 60%, particle size 300-600μm
granules were produced. The average micropore diameter of these granules is 100
nm, and the average small pore diameter was 6 μm. Using 1 g of these granules, the same procedure as in Example 11 was performed to examine the positive rate of T cells and B cells. The results are shown in Table 2.

Comparative Example 3 Ca/P ratio 1. Hydroxyabatite No. 67 was burned at 700''C to produce granules with a porosity of 50% and a particle size of 300 to 600 μm, which had only micropores with an average pore size of 50 nm.

Using 1 g of these granules, the same procedure as in Example 11 was carried out to examine the positive rate of T cells and B cells. The results are shown in Table 2.

In Examples 11, 12, and 13 of Table 2, the amount of B cells adsorbed increases in this order, which is thought to be because the volume of the entire granule increases in that order, but in Example 12 and No. 13 showed good adsorption performance despite having a smaller volume than Comparative Example 3. In addition, Example 1l
Also in Comparative Example 3, higher adsorption was achieved than in Comparative Example 3.

Example 14 Hydroxy abatite with a Ca/P ratio of 1.67 was
Baked at 0℃, porosity 20%, particle size 300-600μm
granules were produced. The average micropore diameter of this granule is 20n
m, and the average small pore diameter was 2 μm. Add glass wool to a syringe with an internal volume of 6all. 0 3
The granules were packed to approximately 11 g, and the above granules lg were packed on top to form a column. Blood IId to which 13% citric acid was added as a blood coagulation inhibitor at the time of blood collection was flowed through the above-mentioned force ram, and the flowed blood was collected. Fluorescently labeled antibodies and CD
After labeling with NH.3 (anti-Leu4) and CD19 (anti-Leul2), NH. c/! Hemolyze the red blood cells with
The positive rate of T1ll cells and B cells was investigated using CS. The results are shown in Table 3. Example 15 After blood collection in the same manner as in Example 14, 2d of blood diluted 2 times with Hank's medium was poured into a column prepared in the same manner as in Example 14. It is shown in Table 3.

Example 16 After blood collection in the same manner as in Example 14, blood 4Id diluted 4 times with Hank's medium was poured into a column prepared in the same manner as in Example 15, and then operated in the same manner as in Example 14, and the results were evaluated. It is shown in Table 3. Table 3 Note that in the above examples, the adsorption performance for influenza viruses and lymph cells was measured, but the adsorption/separation agent of the present invention is effective not only for these viruses and cells but also for other viruses and cells. works similarly.

Furthermore, the adsorption/separation agent of the present invention can also be used as an adsorbent for plant cells such as cedar pollen, which causes hay fever. "Effects of the Invention" As described above, the adsorption/separation agent of the present invention exhibits high adsorption ability for viruses and animal and plant cells, and is effective in adsorption and separation of these. The adsorption/separation agent of the present invention selectively adsorbs B cells and macrophages, but does not affect T cells, so T cells can be recovered with high purity without changing the distribution of subsets. It can be used not only for analysis, but also for immunological research and clinical testing. As clinical tests, T cell distribution and function tests can be performed for cancer, autoimmune diseases, AIDS, and the like. In clinical tests, whole blood may be tested using a testing device as it is after blood collection. This makes it possible to perform high T cell tests.

Furthermore, the adsorption/separation agent of the present invention has sufficient water retention,
Moreover, since it has a fine structure with excellent stability, separation can be performed in a short time with high reproducibility. Furthermore, when performing separation using the adsorption/separation agent of the present invention, no incubation is required, so the operation is extremely simplified.

Claims (1)

[Scope of Claims] 1. A virus characterized by being a calcium phosphate-based porous granule having two types of continuous pore structures: continuous fine pores with an average pore diameter of 20 to 500 nm and continuous small pores with an average pore diameter of 1 to 50 μm; Cell adsorption separation agent. 2. The adsorption/separation agent according to claim 1, which has a porosity of 10 to 75%. 3. The adsorption/separation agent according to claim 1 or 2, which has an average particle diameter of 10 to 2000 μm. 4. The adsorption/separation agent according to claim 1, wherein one or more of biologically derived polysaccharides, mucopolysaccharides, proteins, and derivatives thereof are partially adsorbed on the surface of the granules. 5. A method for separating viruses and cells, which comprises flowing a biological liquid through a column packed with the adsorption/separation agent according to claim 1. 6. The separation method according to claim 5, wherein the biological liquid is blood, serum, urine, saliva, or a suspension of cells and/or viruses. 7. The separation method according to claim 6, wherein the biological liquid is blood to which an anticoagulant was added at the time of blood collection, diluted 1 to 10 times with a culture medium.