Method of separating protective components of bordetella pertussis

a protective component and technology of bordetella pertussis, applied in the direction of antibacterial agents, antibacterial ingredients, peptides, etc., can solve the problems of contaminated human haptoglobin, insufficient satisfaction, and inability to remove endotoxin (et), and achieve high efficiency, high practical value, and high recovery rate

Inactive Publication Date: 2002-08-29
TAKEDA PHARMA CO LTD
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  • Abstract
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Benefits of technology

[0032] The calcium phosphate gel used for the present invention is not a ready-made gel, but preferably calcium phosphate gel formed in a culture or a supernatant to be treated by adding calcium ions to them in the presence of excess phosphate ions (may referred to as the in-side gel forming method). Although prepared calcium phosphate gel (e.g., commercially available hydroxyapatite gel). In comparison with the former method by using the ready-made hydroxyapatite gel mentioned above (may referred to as the out-side gel forming method), the present method by using calcium phosphate gel is higher in both adsorption efficiency for pertussis filamentous hemagglutinin (FHA) and pertussis fimbriae (FIM) and recovery efficiency of them, as shown hereafter. Moreover, the calcium phosphate gel used in the present invention is better in operational efficiency because of the absence of gel pretreatment and regeneration process, and more advantageous in cost. Furthermore, each of protective components of Bordetella pertussis can be selectively absorbed to the calcium phosphate gel by properly selecting the ratio of phosphate ions to calcium ions. If the the culture or the supernatant to be treated with calcium phosphate gel, does not contain a sufficient amount of phosphate ions, a phosphate buffer of appropriate concentration is added to provide phosphate ions before addition of calcium ions. For example, by adding 1 M phosphate buffer, the final phosphate ion, concentration is adjusted to 0.02-0.2 M, preferably 0.05-0.1 M.
[0045] The above-described effect of the present invention can be summarized as follows: The method of the present invention is characterized by the use of the same means of purification for all subject protective components of Bordetella pertussis. This obviates the necessity of different painstaking procedures for the respective components as in prior art methods, thus permitting component purification with high efficiency and high recovery rate, an aspect very advantageous for industrial production. In addition, the endotoxin content, as determined by the Limulus test, is not more than 1 ng per 100 .mu.g total protein, for all protective components of Bordetella pertussis obtained by the present invention, providing very high practical value. It is also possible to produce an improved purified pertussis component vaccine comprising an effective combination of pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP), pertussis fimbriae (FIM) and pertussis toxin (PT).

Problems solved by technology

Pertussis, a communicable respiratory disease caused by infection with Bordetella pertussis, is likely to severely affect patients, especially infants, due to apneic cough with occasional spasm.
For example, acellular pertussis vaccine (ACP vaccine), prepared by extracting protective proteins, such as pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM), from Bordetella pertussis cells, and removing endotoxin (ET), is being into practical application, but is not fully satisfactory, due to the drawbacks described below.
However, human haptoglobin can be contaminated with hepatitis virus, because it is collected from human blood; the same applies when animal sera are used.
Although this method is free of the problem of viral contamination, some problems arise, including vaccine contamination with ceruloplasmin and the high toxicity and potential body retention of sodium thiocyanate and other eluents having protein-denaturing effect.
However, it takes long time for column operation, and is uneconomic due to the high cost of hydroxyapatite.
442 (1985)], but this method is poor in vaccine production efficiency due to low yield.
The vaccine obtained by the method of WO93 / 10216 has a danger of side effects, such as fever and endotoxin-shock, by the endotoxin released into body because of the diluted vaccines.
However, this method formed the calcium phosphate in the presence of a 1M sodium chloride does not absorb the protective components.
This approach is unsuitable to large-scale vaccine production due to painstaking operation, and difficult to apply practically.
Moreover, the customary methods of separating protective components disclosed in prior art have some problems that materials or reagents have pathogenicity or toxity.

Method used

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Examples

Experimental program
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example 1

[0047] Bordetella pertussis Tohama phase I strain was cultured to a final concentration of 2 billion cells / ml by Roux bottle stationary culture (450 ml, 35.degree. C., 5 days) and tank agitating culture (40 l, 35.degree. C., 2 days) using Stainer-Scholte medium, to yield a Bordetella pertussis culture.

[0048] The cell culture was concentrated to a one-tenth volume using an ultrafiltration membrane, after which it was centrifuged to separate the supernatant and cells. To the supernatant, a 1 M phosphate buffer (pH 8.0) was added to a final concentration of 0.1 M, followed by addition of an calcium acetate solution to a final concentration of 1.6 w / v% and stirring at room temperature for 1 hour. This calcium phosphate gel solution was filtered. The resulting filtrate was concentrated and desalinized to an electroconductivity of 200 umho using an ultrafiltration membrane, passed through a sulfopropyl cation exchange chromatography column (produced by Tosoh Corporation), washed with a 0....

reference example 1

[0055] To a control solution prepared by the method described in Example 1, calcium acetate was added to a final concentration of 0.5 w / v%, followed by stirring at room temperature for 1 hour. To the filtrate obtained by filtering this; calcium solution, a half amount of a saturated ammonium sulfate solution was added; the mixture was kept standing at 4.degree. C. for 7 days. This ammonium sulfate salting-out product was centrifuged; the resulting precipitate was collected and resuspended in a 0.025 M phosphate buffer (pH 7.0) supplemented with 0.25 M sodium chloride to yield a starting material. To this starting material, aluminum hydroxide gel, previously prepared to a final concentration of 0.4 mg / ml, was added; to the aluminum hydroxide gel recovered by centrifugation, ammonium sulfate was added to a final concentration of 0, 2, 4 or 8 w / v%, followed by gentle stirring at room temperature for 30 minutes. After completion of the reaction, the aluminum hydroxide gel was removed by...

example 2

[0057] To each of pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM) as obtained in Example 1, a half amount of a saturated ammonium sulfate solution was added, followed by sufficient stirring. After being kept standing at 4.degree. C. for 1 week, the mixture was again centrifuged; the resulting precipitate was collected.

[0058] This precipitate was dissolved in a 0.025 M phosphate buffer (pH 7.0) supplemented with 0.25 M sodium chloride to yield a solution of pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) or pertussis fimbriae (FIM). To each solution, a saturated ammonium sulfate solution was added to a final concentration of 4.0 v / v%. To this mixture, previously prepared, recovered aluminum hydroxide gel was added to a final concentration of 0.4 mg / ml, followed by gentle stirring for 30 minutes at room temperature. After completion of the reaction, the aluminum hydroxide g...

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Abstract

On the basis of differences in adsorbability to calcium phosphate gel formed by adding calcium ions to a Bordetella pertussis culture in the presence of excess phosphate ions, protective components of Bordetella pertussis are separated from the Bordetella pertussis culture. Traditionally, protective components of Bordetella pertussis have been separated using different purification methods for the respective components. According to the present invention, the use of the same means of purification for all subject components makes it possible to purify each component with high efficiency and high recovery rate, an aspect very advantageous for industrial production. It is also possible to efficiently produce an improved purified pertussis component vaccine comprising an effective combination of pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP), pertussis fimbriae (FIM) and pertussis toxin (PT).

Description

[0001] The present invention relates to a method of separating protective components of Bordetella pertussis. The pertussis component vaccine can be produced by suitably mixing the protective components separated by the method of the present invention.[0002] Vaccines are widely used to prevent communicable diseases. Pertussis, a communicable respiratory disease caused by infection with Bordetella pertussis, is likely to severely affect patients, especially infants, due to apneic cough with occasional spasm. To cope with this disease, it has been common practice to use whole cultured cells of Bordetella pertussis after inactivation (inactivated vaccine). However, localized reactions at the site of vaccination and side reactions, such as fever, have been reported, creating a social urge to solve this problem. To solve this problem, there have been a large number of attempts of using protective components separated from Bordetella pertussis as vaccine. For example, acellular pertussis ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/00A61P31/04C07K14/235
CPCA61K39/00C07K14/235A61P31/04C07K1/14C07K14/325
Inventor SUEHARA, AKIHIROYAMAMOTO, EIJIFUJII, SHIGEO
Owner TAKEDA PHARMA CO LTD
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