JPS60226822A - Method of purification of filamentous hemaglutinin - Google Patents

Abstract

PURPOSE:To purify industrially the titled filamentous hemaglutinin useful as various reagents, by bringing a solution containing a filamentous hemaglutinin produced from a mold belonging to the genus Bordetella into contact with a crosslinked polysaccharide sulfuric ester gel derivative, adsorbing the filamentous hemaglutinin on the gel, eluting it. CONSTITUTION:A solution containing a filamentous hemaglutinin (F-Ha for short) produced from a mold belonging to the genus Bordetella, preferably a mold of Bordetella bronchiseptica is brought into contact with a crosslinked polysaccharide sulfuric ester (e.g., epichlorohydrin crosslinked dextran sulfuric ester, epichlorohydrin crosslinked cellulose ester, etc.) gel derivative, F-HA is adsorbed on the gel, eluted from the gel, to purity F-HA. The adsorption is carried out at 6-8pH at 0-30 deg.C at 5-25ms/cm specific conductivity, and the elution is done at 5-10pH, at 25-130ms/cm specific conductivity. A large amount of high-purity F-HA can be isolated and purified by a simple operation.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to filamentous hemagglutinin (Filamentous He+nagHIuti) produced by Bordetella bacteria.
nin; hereinafter abbreviated as F-HA), more specifically, a culture of Bordetella bacteria is brought into contact with a gel derivative of cross-linked polysaccharide sulfate ester, and F-HA
After adsorbing F, high purity F is obtained by elution from the gel.
'-Relating to a method for collecting HA.

Industrial Application Fields Microorganisms belonging to the genus Bordetella include Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica, which produce various biologically active substances. F-HA is one of these biologically active substances, and all bacteria belonging to the genus Bordetella produce F-HA.

Recently, it has been revealed that Bordetella pertussis F-HA plays an extremely important role in protecting against Bordetella pertussis infection and disease onset, and has attracted attention as a protective antigen against Bordetella pertussis infection [5ato , Y et al.: Inf.
ect, Immun, + 31. 1223-123
1 (1981), and Sem1nars 1nInf
ectious Diseases IV, Bact
Erial Vaccine, 380-385 (198
2)]. Furthermore, it was confirmed that the F-HA of each Bordetella species is immunologically identical [Arai, H et al.; Infect, Immun, +32+ (1)+
243-'250 (1981)], it has also been shown that each F-HA may serve as a vaccine component common to Bordetella bacteria. For these reasons, it is desired to develop a method for easily isolating and purifying F-HA, which is a medically effective biologically active substance, in large quantities.

Prior art The conventionally known method for collecting and purifying F-HA is to fractionate the B. pertussis culture supernatant with ammonium sulfate and subject it to sucrose density gradient centrifugation.
Furthermore, there is a method to obtain Bordetella pertussis F-HA by repeating gel filtration twice [5atorY et al.; Infect, Iuu
nun, + 9+ 8 0 1 (1974) 1. Lh
However, this method has many steps and is complicated, and F
-There are drawbacks such as low yield of HA, and it is difficult to adopt it as an industrial purification method.

Similarly, as an example of purifying Bordetella pertussis F-HA, methods using ion exchange chromatography and gel filtration [Ar
ai, H et al; Infect, I++unun, +
25.460 (1979)], but this method has a low yield of F-HA, and it is difficult to remove Bordetella pertussis endotoxin, making it difficult to put to practical use.

As another example, a method combining droxyapatite adsorption chromatography, habtoglobian affinity chromatography, ammonium sulfate fractionation, and gel filtration [Cou+ell, J. L., et al.;
in Infectious Diseases I
V+Bacterial Vaccine, , 37
+ 1 (1982)], hydroxyapatite adsorption chromatography, and specific antibody/affinity chromatography.

There is a method that combines sucrose density gradient ultracentrifugation [Flow-through; Japanese Journal of Bacteriology, 3'8.423 (1983)], but these methods have very long and complicated steps, and also The yield of HA is low, hydroxyapatite is expensive, the affinity chromatography gels used above are not commercially available, and their preparation is extremely labor-intensive. Very expensive. Due to these various drawbacks, the above method cannot be used as an industrial and inexpensive method for collecting F-HA.

Purpose of the Invention The present inventors have conducted various studies in order to find an industrial method for isolating and purifying F-)-IA. As a result, the present inventors have found that a culture of Bordetella bacteria is brought into contact with a gel derivative of cross-linked polysaccharide sulfate ester. They discovered that highly pure F-HA could be obtained very easily and in a very high yield by adsorbing F-HA, separating it from contaminants, and eluting it from the gel, and completed the present invention. I ended up doing it.

That is, an object of the present invention is to provide a method for industrially and simply purifying F-HA, which is a biologically active substance that is extremely useful medically, in large quantities to extremely high purity.

Structure and effects of the invention.

In the present invention, a culture of Bordetella bacteria is brought into contact with a gel derivative of cross-linked polysaccharide sulfate ester, and F-HA
F is characterized by being eluted from the gel after being adsorbed.
- A method for purifying HA.

In the present invention, the starting material Bordetella culture includes cultures of Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica. A preferred culture in the present invention is a B. pertussis culture, in which B. pertussis is cultured statically or with shaking in a conventional medium, such as a Zuen-Willer medium or a liquid medium such as a Tenner-Scholte medium. Alternatively, it is a culture obtained by aeration agitation culture. This culture is subjected to the method of the present invention in the form of a culture supernatant from which bacterial cells have been removed by centrifugation, a centrifuged supernatant of disrupted bacterial cells, or a partially purified preparation thereof. According to the method of the present invention, there is no need to carry out preliminary partial purification treatments such as salting out, extraction, and ultracentrifugation, and the culture supernatant can be directly subjected to polysaccharide sulfate ester del adsorption chromatography, thereby simplifying the process. It's extremely simple.

The cross-linked polysaccharide sulfate ester used in the present invention is obtained by cross-linking polysaccharides such as dextran, cellulose, and agarose with a cross-linking agent such as epichlorohydrin, nochlorohydrin, dibromhydrin, and ethylene glycol bisepoxypropyl ether. It is obtained by sulfuric acid esterification of crosslinked polysaccharide. Cross-linked polysaccharides are already commercially available; examples of cross-linked dextran include Sephadex G-10, G-25, G-50, and G-100 (manufactured by Pharmacia), and cross-linked agarose includes Sephadex G-10, G-25, G-50, and G-100 (manufactured by Pharmacia);
Examples of crosslinked cellulose include Cellulofine GCL-25 and GGL-90 (manufactured by Chisso Corporation). By treating these gels with chlorosulfonic acid, sulfuric anhydride, or the like in the presence of an organic solvent such as pyridine, the desired crosslinked polysaccharide sulfate ester can be obtained.

In the present invention, the following method is used to purify and collect F-HA in a culture of Bordetella bacteria using a crosslinked polysaccharide sulfate gel.

The crosslinked polysaccharide sulfate ester gel is prepared by adding 0.2M sodium chloride in advance (0°f) i M
F-)( A is subjected to adsorption treatment.

A series of purification procedures such as adsorption of F-HA onto cross-linked polysaccharide sulfate ester gel, washing of the gel, and elution of F-HA are carried out using operational force methods commonly used in industry, such as batch methods and column methods. Let's do it. When performing the batch method, add the cross-linked polysaccharide sulfate gel to a culture of Bordetella bacteria, and slowly incubate at a temperature of 0 to 30°C for about 10 to 60 minutes at a pH of about 6.0 to 9.0. Stir to adsorb F-HA. At this time, the Bordetella genus bacterial culture is appropriately concentrated or diluted so that the specific conductivity thereof is approximately 5.0-25.0 ms/am for use in the adsorption operation.

After the adsorption is completed, the culture-gel mixture is filled into a filter and filtered by suction to separate the gel from the oral fluid. The separated gel has a specific conductivity of about 5 to 25 ms/can and a pH of 5.
A suitable buffer solution having a concentration of about 0 to 10.0, for example, 0.2M
Added 0.02M sodium chloride.

Kirvene's (Me I 1vaine's) buffer, 0.
3M sodium chloride addition 0.01M phosphate buffer or 0°3) 14 sodium chloride addition 0.01. Wash by pouring M Tris-HCl buffer, etc., and suctioning.

After this, the pH is about 560 to 10.0 and the specific conductivity is 2.
5 to 130 m5/gloss (greater than the specific conductivity of the bipedal washing buffer). For example, 1.5M sodium chloride-added pine kilbene buffer, 1.5M lium-added phosphate buffer. F-HA that is adsorbed by pouring liquid etc.
elute.

Column method 1 When carrying out the method of the present invention, the raw material liquid,
The conditions for the washing buffer and elution buffer may be the same as in the batch method, and the flow rate of these solutions is 10 ml/cm2/
It is preferable to adjust it to about Hr-50-Oi1/c+++2/)lr.

According to the purification method of the present invention, F-HA in B. pertussis culture
has excellent specific adsorption ability, the degree of purification of F-HA reaches several tens of times, and the recovery rate of F-HA reaches 90% or more and nearly 100%. The specific activity of the purified F-HA obtained is 4-8X
Extremely high at 10'HA units/l11g protein,
A single band was formed in polyacrylamide disk electrophoresis (pH 4, 5) analysis, and Bordetella pertussis endotoxin was almost completely removed.

As described above, according to the method of the present invention, the desired F-HA can be collected in high yield and purity from the B. pertussis culture as the starting material, and its repetition is extremely simple, and it is easy to purify it. The chromatography adsorbent can be prepared at a low cost, shows no deterioration during repeated use, and is extremely economical.

Therefore, the method of the present invention is an extremely excellent method for industrially purifying high-purity F-HA.

The method of the present invention can also be combined with conventional techniques such as sucrose density gradient ultracentrifugation or ion exchange chromatography, and in that case, the results obtained are significantly better than those obtained with conventional methods. You can get excellent results.

F-HA obtained by the method of the present invention is highly purified and does not contain other proteins, lipids, sugars, etc., and endotoxins are almost completely removed. Therefore, various reagents utilizing its biological activity can be used. It is useful in the preparation of pharmaceuticals and also in the preparation of Bordetella pertussis vaccines.

The present invention will be explained in more detail with reference to the following Preparation Examples and Examples.

Preparation Example 1 Add 11 parts of chlorosulfonic acid dropwise to 200 J of pyridine at a temperature below 0°C and mix. After the dropwise addition is completed, the mixed liquid is heated to a temperature of 65 to 70°C.

7.5 g of Sephadex G-50 (manufactured by 7Fulmacia), which is an epichlorohydrin crosslinked dextran, is added to the mixture, and the mixture is maintained at 65 to 70° C. for 4 hours while stirring. After the reaction is completed, it is cooled and neutralized by adding an aqueous sodium hydroxide solution. The gel is separated by filtration and thoroughly washed with 0, OIM + 7 phosphate buffered saline to obtain cross-linked dextran sulfate.

Preparation Example 2 7.5 g of dried cellulofine GCL-25 (manufactured by Chisso Corporation), which is a crosslinked cellulose gel, was added to 210 IlIl of a pyridine-chlorosulfonic acid mixture prepared in the same manner as in Preparation Example 1, and the mixture was heated to 65 to 70°C. and react for 4 hours.

After the reaction is completed, it is cooled and neutralized by adding an aqueous sodium hydroxide solution. The gel was overseparated at 0. 7. Wash thoroughly with OIM phosphate buffered saline to remove cross-linked cellulose sulfate.
Obtain 2g.

Preparation Example 3 To 210 liters of a pyridine-chlorosulfonic acid mixture prepared in the same manner as in Preparation Example 1, 30 liters of a pyrinone inclusion body of Cef70-S CL-6B (manufactured by 7Furmacia), which is a cross-linked agarose gel, was added. React at 65-70°C for 4 hours. After the reaction is completed, it is cooled and neutralized by adding an aqueous sodium hydroxide solution.

The gel is separated by filtration and thoroughly washed with 0.01M phosphate buffered saline to obtain crosslinked agarose sulfate ester 23P.

Example 1 Sephadex G- prepared in the same manner as in Preparation Example 1
Column (16 mm φ x 100 m)
m), and add 0.2M sodium chloride to this.
01M phosphate buffer (pH 8°0, specific conductivity approximately 17,
5 ms/cm) to equilibrate. 800 ml of a static culture supernatant of pertussis I phase bacteria Higashihama strain was diluted and the solution adjusted to a specific conductivity of about 17.5 ms/cm 2 and a pH of 8.0 was passed through this column. After passing through the solution, wash with the above buffer solution,
Wash out contaminants. Then, it is eluted with a phosphate buffer solution (pH 7.6) containing 1.5 M sodium chloride to obtain 30 pieces containing F-HA.

The analysis results of the culture supernatant, both the flow-through fraction and the purified F-HA are shown in Table 1.

The recovery rate of F-HA was 93.8%, and the purity (purified F-H
The specific activity of fraction A/specific activity of culture supernatant) reached 34 times. The LPF-HA activity of the purified F-HA fraction was determined by the hub FEL
ISA method [Tsumi Sato Proceedings of the 28th Toxin Symposium 14
1 (1981)].
/ml or less.

Table 1 1) F-HA hemagglutination test [5ato, Y, et a
l, Infect.

1+nmun, +7+929-999 (1973)]
2) Displayed as protein content using the Kirsol method protein nitrogen measurement value
It was conducted in accordance with the Biological Products Standards (Yakuhatsu No. 287, 1981).

Example 2 Cellulofine GCL- obtained in the same manner as Preparation Example 2
25 sulfuric acid ester in a column (16 mmφ
0 mm), and equilibrated by passing 0.01 M phosphate buffer (pH 8.0) containing 0.14 M sodium chloride through it. Through this column, 800+++ liters of culture supernatant of Pertussis spp. Higashihama strain Fermenter from the same lot as that used in Example 1 was diluted to a specific conductivity of approximately 10+ns/cm and adjusted to pH 8.0. do. After passing through the solution, the column is washed with the above buffer, and then eluted with 1.5 M sodium chloride-added phosphate buffer (+) 17.6) to obtain 30 ml of purified F-HA-containing fraction.

The analysis results of the culture supernatant, the flow-through fraction, and the purified F-HA fraction are shown in Table 2.

The recovery rate of F-HA was 75%, and the degree of purification reached 23 times.

Table 2 1), 2), 3), 4), 5) Same Example 3 as in Table 1 Cef70-S CL-6B obtained in the same manner as Preparation Example 3
Column (16+nmφX 100
mto) and added 0.2M sodium chloride to this. OIM phosphate buffer (pH 8,0) is passed through to equilibrate. This column was filled with 80% of the culture supernatant of Rondo's pertussis spp. Higashihama strain Fermenter, the same as that used in Example 1.
hl was diluted to have a specific conductivity of about 17, 5 ms/cm, and a pH of 8.0. After passing through the solution, wash with the above buffer solution to wash out contaminants. Next, a phosphate buffer solution (pH 7.6) containing 1.5M sodium chloride was added.
) to obtain 28 ml of both fractions containing F-HA.

Table 3 shows the analysis results of the culture supernatant, the flow-through fraction, and the purified F-HA fraction.

The recovery rate of F-HA was 87.5%, and the degree of purification reached 33 times. The main LPF-HA activity was less than 10 ELISA units)/+Ill.

Using this purified product, we conducted a mouse weight loss test, a mouse white blood cell increase test, a heat-labile toxin test, and a mouse histamine sensitization test in accordance with the biological product standard "pertussis vaccine" (see Yakuhatsu No. 287, 1981). The results were comparable to the control group inoculated with physiological saline, and no side effects were observed.

Table 3

Claims (10)

[Claims] (1) When purifying and obtaining fibrillar hemagglutinin produced by Bordetella bacteria, the fibrillar hemagglutinin-containing solution is
A method for purifying fibrillar hemagglutinin, which comprises bringing it into contact with a gel derivative of cross-linked polysaccharide sulfate ester, adsorbing fibrillar hemagglutinin, and then eluting the adsorbed fibrillar hemagglutinin from the gel. (2) The method according to item (1), wherein the crosslinked polysaccharide sulfate is one selected from crosslinked dextran sulfate, crosslinked agarose sulfate, and crosslinked cellulose sulfate. (3) Item (2) above, wherein the crosslinked dextran sulfate is epichlorohydrin crosslinked dextran sulfate.
The method described in section. (4) The method according to item (2) above, wherein the crosslinked agarose sulfate is epichlorohydrin crosslinked agarose sulfate. (5) The method according to item (2) above, wherein the crosslinked cellulose sulfate is epichlorohydrin crosslinked cellulose sulfate. (6) The gel derivative of the polysaccharide sulfate ester has a pH of 6.9 to 9.0, a specific conductivity of S, O to 2S, O
The method according to any one of items (1) to (5) above, wherein the method is subjected to an adsorption treatment after being equilibrated by a force comparison treatment with a buffer solution of ms/cm. (7) The adsorption treatment was carried out at a pH of 6.0 to 8. O1 temperature θ~3
The method according to any one of items (1) to (6) above, which is carried out under conditions of 0° C. and a specific conductivity of 5.0 to 25.0 ms/cm. (8) The elution of fibrillar hemagglutinin from the gel was 1.8
5.0-10.0, specific conductivity 25.0-130m5/c
Any one of the above methods (1) to (7) is carried out using a buffer solution of m. (9) Prior to the elution treatment, the adsorption gel was adjusted to pH 5.0.
-10,0, specific conductivity S, 025. The method according to item (8) above, in which washing is performed with a buffer solution of Oms/cm. (10) The method according to any one of items (1) to (9) above, wherein the filamentous hemagglutinin-containing liquid is a culture of Bordetella pertussis.