JPH0720984B2 - How to remove endotoxin - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for removing endotoxin, and more particularly to a method for selectively and efficiently separating and removing endotoxin contained in a protein solution containing a protein and endotoxin. .

[Prior art] In the purification of pharmaceuticals such as proteins produced by biotechnology, several pg of impurities such as endotoxin are used.
It is necessary to remove from / ml to several tens of pg / ml. Conventionally, as a technique for removing endotoxin, a method using low molecular weight chitosan has been proposed (JP-A-63-56300). That is, when low molecular weight chitosan is adsorbed on a carrier and packed in a column, and a liquid to be treated is passed through this column, low molecular weight chitosan and endotoxin are specifically adsorbed and endotoxin in the liquid is removed. In addition, JP-A-63-287503 discloses that
It is disclosed that an endotoxin aqueous solution is brought into contact with a polyamide microporous membrane cross-linked with chitosan to adsorb and separate endotoxin. Furthermore, as a method for removing endotoxin to a low concentration, ion chromatography, gel chromatography, affinity chromatography (Japanese Examined Patent Publication No. 1-16389) and the like are being studied.

[Problems to be Solved by the Invention] In the method using low molecular weight chitosan disclosed in JP-A-63-56300, the adsorption efficiency of endotoxin is not sufficient,
It is effective to remove mg / ml order up to several + ng / ml order, but it is difficult to remove it to a very low concentration of 8.5 ng / ml or less. That is, the chitosan immobilized on the low molecular weight chitosan-immobilized carrier disclosed in Japanese Patent Laid-Open No. 63-56300 is a low molecular weight substance, and the chitosan content in the carrier used for adsorbing endotoxin is small. Small quantity. Therefore, it is not practically suitable for the purpose of removing endotoxin in the protein solution to the order of pg / ml. Incidentally, in the examples of JP-A-63-56300, endotoxin is 270 to 57 ng / ml and several hundred ng / ml to several.
Only 10 ng / ml has been removed.

In the chitosan-immobilized membrane disclosed in JP-A-63-287503, since the chitosan content in the membrane is very low at 10% or less,
After all, the equilibrium adsorption amount of endotoxin is small, and it is considered impossible to remove endotoxin to an extremely low concentration. JP-A-63-287503 exemplifies removal of endotoxin in water, but as described above, it is not practically suitable for removal of endotoxin in a protein solution because the endotoxin equilibrium adsorption amount of the membrane is small. is there.

Ion chromatography, gel chromatography,
Affinity chromatography has been studied for the purpose of removing up to pg / ml order,
Each method has its own drawbacks and is not a practically advantageous method.
That is, since gel chromatography has a very broad molecular weight distribution of endotoxin, it is difficult to separate it from high molecular weight substances such as proteins, and ion chromatography and affinity chromatography have small equilibrium adsorption amount. It is difficult to set conditions such as pH, ionic strength, and regeneration conditions.

In the past, in particular, for the purpose of purifying a protein, a technique for removing endotoxin in a protein solution has not been clearly set, so that the endotoxin in the protein solution can be selectively and efficiently removed. I couldn't.

An object of the present invention is to solve the above conventional problems and provide a method for removing endotoxin capable of selectively and efficiently removing endotoxin in a protein solution to an extremely low concentration.

[Means for Solving the Problems] The method for removing endotoxin of the present invention is a method for removing endotoxin from a liquid containing a protein and endotoxin, after adjusting the pH of the liquid to be equal to or lower than the isoelectric point of the protein, SV0.2 ~ 2BV in column packed with crosslinked chitosan granules
・ The residual concentration of endotoxin is increased by passing the solution at the passing rate of hr ^-1
It is characterized in that a purified protein-containing solution of 8.5 ng / ml or less is obtained.

That is, the present inventors conducted a study on a method for removing endotoxin in a protein solution, and found that crosslinked chitosan granules as an adsorbent have a large chitosan content and a large endotoxin equilibrium adsorption amount, and thus are extremely effective. I found out that. Then, in the method using the crosslinked chitosan granules, as a result of further studies to selectively and efficiently remove only endotoxin, the pH of the liquid was set to be equal to or lower than the isoelectric point of the protein, and the column flow rate was increased. The present inventors have completed the present invention by finding that endotoxin can be selectively and efficiently removed by adopting a treatment condition of controlling, which has never been suggested in the prior art.

The present invention will be described in detail below.

First, the crosslinked chitosan granular material used as the endotoxin adsorbent in the present invention will be described.

The crosslinked chitosan granules according to the present invention can be easily produced by subjecting ordinary chitosan or low molecular weight chitosan to a crosslinking treatment by a known method. For example,
Examples of the method for crosslinking chitosan include a method in which a hydrophobic solvent containing an emulsifier is added to an acidic chitosan solution to form an emulsion, and then the mixture is granulated with stirring in an alkaline solution (Japanese Patent Publication No. 59-30722). Further, as a method for crosslinking the low molecular weight chitosan, a method of dissolving the low molecular weight chitosan in an acidic solution, then dropping it into a basic solution to granulate it, and performing a crosslinking treatment (Japanese Patent Publication No. 63-54285) can be mentioned. .

An example of the method for producing the crosslinked chitosan granular material according to the present invention will be described below.

Chitosan is obtained by deacetylating chitin, which is contained in the shells of crustaceans such as crab and shrimp, by heating with concentrated alkali. The chitosan thus obtained usually has a molecular weight of 5,000 to 1,000,000 and an intrinsic viscosity (30 ° C., 0.2 M acetic acid + 0.1 M sodium acetate) [η] = 0.25
30 dl / g-chitosan, colloid equivalent 1.0-6.2 meq / g-chitosan. The degree of deacetylation is not particularly limited as long as it is deacetylated to the extent that it can be dissolved with an acid, and the degree of deacetylation is generally preferably about 50 to 100 mol%. Further, it is preferable that the average particle size of chitosan is granular with about 9 to 300 mesh.

Among the chitosan thus obtained, in this production example, as the low molecular weight chitosan, a molecular weight of 10,000 to 500,000,
A deacetylation degree of 50 to 100 mol% is used. Then, this low molecular weight chitosan is first dissolved in an acidic solution to obtain a chitosan acidic aqueous solution. In this case, any acid can be used as long as it can dissolve chitosan, but typical ones include acetic acid, formic acid, propionic acid, butyric acid, valeric acid, isopropionic acid, isobutyric acid, and isobutyric acid. Examples thereof include organic acids such as valeric acid, benzoic acid, cinnamic acid, salicylic acid and phthalic acid, and mineral acids such as hydrochloric acid and nitric acid.

It is preferable that the amount of acid used is as small as possible, and it is used in the minimum amount capable of dissolving chitosan. Generally, 0.2 to 5 times (weight) of acid is added to chitosan.

The concentration of chitosan in the acidic aqueous chitosan solution is determined appropriately depending on the molecular weight of chitosan and the degree of deacetylation, but is usually preferably 2 to 20% by weight.

The obtained chitosan acidic aqueous solution is dropped into the basic solution with stirring to be granulated. As the basic substance of the basic solution used here, sodium hydroxide, potassium hydroxide,
Examples of the solvent include alkaline substances such as sodium carbonate, potassium carbonate, ammonia, and ethylenediamine. Examples of the solvent include water or a polar alcohol such as methanol and ethanol, or a mixed solvent of these polar solvents and water. The concentration of the basic solvent is not particularly limited,
Usually, a concentration of 1 to 20% by weight is used.

Granular chitosan obtained by granulation is usually 1 μm.
Although it is a porous chitosan granular material having a size of about m to 1 mm, it is subjected to a crosslinking treatment after washing with water to remove water.

The cross-linking treatment can be carried out by adding a cross-linking agent such as epichlorohydrin, glutaraldehyde, organic diisocyanates, etc. to chitosan in a suitable solvent, if necessary.

As the solvent used for the crosslinking treatment, methanol, ethanol, alcohols such as isopropyl alcohol,
Ketones such as acetone and methyl ethyl ketone, amides such as dimethylformamide and dimethylacetamide, and dimethyl sulfoxide can be used. These solvents may be used alone or in combination of two or more. Examples of organic diisocyanates include 4,4'-diphenylmethane diisocyanate and 1,4-
Examples thereof include diphenylene diisocyanate, 2,4-tolylene diisocyanate, naphthalene diisocyanate, 4,4'-dicyclohexylmethane diisocyanate, xylylene diisocyanate, isophorone diisocyanate, and hexamethylene diisocyanate.

The amount of the cross-linking agent used is not particularly limited, but it is preferable that the amount of the cross-linking agent is 0.2 to 0.8 equivalent to 1 equivalent of the amino group of chitosan. When a solvent is used, its amount is preferably 1 to 5 times the volume of the chitosan granules.

The cross-linking treatment conditions vary depending on the type of cross-linking agent and solvent used, but in general, it is carried out at 0 to 60 ° C for 0.05 to 5 hours.

When using the crosslinked chitosan granules produced in this manner, first, the crosslinked chitosan granules are washed with an alkali by bringing them into contact with a basic solution to deactivate and remove the adsorbed endotoxin. As the basic solution used for the alkali washing, an aqueous solution of NaOH, KOH, NH ₃ or the like or a mixed solution of these with an organic solvent and having a concentration of 0.01 to 1 N can be used. After that, sterilized water containing no endotoxin (endotoxin-free water) is used to wash with water to remove excess alkali. After washing with water, it can be used as it is for the removal of endotoxin, but if necessary, an aqueous solution of the acid used in the preparation of the acidic aqueous solution of chitosan or a mixed solution thereof with an organic solvent at a concentration of 0.01-
The amino group of chitosan can also be used as a dissociated type by contacting with 1N. When it is contacted with an acid, it is then washed with sterile water containing no endotoxin to wash off excess acid.

Next, the method for removing endotoxin of the present invention using such a crosslinked chitosan granular material as an endotoxin adsorbent will be described.

In the method of the present invention, the crosslinked chitosan granules are packed in a column, and a liquid containing a protein to be treated and endotoxin in this column is adjusted to a pH on the acidic side below the isoelectric point of the protein. Pass the liquid through and collect the liquid at the outlet of the column. If the pH of the liquid is more basic than the isoelectric point of the contained protein, the protein will also be adsorbed by the crosslinked chitosan granules, and the protein recovery rate will decrease. The pH of the liquid is preferably 0.5 or more lower than the isoelectric point of the protein.

In the present invention, the pH can be easily adjusted by changing the concentration of acid and base in the buffer that dissolves the protein. If necessary, salt such as salt may be added.

In the present invention, the liquid passing rate is SV 0.2 to 2 BV · hr ⁻¹ , preferably SV 0.5 to ¹ BV · hr ⁻¹ . This flow rate has a great influence on the endotoxin removal efficiency, and the endotoxin removal rate significantly decreases even if the SV exceeds 2BVhr- ¹ and less than 0.2Vhr- ¹ , and the endotoxin concentration in the recovered fluid is reduced. 8.5ng /
It is difficult to achieve extremely low concentrations of less than ml.

In the method of the present invention, the crosslinked chitosan granules used for the above endotoxin removal can be regenerated and reused by carrying out alkaline washing and water washing with the same basic solution as described above.

In the present invention, the protein to be treated as a control and the protein in the endotoxin-containing solution are not particularly limited, and any protein that is stable at a pH below its isoelectric point can be applied.

According to the present invention, the endotoxin concentration selectively and efficiently removes endotoxin from a liquid having a relatively low concentration,
The endotoxin concentration can be reduced to an extremely low concentration of 8.5 ng / ml or less. Therefore, the method of the present invention is extremely effective especially for a liquid having an endotoxin concentration of 1000 ng / ml or less.

[Operation] The crosslinked chitosan granular material according to the present invention has a chitosan content of 80.
%, The balance is a cross-linking agent, and the amount of endotoxin equilibrium adsorption is large. Therefore, endotoxin can be efficiently adsorbed, separated and removed, and the endotoxin concentration can be reduced to an extremely low concentration. Incidentally, the polyamide microporous membrane disclosed in JP-A-63-287503 has a chitosan content of 10% or less, and also JP-A-63-5630.
Even with the low molecular weight chitosan-immobilized carrier disclosed in No. 0, a high endotoxin equilibrium adsorption amount cannot be obtained as in the present invention because the chitosan content is low, and therefore it is difficult to reduce the endotoxin to an extremely low concentration. .

Moreover, in the present invention, using such a crosslinked chitosan granular material, at a pH below the isoelectric point of the protein, and SV0.
Since the treatment is carried out at a column flow rate of 2 to 2 BV · hr ⁻¹ , the protein is hardly adsorbed by the crosslinked chitosan granules, and only endotoxin can be selectively and efficiently removed.

[Examples] Hereinafter, the present invention will be specifically described with reference to Examples and Comparative Examples, but the present invention is not limited to the following Examples unless it exceeds the gist.

In the following Examples and Comparative Examples, endotoxin was quantified by using a Toxicolor system manufactured by Seikagaku Corporation, and albumin was quantified by measuring the absorbance of ultraviolet rays (280 nm).

Examples 1 to 4 and Comparative Example 1 Chitosan 7 having a deacetylation degree of 90 mol% and an average molecular weight of 50,000
A solution obtained by dissolving 0 g in 930 g of water containing 70 g of acetic acid was dropped into a 10 wt% sodium hydroxide aqueous solution with stirring to cause solidification and precipitation. Then, it was thoroughly washed to obtain a porous chitosan having an average particle size of 0.1 mm. Next, after removing water, it was added to acetone and crosslinked with hexamethylene diisocyanate.

The following tests were carried out using the crosslinked chitosan granules thus obtained. At the time of use, the crosslinked chitosan granules were washed with 0.2N NaOH aqueous solution with an alkali and then thoroughly washed with endotoxin-free water.

A sample solution containing 5 mg / ml bovine serum albumin (isoelectric point 4.8) and the amount of endotoxin shown in Table 1 was filled with the crosslinked chitosan granular material obtained above in a column having an inner diameter of 10 mm and a length of 100 mm. Was passed through. The liquid passing conditions are as shown in Table 1. Table 1 shows the endotoxin concentration of the recovered liquid. In each test, the albumin recovery rate was 95% or more as a result of UV absorption (wavelength 280 mm).

As is clear from Table 1, the pH of the solution was adjusted to be below the isoelectric point of the protein, and the column flow rate was SV0.2-2BV ・ hr.
By setting ^-1 , endotoxin can be removed to an extremely low concentration of 8.5 ng / ml or less.

On the other hand, in Comparative Example 1 in which the flow rate was SV0.1BV · hr ^-1 , endotoxin could be removed to some extent, but 8.5n
It cannot be removed to an extremely low concentration below g / ml. After each test, the crosslinked chitosan granules in the column were
Regeneration was carried out by contacting with NaOH for 30 times, and after washing with water, the same treatment was repeated. In all cases, almost the same treatment result was obtained.

Comparative Example 2 A test was performed in the same manner as in Example 3 except that the pH was 5.3 among the liquid passing conditions.

As a result, the endotoxin concentration in the recovered solution was 0.9 ng / ml, the albumin recovery rate was 10% or less, and selective and efficient removal of endotoxin was not achieved.

[Effects of the Invention] As described in detail above, according to the method for removing endotoxin of the present invention, from a liquid containing a protein and endotoxin,
Only endotoxin can be easily removed selectively and efficiently, and an extremely low concentration treatment solution with a residual endotoxin concentration of 8.5 ng / ml or less can be obtained. Moreover, since the crosslinked chitosan granular material used as the adsorbent has excellent alkali resistance, it can be recycled and reused by washing with an alkali, which is extremely economical.

The method for removing endotoxin of the present invention is industrially extremely useful as a method for purifying a protein solution for pharmaceuticals.

Claims (1)

[Claims] 1. A method for removing endotoxin from a liquid containing a protein and endotoxin, which comprises adjusting the pH of the liquid to the isoelectric point of the protein or lower, and then applying SV0.2 to a column packed with crosslinked chitosan granules. A method for removing endotoxin, which comprises passing a solution at a rate of 2 BV · hr ⁻¹ to obtain a purified protein-containing solution having a residual endotoxin concentration of 8.5 ng / ml or less.