JP2009039038A - Method for separating substance to be separated and method for purifying the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new means for enhancing efficiency of separation/purification using poorly water-soluble inorganic compounds for separation of the substance consisting of a peptide, a nucleic acid and their complex. <P>SOLUTION: The method for separating substance to be separated consisting of the peptide, the nucleic acid and their complex comprises reacting at least one kind of cation selected from the group consisting of barium ion, calcium ion, magnesium ion and aluminum ion, with an anion reacting with the cation, forming a poorly water-soluble salt in an aqueous solution to be processed containing the substance to be separated, and making the substance to be separated adsorbed on the deposited poorly water-soluble salt. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  Insoluble inorganic compounds such as sulfates, phosphates, hydroxides, carbonates and aluminum phosphates or hydroxides belonging to Group 2 of the periodic table of biological materials are biologically It is known that inactivation is unlikely to cause inactivation of physiologically active substances. Since poorly water-soluble inorganic compounds have high affinity with organic substances such as biologically active proteins and nucleic acids, they are applied to the purification of vaccines, blood proteins, growth hormones, food additives, interferons, etc. (Patent Document 1, Non-Patent Document 1) Literature 1-6).

  In many cases, a purification method for a substance to be separated that uses a poorly water-soluble inorganic compound as an adsorption carrier is used by adding a powdery adsorption carrier to a test solution, and is generally used for solid-liquid separation during the purification process. A centrifuge is used. When increasing production, the amount of carrier used increases, so the test solution must be divided into a plurality of lots and a plurality of batches of purification steps must be performed. In addition, since the amount of industrial waste such as adsorption carriers increases due to increased production, much time must be spent on post-treatment.

  If the adsorption performance and elution performance of the poorly water-soluble inorganic compound used as the adsorption carrier can be further improved, the efficiency of separation / purification of the substance to be separated can be further improved. Can be reduced. To date, however, no satisfactory and useful means have been provided.

Japanese Unexamined Patent Publication No. 2000-262280 Voss, D.:Barium sulphate adsorption and elution of the 'prothrombin complex' factors., Scand J Clin Lab Invest. 1965; 17: Suppl 84: 119-128. C.B.Reimer et.al.:Purification of Large Quantities of Influenza Virus by Density Gradient Centrifugation., Journal of Virology., Dec. 1967. 1207-1216 H.Prydz: Studies on Proconvertin (Factor VII) IV. The Adsorption on Barium Sulphate, Scandinav. J. Clin. & Lab. Investigation, 16, 1964, 409-414 Andrzej G.: Inhibition of influenza A virus hemagglutin and induction of interferon by synthetic sialylated glycoconjugates, Can.J.Microbiol., Vol37, 1991,233-237 Glycotechnology First Edition 1st Edition Pages 549-552 (Industrial Research Institute Biotechnology Information Center) Tsutomu Kawasaki: Hydroxyapatite as a liquid chromatographic packing., Journal of Chromatography, 544. 1991.147-184

  Accordingly, an object of the present invention is to provide a novel means that can further improve the efficiency of a separation / purification method using a poorly water-soluble inorganic compound.

  As a result of diligent research, the inventors of the present application have added a suitable ion to a solution containing the substance to be separated in the separation process of the substance to be separated to form a poorly water-soluble salt in the solution. The present invention has been completed by finding that the substance to be separated is efficiently adsorbed to the salt.

  That is, the present invention provides a reaction between at least one cation selected from the group consisting of barium ion, calcium ion, magnesium ion and aluminum ion in an aqueous treatment liquid containing a substance to be separated, and the cation. Reacting with at least one kind of anion capable of forming a hardly water-soluble salt to cause precipitation of the hardly water-soluble salt in the liquid to be treated, and adsorbing the substance to be separated to the precipitated slightly water-soluble salt A method for separating a substance to be separated is provided. The present invention also provides a method for purifying the substance to be separated, which comprises allowing an eluate to act on the poorly water-soluble salt adsorbed with the substance to be separated obtained by the method of the present invention.

  According to the present invention, a novel method for separating and purifying a substance to be separated using a hardly water-soluble salt is provided. According to the method of the present invention, a substance to be separated can be efficiently separated with a less water-soluble salt than in the conventional method. Therefore, the separation / purification process can be made more efficient, and the amount of used hardly water-soluble salt discarded can be reduced.

  In the separation method of the present invention, a hardly water-soluble salt is precipitated in an aqueous solution (aqueous liquid to be treated) containing a substance to be separated, and the substance to be separated is adsorbed on the hardly water-soluble salt. As a result, an adsorbent composed of the same poorly water-soluble salt is prepared, and it exhibits better adsorptivity with a small amount of the poorly water-soluble salt than when it is used by adding it to the liquid to be treated (Examples below). reference). Although the detailed principle is unknown, in the separation method of the present invention, since the substance to be separated and the poorly water-soluble salt are kept in contact with each other from the start of formation of the slightly water-soluble salt fine particles, a separately synthesized poorly water-soluble The contact area between the substance to be separated and the sparingly water-soluble salt can be increased as compared with the case where salt is added, and it is considered that adsorption is performed efficiently. The “aqueous solution” refers to a solution in which 50% or more of the solvent is water. The aqueous liquid to be treated may contain a solvent other than water, such as a hydrophilic organic solvent, as long as 50% or more of the solvent is water, but an aqueous solution containing only water is preferable.

  Here, “slightly water-soluble salt” refers to an inorganic compound having a solubility in 100 g of water at 25 ° C. of 50 mg or less, preferably 25 mg or less, more preferably 10 mg or less. Information regarding the solubility of the compound can be easily obtained by those skilled in the art, and can be easily obtained by examining the amount actually dissolved in 100 g of water at 25 ° C. Therefore, the solubility of the hardly water-soluble salt to be precipitated in the liquid to be treated can be easily known.

  The sparingly water-soluble salt to be formed in the liquid to be treated is not particularly limited as long as it is a sparingly water-soluble inorganic compound capable of adsorbing the material to be separated, which will be described later. Examples include barium, barium carbonate, calcium phosphate, calcium carbonate, magnesium carbonate, magnesium hydroxide, aluminum phosphate, and aluminum hydroxide.

  In order to form a hardly water-soluble salt, the cation to be present in the liquid containing the substance to be separated (processed liquid) is at least one selected from the group consisting of barium ions, calcium ions, magnesium ions and aluminum ions. The cation.

  On the other hand, the anion which should exist in a to-be-processed liquid should just be a thing which can form a hardly water-soluble salt by the coupling | bonding with at least 1 type of the said cation in a to-be-processed liquid. Specifically, the anion is selected from the group consisting of phosphate ion, monohydrogen phosphate ion, dihydrogen phosphate ion, carbonate ion, bicarbonate ion, sulfate ion, hydrogen sulfate ion, and hydroxide ion. At least one anion. Among these, at least one selected from the group consisting of sulfate ion, phosphate ion, carbonate ion and hydroxide ion is preferable.

  The concentration of the above cation and anion in the liquid to be treated can be appropriately selected according to the amount of the poorly water-soluble salt to be precipitated in the liquid to be treated, and is not particularly limited, but usually 0.001 mM each. About 3M, especially about 0.01mM to 1M. When using a some cation, the total amount should just be in the said range. Similarly, when a plurality of anions are used, the total amount may be within the above range.

  As a method of including the cation and the anion in the liquid to be treated, for example, a water-soluble compound that generates desired ions in an aqueous solution by dissolution (hereinafter referred to as “ion supply source”) is directly added to the liquid to be treated. Examples thereof include a method and a method of adding an ion supply source to the liquid to be treated in the form of an aqueous solution, but are not limited thereto. The order of adding the cation source and the anion source is not particularly limited, and the cation source may be added first, and then the anion source may be added, or vice versa. Further, when one of the cation and the anion is already present in a desired concentration in the liquid to be treated, only the other is added to the liquid to be treated. For example, when the solution to be treated is a phosphate buffer solution, phosphate ions in the solution to be treated can be used, so that at least a poorly water-soluble salt can be generated by binding with phosphate ions. By adding one kind of cation supply source to the liquid to be treated, a poorly water-soluble phosphate can be generated in the liquid to be treated. In addition, when the substance to be separated is a virus or the like, and the liquid to be treated is a culture medium such as a virus, carbon dioxide is often used as a pH adjuster for the culture liquid. Since carbonate ion is contained in, this carbonate ion can be used for forming a hardly water-soluble salt.

  In the present invention, “adding an ion source” means not only adding the water-soluble compound as an ion source in the form (solid, liquid or gas) but also in the form of an aqueous solution. To include. For example, when the ions to be supplied into the liquid to be processed are carbonate ions, “adding a carbonate ion source” means that a solid such as sodium hydrogen carbonate is directly dissolved in the liquid to be processed, sodium hydrogen carbonate And the like are once dissolved in water to prepare an aqueous solution, the aqueous solution is added, and carbon dioxide gas is passed through the liquid to be treated.

  The pH of the liquid to be treated is not particularly limited, but is desirably near neutral so that the properties of the substance to be separated are not impaired, preferably about pH 5 to 9, more preferably about pH 6 to 8.

  The reaction for precipitating the sparingly water-soluble salt may be carried out at any temperature, and is not particularly limited, but is desirably carried out under temperature conditions that do not impair the properties such as biochemical activity of the substance to be separated. About room temperature is preferable. When the substance to be separated is not a particularly unstable substance and it is not necessary to maintain a low temperature, it is convenient and preferable to carry out the reaction at about room temperature.

  The substance to be separated to be separated by the separation method of the present invention is not particularly limited as long as it is adsorbed to a hardly water-soluble salt. For example, it is conventionally separated using an adsorbent composed of a hardly water-soluble salt such as barium sulfate. A purified substance is preferable, and biological substances such as peptidic substances, nucleic acids, and complexes thereof are preferable.

  “Peptidic substance” means a substance composed of molecules in which a plurality of (two or more) amino acids are linked by peptide bonds. In addition to substances consisting of many molecules, it also includes full-length proteins. The “peptidic substance” also includes substances that have undergone various modifications (sugar chain modification, chemical modification, etc.). Specific examples of peptidic substances include, but are not limited to, enzymes, antibodies, antigens, glycoproteins, lipoproteins and labeling enzymes, and fragments thereof.

  The nucleic acid may be RNA or DNA, and any nucleic acid that is usually classified as a nucleic acid in the biochemical field and the medical field. Specific examples include, but are not limited to, ssDNA, dsDNA, RNA, plasmid DNA and the like.

  The complex of peptidic substance and nucleic acid refers to a combination of the above peptidic substance and nucleic acid. Representative examples include nucleoproteins, and specific examples include, but are not limited to, viruses and phages.

  A hardly water-soluble salt ([substance to be separated-water-insoluble salt] complex) adsorbed to a substance to be separated formed in a liquid to be treated by the separation method of the present invention is a general centrifugal method, filtration. Solid-liquid separation can be performed by a method or the like.

  The substance to be separated can be recovered by allowing the substance to be separated to elute by allowing the eluate to act on the solid-liquid separated [substance to be separated-water-insoluble salt] complex. That is, the present invention also provides a method for purifying the substance to be separated, which comprises allowing an eluent to act on the poorly water-soluble salt adsorbed with the substance to be separated obtained by the separation method of the present invention.

  As the eluent, any known eluent that is usually used in a separation / purification method using an adsorbent composed of a hardly water-soluble salt can be preferably used. Examples of such a known eluate include a phosphate solution, a citrate solution, and a mixed solution thereof. Although not particularly limited, the salt concentration of the eluate is usually about 0.005 M to 3 M (preferably 0.1 M to 2 M) (when a plurality of salts are included, the total is indicated). Specifically, 1M glucose-12% sodium citrate-6% sodium chloride eluate (pH 7.3) used in the following examples can be used, but is not limited thereto.

  The pH of the eluate is not particularly limited. However, when the substance to be separated is a biological substance, the pH is preferably about 5 to 9, more preferably about 6 to 8 when the biochemical activity is to be maintained. It is desirable. By using an eluate near neutrality, elution can be performed without impairing the activity of the substance to be separated. As a method of allowing the eluate to act on the [substance to be separated-slightly water-soluble salt] complex, the complex is suspended in the eluate, left for about 15 minutes to 2 hours, and then solid-liquid by centrifugation or filtration. Known conventional methods such as a method of separating and a method of passing an eluate while holding the complex on a filtration membrane are exemplified, and are not particularly limited.

  The temperature at which the eluate is allowed to act is not particularly limited, but it is desirable that the temperature is such that the properties such as biochemical activity of the substance to be separated are not impaired, and about 4 ° C. to room temperature is preferable. When the substance to be separated is not a particularly unstable substance and it is not necessary to maintain a low temperature, it is convenient and preferable to carry out the reaction at about room temperature.

  Prior to the elution of the substance to be separated, a liquid having a low elution effect of the substance to be separated from the [substance to be separated-water-insoluble salt] complex is used as a washing liquid, and the washing liquid is allowed to act on the complex by Contaminants can be removed from the complex. By further including such a washing step, the degree of purification of the separated substance to be recovered can be increased. As the cleaning liquid, a known cleaning liquid that is usually used in a separation / purification method using an adsorbent composed of a poorly water-soluble salt can be used. Specific examples include, but are not limited to, an aqueous solution having a lower salt concentration than water and an eluate (although not particularly limited, the total salt is generally about 0M to 1M). The pH conditions of the washing liquid, the method for operating the washing liquid and the temperature conditions are the same as those in the elution step described above.

  In the above purification method, the salt concentration is increased stepwise or by a linear gradient or a curve gradient from the low salt concentration pretreatment solution to the eluate in the [substance to be separated-poorly water-soluble salt] complex. By acting on the complex, among substances adsorbed on the poorly water-soluble salt, substances having weak adsorptive power can be sequentially eluted. As a result, it is possible to selectively separate and purify the target substance to be separated and contaminants, which is advantageous when a plurality of kinds of target substances to be separated are adsorbed on the poorly water-soluble salt. .

  Hereinafter, the present invention will be described more specifically based on examples.

1. Virus Adsorption and Recovery Test Adsorption of influenza virus to sparingly water-soluble salts, solid-liquid separation of virus- sparingly water-soluble salt complex, and recovery of viruses were performed according to the following procedure. The cation supply sources for generating poorly water-soluble salts in the used adsorption carrier or test solution are shown below.

Adsorption carrier used in conventional method (Comparative Examples 1-4)
Reagent A: Barium sulfate (BaSO ₄ : MW 233.39) Japanese Pharmacopoeia Fushimi Manufacturing Co., Ltd.
(Use a suspension with 25% water / water for injection and autoclaved at 121 ° C for 20 minutes)
Cation source for producing a poorly water-soluble salt in the test solution (Examples 1 and 2)
Reagent B: Barium chloride dihydrate (BaCl ₂ · 2H ₂ O: MW 244.26) WAKO reagent special grade Reagent C: Calcium chloride dihydrate (CaCl ₂ · 2H ₂ O: MW 147.01) WAKO reagent special grade

(1) Adsorption of virus from culture solution (removal from culture solution)
Add 0.9 mmol of any of reagents A to C to 4 mL of H1N1 A / New Caledonia strain influenza virus culture solution (carbonate concentration 20 mmol / L) obtained by the conventional cell culture method, and gently 60 Stir for minutes.
(2) Solid-liquid separation of virus-poor water-soluble salt complex After centrifuging the suspension (2000 rpm, 10 minutes, 4 ° C), the supernatant (adsorption process supernatant) is removed by the gradient method, and the sediment is collected. did.
(3) Virus recovery Add 2 mL of 1M glucose-12% sodium citrate-6% sodium chloride eluate (pH 7.3) to the virus-poor water-soluble salt complex obtained in the solid-liquid separation step (2). Stir gently at room temperature for 30 minutes.
(4) Solid-liquid separation After centrifuging the suspension (2000 rpm, 10 minutes, 4 ° C), the supernatant (elution step supernatant) was collected by a gradient method.

2. Evaluation of virus removal rate and recovery rate Adsorption process supernatant, elution process supernatant, and culture solution before addition of reagents, using an antibody against influenza virus nucleoprotein (NP) (manufactured by Denka Seiken Co., Ltd.) based on conventional methods -ELISA was performed to measure the amount of influenza virus NP antigen. The amount of NP antigen in the culture solution is taken as 100%, and the amount of NP antigen in each supernatant is calculated as the relative value. The virus residual rate (the amount of virus not adsorbed on the carrier,%) and the recovery rate (eluted virus) The ratio of the amount to the amount of virus in the culture solution,%) was determined. The results are shown in Table 1.

  Moreover, the virus residual rate and the recovery rate were calculated | required by the method similar to the above except the addition amount of the reagent A having been 0.08 mmol (comparative example 2).

  In addition, regarding the conventional method, the amount of non-adsorbed virus (virus residual rate) was determined in the same manner as above except that the amount of barium sulfate added was changed. A correlation was seen (Table 2).

Adsorption process In Examples 1 and 2 and Comparative Example 1, since the virus remaining rate in the test solution in the adsorption process is almost close to 0, almost all of the virus in the test solution is adsorbed by the hardly water-soluble salt. I understand that. Moreover, when visually confirmed, the amount of the hardly water-soluble salt residue recovered by solid-liquid separation in Examples 1 and 2 was significantly smaller than the amount of barium sulfate residue recovered by solid-liquid separation in Comparative Example 1.

  In Examples 1 and 2, since a test solution containing 0.08 mmol of carbonate ions (4 mL of a culture solution having a carbonate ion concentration of 20 mmol / L) was reacted with 0.9 mmol of the cation supply source, It can be estimated that the slightly water-soluble salt produced in is 0.08 mmol. The virus remaining rate in the test solution in the adsorption steps of Example 1 and Example 2 was almost zero. On the other hand, when 0.08 mmol of barium sulfate was added by the conventional method (Comparative Example 2), the virus residual rate was as high as 30% or more. From this, the method of generating a poorly water-soluble salt in the treatment liquid can achieve the same virus removal rate with a smaller amount of the poorly water-soluble salt compared to the method of adding barium sulfate to the treatment liquid. all right.

Elution process In Examples 1 and 2, the virus recovery rate after elution was lower than that of Comparative Example 1 which is a conventional method. However, since almost all of the virus present in the test solution is adsorbed and removed in the adsorption step, elution is performed while a part of the virus remains immobilized on the poorly water-soluble salt complex under the above conditions. It is thought that it is not.

Claims (8)

  A water-insoluble salt is formed by reacting at least one cation selected from the group consisting of barium ions, calcium ions, magnesium ions, and aluminum ions with the cation in an aqueous treatment liquid containing a substance to be separated. Reacting with at least one kind of anion that can be formed, precipitating a hardly water-soluble salt in the liquid to be treated, and adsorbing the substance to be separated to the precipitated poorly water-soluble salt, Separation method.   The anion is at least one anion selected from the group consisting of phosphate ion, monohydrogen phosphate ion, dihydrogen phosphate ion, carbonate ion, bicarbonate ion, sulfate ion, hydrogen sulfate ion and hydroxide ion. The method according to claim 1, which is an ion.   3. The method according to claim 2, wherein the anion is at least one selected from the group consisting of sulfate ion, phosphate ion, carbonate ion and hydroxide ion.   The method according to claim 3, wherein the cation is barium ion and / or calcium ion, and the anion is carbonate ion.   The sparingly water-soluble salt comprises: (1) at least one water-soluble compound that generates the at least one cation in an aqueous solution; and (2) the at least one anion in an aqueous solution. The method according to any one of claims 1 to 4, wherein at least one of at least one water-soluble compound that generates water is added to the liquid to be treated.   The method according to any one of claims 1 to 5, wherein the substance to be separated is at least one selected from the group consisting of peptide substances, nucleic acids, and complexes of peptide substances and nucleic acids.   The method according to claim 6, wherein the complex of the peptidic substance and the nucleic acid is a virus.   A method for purifying the substance to be separated, which comprises allowing an eluate to act on the poorly water-soluble salt adsorbed with the substance to be separated, obtained by the method according to claim 1.