JP3383332B2 - Novel peptide and endotoxin removal method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel polypeptide having a specific binding ability to endotoxin, an insoluble carrier to which the polypeptide is bound as a ligand,
And a method for removing endotoxin using the same.

[0002]

2. Description of the Related Art Endotoxin, also called endotoxin, is a general term for toxic substances existing in bacterial cell components. Endotoxins cause a strong fever when contaminated in the blood and may cause shock death if the contamination is excessive. The presence of this endotoxin causes problems during contamination during the manufacturing process of pharmaceuticals,
In consideration of this, the Japanese Pharmacopoeia strictly regulates the amount of endotoxin in the preparation.

However, once this endotoxin is mixed in the preparation, its removal is very difficult. Various methods have been tried so far with the intention of removing endotoxin from the preparation, but the effects by these methods have not always been satisfactory. For example,
Although a method of treating the preparation with an ion exchange resin, an ultrafiltration membrane, activated carbon or the like has been tried, such a method has not been sufficient in terms of endotoxin removal ability itself. Although heat treatment is certainly an effective method for removing endotoxin, there is a high possibility that the drug itself will be denatured under the temperature and time conditions that completely inactivate endotoxin. Furthermore, attempts have been made to remove endotoxin by immobilizing a nitrogen-containing heterocyclic compound (Japanese Patent Publication No. 03-7681, JP-A No. 01-317502). However, the bond between the nitrogen-containing heterocyclic compound and endotoxin is not so specific, and the removal ability itself depends largely on the ion-exchange property of the nitrogen-containing heterocyclic compound. Therefore, this method has a big problem that it tends to nonspecifically adsorb substances other than endotoxin, particularly the drug itself.

[0004]

The object of the present invention is to establish means for specifically adsorbing endotoxin and removing it.

[0005]

Means for Solving the Problems As a result of extensive studies on the means for solving the above problems, the present inventor has found that in the blood cell components of horseshoe crab (T. tridentatus), which are mainly distributed in China,
The present invention has been completed by finding a novel peptide (hereinafter referred to as TTLBP) that specifically binds to endotoxin. That is, the present application provides the following inventions. (1) It has an amino acid sequence represented by SEQ ID NO: 1 as an amino acid sequence from the N-terminus of a polypeptide having the following properties.

The molecular weight is around 8000. (2) An insoluble carrier to which the polypeptide according to (1) or a salt thereof is bound as a ligand. (3) An insoluble carrier in which the polypeptide according to (1) or a salt thereof is bound as a ligand via a spacer. (4) The insoluble carrier according to (2) or (3),
A method of contacting a liquid containing endotoxin to adsorb the endotoxin to the insoluble carrier to remove endotoxin from the liquid containing.

The present invention will be described in detail below. A. The polypeptide of the present invention can be obtained by extracting and purifying from the blood cell component of horseshoe crab (T. tridentatus), which is mainly distributed in China. The extraction
The details of the purification method are described in detail in the Examples below.However, the supernatant of the T. tridentatus hemocyte crushed product obtained by centrifugation is generally subjected to various treatment operations using the physical and chemical properties of TTLBP. Executable (for example, "Biochemistry Data Book II" pp1175 to 1259, 1st edition, 1st printing, June 1980)
23rd, see Tokyo Kagaku Doujinshi). As the method, specifically, for example, treatment with a usual protein precipitating agent, ultrafiltration, molecular sieve chromatography (gel filtration)
, Liquid chromatography, centrifugation, electrophoresis, affinity chromatography, dialysis method, or a combination thereof can be adopted.

The N-terminal amino acid sequence of purified / isolated TTLBP can be determined by a generally known method, for example, a commercially available protein sequencer. The molecular weight of TTLBP can be determined by a generally known method such as SDS-PAGE. B. The insoluble carrier of the present invention can be prepared by immobilizing TTLBP on a commonly known carrier.

As a base material of the insoluble carrier of the present invention, generally, a carrier generally used in affinity chromatography can be widely used, TTLBP can be immobilized, and activity of TTLBP can be fixed. The type is not particularly limited as long as it does not hurt. However, among these carriers, cross-linked agarose and polymethacrylate resin are particularly preferable in that they are mechanically strong and have less non-specific adsorption to the carrier itself.

The method of immobilizing TTLBP on the carrier is not particularly limited, and generally known methods can be widely adopted. Specifically, it can be immobilized according to a CNBr activation method, a carbodiimide coupling method, an epoxy activation method, or the like. In addition, the endotoxin intended to be adsorbed on the insoluble carrier of the present invention becomes difficult to bind to the insoluble carrier due to steric hindrance of the endotoxin itself when adsorbing the endotoxin on the insoluble carrier unless a spacer due to a polymeric substance is intervened. Therefore, it is particularly preferable to interpose a spacer to prevent steric hindrance during the immobilization.
As such a spacer, a spacer depending on the material used as the carrier can be adopted. For example, when the compound represented by the formula (1) is used as a spacer in the above crosslinked agarose;

[0011]

## STR00001 ## --OCH ₂ --CH (OH)-CH ₂ O (CH ₂ ) ₄ O--CH ₂ --NH-- Formula (1) Further, the compound represented by Formula (2) is added to the polymethacrylate resin as a spacer. When used as

[0012]

Embedded image —OCH ₂ —CONH (CH ₂ ) ₂ NHCO (CH ₂ ) ₂ O—CO—NH— Formula (2) and the like can be mentioned as a particularly preferred embodiment. The insoluble carrier of the present invention thus prepared is packed in a column, equilibrated in an appropriate buffer and equilibrated with a sample, or a column method in which the sample is added, or a sample solution having a volume of 5 to 10% is used. It can be used for both the batch method in which the carrier is added and the treatment liquid is recovered after stirring for 1 to several hours.

[0013]

EXAMPLES The present invention will be described below with reference to examples.

[0014]

Example 1 12 g of blood cells were isolated from 500 ml of blood of Chinese horseshoe crab (T. tridentatus) by centrifugation. Next, 100 ml of 30% acetic acid was added to the blood cells to pulverize them, which was then centrifuged at 4 ° C. (3500 rpm, 20 minutes) to separate the supernatant. This operation was repeated twice more.

The supernatant separated as described above was equilibrated with 50 mM ammonium acetate buffer (pH 5.5) to Sephacryl S-20.
This was added to a 0 HR column (φ26 × 900 mm) (Pharmacia) and eluted with the same buffer. The eluate was fractionated by 10 ml, and the absorbance at 280 nm of each fraction was measured (
(Fig. 1). Fractions numbered 36 to 39 were collected from this, added to a Mono-S column (φ5 × 50 mm), and the concentration of sodium chloride was changed from 0 to 1 M in 50 mM ammonium acetate buffer (pH 5). .5), and the absorbance at 280 nm of each fraction was measured (Fig. 2). In Fig. 2, the fraction I was collected and was collected on a Cosmosir 5C ₄ -300 column (φ4.6 × 150m).
m) (manufactured by Nacalai Tesque, Inc.) and eluted with a 0.1% trifluoroacetic acid solution in which the concentration of anitrile was changed from 0 to 80% (FIG. 3). Fractions II in FIG. 3 were collected and freeze-dried to obtain 850 μg of a purified product of the polypeptide of the present invention (TTLBP).

The structure of this TTLBP was determined as follows. That is, TTLBP obtained above was subjected to reductive S-pyridylethylation and then subjected to protein sequencer 477 / 120A.
(Manufactured by Applied Biosystems) was used to determine the amino acid sequence from the N-terminal to the 26th residue. further,
Pyridylethylated TTLBP was digested with lysyl endopeptidase and endopeptidase ASP.n and isolated for each fragment by reverse phase HPLC. Next, the amino acid sequence of each fragment was determined using the protein sequencer 477 / 120A to finally determine the total amino acid sequence from the N-terminal to the 73rd residue.
The row was determined (SEQ ID NO: 1).

The molecular weight of TTLBP is 23% for gel concentration.
SDS-PAGE revealed that it was around 8000.

[0018]

Example 2 Preparation of insoluble carrier (1) Affiprep 10 which is a carrier for immobilizing activated ligand
(BIORAD) 1 ml of 10 mM sodium acetate buffer (pH 4.5) 5
After washing with 0 ml, add 10 mM HEPES buffer (pH 7.2) 3.0
It was washed with ml. The polypeptide of the present invention obtained in Example 1
5 mg of (TTLBP) was dissolved in 1 ml of 10 mM HEPES buffer (pH 7.2), and this was added to the above Affiprep 10 and stirred at 4 ° C. for 6 hours.

After completion of the stirring, the unbound added polypeptide of the present invention was washed with 10 mM HEPES buffer (pH 7.2), and 3 ml of 1 M ethanolamine (pH 8.0) was added. This was stirred overnight at 4 ° C. to block residual active groups.

[0020]

Example 3 Preparation of Insoluble Carrier (2) 10 ml of Sepharose CL-4B (Pharmacia) was thoroughly washed with distilled water, and then 14 mg of sodium tetrahydroborate, 7 ml of 1N sodium hydroxide aqueous solution, and 1,4-butanediol. The mixture was added to a mixed solution of 7 ml of diglycyl ether and reacted at 23 ° C for 10 hours. After the reaction, after filtering on a glass filter,
The reacted Sepharose CL-4B was thoroughly washed with chilled distilled water to obtain epoxy-activated Sepharose.

8 ml of this epoxy-activated Sepharose
After thoroughly washing with 0.2 M sodium carbonate buffer (pH 11.0), 3 mg
/ Ml of the polypeptide solution of the present invention obtained in Example 1 (5 ml) was added, and the mixture was stirred at 30 ° C for 16 hours. After stirring, it was thoroughly washed with distilled water, a 0.1 M sodium carbonate buffer solution (pH 8.0), and a 0.1 M acetic acid solution. Further, add 16 ml of 1M ethanolamine, and add 4
The remaining active groups were blocked by stirring at ℃ for 1 hour.

[0022]

Example 4 Examination of Endotoxin Adsorption Capacity of Insoluble Carrier (1) Using the insoluble carrier prepared in Example 2 and Example 3,
The ability to remove endotoxin from the buffer was examined. As the endotoxin solution, standard Japanese Pharmacopoeia endotoxin derived from E. coli UKT-B strain was dissolved in 50 mM sodium acetate buffer (pH 5.0) at a concentration of 100 EU / ml and used.

1 ml of endotoxin was taken in a tube, 20 ml of endotoxin solution was added, and this was shaken at room temperature for 3 hours. After permeation, the endotoxin concentration in the buffer was quantified using Limulus reagent. The results are shown in Table 1.

[0024]

[Table 1]

[0025]

Example 5 Investigation of Endotoxin Adsorption Capacity of Inert Carrier (2) Using the insoluble carrier prepared in Example 2 and Example 3,
The ability to remove endoxin from the protein solution was examined. 100 EU / ml Japanese Pharmacopoeia standard endotoxin, 5 mg
/ Ml bovine serum albumin and 50 mM NaCl containing 50 mM
A sodium acetate buffer (pH 5.0) was used as a sample.

1 ml of the insoluble carrier was placed in a tube, 20 ml of the protein solution was added, and the mixture was shaken at room temperature for 3 hours. After shaking, the endotoxin concentration in the buffer was quantified using the Limulus reagent, and the protein concentration was quantified by the Lowry method. The results are shown in Table 2.

[0027]

[Table 2]

As a result, almost both endotoxins were adsorbed on both adsorbents, and bovine serum albumin, which is a general protein, was hardly adsorbed.

[0029]

Example 6 Examination of Endotoxin Adsorption Capacity of Insoluble Carrier (3) Using the insoluble carrier prepared in Example 2 and Example 3,
Adsorption of endotoxin from various strains was examined. 50 mM
An endotoxin solution of 100 EU / ml of each strain was prepared with a sodium acetate buffer (pH 5.0), and 20 ml thereof was added to 1 ml of the adsorbent. After shaking at room temperature for 3 hours, the concentration of endotoxin in the buffer solution was quantified using Limulus reagent. The results are shown in Table 3.

[0030]

[Table 3]

As a result, it was revealed that the insoluble carrier of the present invention has a strong adsorptivity to endotoxins derived from different strains.

[0032]

INDUSTRIAL APPLICABILITY The present invention provides a polypeptide useful for removing endotoxin, an insoluble carrier for endotoxin removal using the polypeptide as a ligand, and a method for removing endotoxin using the carrier.

[0033]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 73 Sequence type: Amino acid topology: Unknown Sequence type: Polypeptide Sequence origin: Tachypleus tridentatus blood cell sequence: Tyr Leu Ala Phe Arg Cys Gly Arg Tyr Ser 10 Pro Cys Leu Asp Asp Gly Pro Asn Val Asn 20 Leu Tyr Ser Cys Cys Ser Phe Tyr Asn Cys 30 His Lys Cys Leu Ala Arg Leu Glu Asn Cys 40 Pro Lys Gly Leu His Tyr Asn Ala Tyr Leu 50 Lys Val Cys Asp Trp Pro Ser Lys Ala Gly 60 Cys Thr Ser Val Asn Lys Glu Cys His Leu 70 Trp Lys Thr 73

[Brief description of drawings]

FIG. 1 is an elution curve of a polypeptide of the present invention on a Sephacryl S-200 HR column.

FIG. 2 is an elution curve of the polypeptide of the present invention on a Mono-S column.

FIG. 3 is an elution curve of the polypeptide of the present invention using a Cosmoseal 5C ₄ -300 column.

─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-2-204500 (JP, A) JP-A-2-221863 (JP, A) JP-A-2-216462 (JP, A) JP-A-2- 207098 (JP, A) JP-A-2-53799 (JP, A) JP-A-2-152987 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) BIOSIS (DIALOG) WPI ( DIALOG) SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / GeneSeq PubMed

Claims (4)

(57) [Claims] From 1. A amino acid sequence shown in SEQ ID NO: 1
Consisting of the polypeptide. 2. An insoluble carrier to which the polypeptide according to claim 1 or a salt thereof is bound as a ligand. 3. An insoluble carrier in which the polypeptide according to claim 1 or a salt thereof is bound as a ligand via a spacer. 4. A method for removing endotoxin from the solution containing the endotoxin by contacting the solution containing endotoxin with the insoluble carrier according to claim 2 or 3 to adsorb the endotoxin to the insoluble carrier.