JPS64930B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is directed to the infection-protective antigen HA fraction (F-HA: Filamentous Hemagglutinin) of Bordetella pertussis.
LPF―HA：Leucocytosis―Promoting Faotr
The method for producing a fraction containing Hemagglutinin, more specifically, involves culturing Bordetella pertussis with aeration in a liquid medium containing cyclodextrin or its derivatives, controlling the culture temperature and amount of dissolved oxygen within a specific range. This invention relates to a method for producing a large amount of HA fraction, an antigen that protects against Bordetella pertussis infection, by carrying out the process under antifoaming conditions. Industrial Application Fields Pertussis is designated as a contagious disease in Japan, and is an infectious disease of public health importance that frequently occurs in infants and young children. Infants in particular often develop a severe course, and sometimes even die. It has been known for a long time that this disease can be effectively prevented by vaccination, and inactivated vaccines containing the whole body of the causative bacteria, the pertussis fungus, have been widely used. However, such inactivated bacterial vaccines have strong side effects, and as a result, vaccination was discontinued for a period of time. On the other hand, the disease of infants and children caused by pertussis has become a major problem, and the production of a vaccine without side effects has been eagerly awaited. Prior Art Earlier, Sato et al. succeeded in producing a purified precipitated pertussis vaccine, which is an epoch-making component vaccine, based on basic research on infectious protective antigens (see Japanese Patent Publication No. 57-5203). This vaccine uses the HA fraction containing F-HA and LPF-HA as the main infection-protective antigen, exhibits almost no side effects, has an excellent preventive effect, and has already been put into practical use. To manufacture this practical vaccine, pertussis bacteria are inoculated into a suitable medium, cultured for 5 days at around 35°C, the culture solution is centrifuged, and the supernatant is saturated with ammonium sulfate to approximately 50% saturation. Add or add alcohol so that the resulting precipitate is
The precipitate was separated by centrifugation for 30 minutes, extracted with a buffer solution containing sodium chloride, and the extracted fraction was subjected to sucrose density gradient centrifugation using a conventional method to recover the pertussis HA fraction, which was detoxified with formalin and used as a vaccine. If desired, diphtheria toxoid and tetanus toxoid are added thereto, and if necessary, aluminum adjuvant treatment is performed, and stabilizers such as gelatin and glucose are added to obtain a sedimentation-purified pertussis/diphtheria/tetanus combined vaccine. However, this method has particular difficulties in culturing, making large-scale culture impossible and making mass production of vaccines difficult. In other words, in this known production method for purified precipitated pertussis vaccine, a small container such as a Roux bottle containing approximately 100 to 300 ml of liquid medium is placed in a lying position.
The culture is statically cultured at around 35°C for 5 days, which requires a very small scale and a long period of time. Generally, a stirring culture method using a liquid medium is often adopted for mass culture of microorganisms. When B. pertussis is cultured with shaking in a liquid medium, the bacterium itself can grow to a certain extent, but the production of the F-HA fraction, for example, is said to be extremely low (Arai, H &
Munoz, JJ, Infect. Immun. 25 764―767, 1976
). This suggests that at least one of the components of a purified vaccine is difficult to mass produce. Therefore, although the pertussis vaccine of Sato et al. is an innovative vaccine, its production must rely on static culture, which is small-scale and takes a long time, and improvements in the production are eagerly awaited. Recently, Suzuki et al. have attempted to search for additives that can promote the growth of pertussis bacteria and the production of LPR-HA.
In particular, methylated β-cyclodextrin (2,6
-di(O-methyl)-β-cyclodextrin,
Stainer, DW & Scholte, MJ; J.Gen.
Microbiol. 63 , 211-220, 1971) was reported to promote bacterial growth and LPF-HA production in agitation culture, and also to contribute to the stability of LPF-HA in the culture medium. (Suzuki et al., Proceedings of the 29th Toxin Symposium, 1-5,
(see 1982). However, when this method is applied to the industrial-scale production of B. pertussis infection-protective antigen using 10 or more scale fermenters, results that cannot be inferred from the knowledge based on conventional agitated culture are obtained. It was done. In other words, we learned that while shaking culture under constant stirring conditions may increase the number of bacteria, the amount of LPF-HA produced is not sufficient. In addition, the WHO's 1977 publication (Manual
for the production and control of vaccines,
Pertussis vaccine (see WHO) describes the mass cultivation of B. pertussis in fermenters for the production of pertussis vaccines, in which air is introduced into the medium by surface aeration from above or through a grid, and special It is said that pertussis bacteria can be obtained by stirring with a blade and incorporating it into the culture solution through constant aeration and agitation.
However, at the culture scale of Steiner-Schjorte medium or its modified medium 10 described below, the aeration rate from the bottom of the tank was set at 0.2VVM (air flow rate ()/medium volume ()/hour) according to the WHO description. (minutes)), set the number of revolutions of the blade to 500 or
When we investigated a so-called constant aeration agitation culture system from the bottom of the tank at a constant speed of 600 rpm, an increase in the number of bacteria could be expected, but the production of the B. pertussis HA fraction was insufficient, making it impossible to produce a purified pertussis vaccine. I learned that it is not suitable for industrial production. Therefore, the present inventors conducted various studies in order to find culture conditions suitable for bacterial growth and mass production of the desired HA fraction even in aerated agitation culture using large-scale culture equipment, especially ordinary fermenters. As a result of repeated experiments, the amount of dissolved oxygen (hereinafter sometimes abbreviated as DO) can be controlled within a specific range at a certain range of culture temperature, while antifoaming treatment is performed, and more preferably, the culture can be carried out under PH controlled conditions. By doing so,
The present inventors have discovered that even in large-scale culture, particularly in aerated agitation culture using a conventional fermenter, B. pertussis can significantly proliferate and the B. pertussis HA fraction can be significantly increased, leading to the completion of the present invention. Structure and Effects of the Invention According to the present invention, Bordetella pertussis is inoculated into a liquid medium supplemented with cyclodextrin or its derivative, and the amount of dissolved oxygen is reduced to 0.7 to 6.0 ppm at a culture temperature of 20 to 37° C. while being subjected to defoaming treatment. Infection with the desired B. pertussis can be achieved by culturing with aeration and agitation at a pH of, for example, 6.0 to 9.0, and collecting the infection-protective antigen HA fraction during the logarithmic growth phase or stationary phase of bacterial growth. The protective antigen HA fraction is mass-produced on an industrial scale. The pertussis strain used in the present invention may be any strain that is commonly known as a vaccine strain, and in general, Bordet-Jeyang medium-subcultured bacteria or shake-cultured bacteria of its compatible bacteria are used.
This is used as a seed fungus to inoculate liquid medium. In addition, pertussis bacteria can also be applied. The amount of inoculation is not particularly limited, but usually the final concentration is 0.2
~10IOU/ml (IOU: International opacity
unit, Biological Products Standards, 238, 1979, Ministry of Health and Welfare), preferably about 1.0 IOU/ml. Any known medium can be used as the liquid medium, but Steiner-Scholte medium is preferably used.
Particularly preferred is a Steiner-Schjorte improved medium (hereinafter referred to as "Steiner-Scholte improved medium") based on the Steiner-Schjorte medium, to which 0.1 to 20 g of casamino acid is added, ascorbic acid is adjusted to 0.01 to 1 g/, and glutathione is adjusted to a range of 0.1 to 5 g/. (simply referred to as improved medium) is used. Cyclodextrin or its derivatives added to the medium include the above-mentioned methylated β-CD,
Methylated α-cyclodextrin, Methylated γ-
Ether derivatives such as cyclodextrin, α-
Examples include isomers such as cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, as well as aminated derivatives and esterified derivatives, which may be used alone or in combination of two or more. Among these, methylated β-CD exhibits the best addition effect. The amount added is not particularly limited, but is usually 0.001 to 5 g/, preferably about 0.5 to 2.5 g/. The present inventors were the first to recognize that culture temperature and DO control are major factors in bacterial growth and increase in F-HA and LPF-HA production in large-scale culture of Bordetella pertussis. Furthermore, it was revealed that controlling the pH of the culture medium also has a large effect. These results will be explained below. Regarding the culture temperature, a strain of Higashihama strain of Bordetella pertussis subcultured in Bordet-Jeyang medium was used as a seed, and inoculated at 0.2 IOU/ml into 10 ml of an improved medium supplemented with 1.0 g/ml of methylated β-CD. , using temperature gradient culture device TN112D (manufactured by Toyo Kagaku Sangyo),
Shake culture 60 times/with culture temperature ranging from 17℃ to 42℃
The optimal range was investigated by culturing with shaking for 48 hours. The number of bacteria grown was measured using a photoelectric colorimeter Coleman Giunia 6D.
It was calculated by converting the measured value at OD650 using a mold (manufactured by Coleman). Although this experiment was conducted on a small scale in the laboratory using shaking culture, the same tendency can be observed in large-scale aerated culture with regard to culture temperature. The results are shown in Figure 1, and the growth of bacteria is 20~
Desirably a range of 37°C, more preferably 23-37°C
It was hot. The amount of dissolved oxygen (DO) in the medium is maintained in the range of 0.7 to 6.0 ppm, preferably 1.0 to 5.5 ppm. By controlling within this range, the growth of Bordetella pertussis will increase and the desired LPF-HA and F
- Production of hyaluronan also increases significantly. Note that DO control can be carried out in combination with controlling the aeration rate and stirring speed, and the aeration rate and stirring speed are not particularly limited, but when using a normal aeration stirring tank, the air aeration rate is 3VVM or less,
Usually ranges from 0.1 to 2 VVM, preferably from 0.1 to 1.5 VVM, stirring speed is below 600 rpm, usually from 50 to
350 rpm, preferably in the range of 100-250 rpm.
However, if pure oxygen is used in combination, the aeration amount or stirring speed can be reduced. In addition, the increase in the number of bacteria in the culture solution and the yield of F-HA and LPF-HA are greatly affected by the presence or absence of defoaming operation, and when cultured under the same experimental conditions as in Example 1 described below, If defoaming is not performed, the bacterial cells attached to the foam may accumulate on the tank wall or flow out from the exhaust nozzle, reducing the number of bacteria in the culture solution and F.
- HA and LPF - A decrease of several to 80% in both HA amounts was observed. For defoaming, both mechanical defoaming and chemical defoaming agents can be used. For example, known defoaming devices such as a rotating disk type or a spray nozzle type can be used, or conventional fatty acid ester-based or silicone-based defoaming agents can be used. Chemical antifoaming agents such as , alcohol-based, etc. can be used. In addition, from the culture solution
From the viewpoint of collection and purification of the HA fraction, it is more preferable to use mechanical defoaming means. In order to find out the optimal range of medium PH, we examined bacterial growth by varying the PH. Culture was carried out under the same experimental conditions as in Example 1 described below, except that DO was kept constant at 2.5 ppm. Bacterial growth was achieved in the pH range of 6.0 to 9.0, and a slight increase in bacterial growth rate was observed in the pH range of 6.5 to 8.5, particularly PH6.8 to 7.5. Control of culture temperature, amount of dissolved oxygen, defoaming, pH, etc. according to the present invention can be carried out either automatically or manually. Additionally, in order to obtain the desired HA fraction at a high yield, it is important to check the culture state of the bacteria, and it is important to collect the bacteria during the growth stage from the logarithmic growth phase through the conversion phase to the stationary phase. Desirably, this will vary depending on the inoculum, but usually corresponds to 7 to 40 hours, for example, for an inoculum of 1.0 IOU/ml, usually 24 to 35 hours. EXAMPLES Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. Example 1 Methylated β-CD was added to an improved medium having the composition shown in Table 1 below in 50 fermenters (manufactured by Marubishi Rika Co., Ltd.).
Medium 35 to which was added at a final concentration of 1.0 g/
The pertussis bacteria was inoculated at an amount of 1.0 IOU/ml, and the control range of DO was varied in a culture system with aeration from the bottom of the tank using a sparger.
℃ and pH 7.2, and cultured for 24 hours using mechanical defoaming as a defoaming means.

[Table]
The number of bacteria in the obtained culture solution was measured in the same manner as before, and F-HA was subjected to a hemagglutination test (Sato,
Y.et.al.Infect.Immun. 7 , 929-999, 1973).
The unit (abbreviated as LPEu/ml) was measured by the ELISA method (see Sato et al., Proceedings of the 28th Toxin Symposium, 141-144, 1981), and in vivo
LPF-HA using dd/Y mice (4 weeks old, female)
Method for counting white blood cells 3 days after intravenous injection (Suzuki et al., Proceedings of the 29th Toxin Symposium, 1-5,
1982). The result is the second
As shown in the figure. As is clear from Figure 2, bacterial growth and
High unit production of HA fraction is observed at DO of 0.7.
~6.0ppm, and especially good results were obtained at DO1.0~5.5ppm. Comparative Examples 1 to 10 and Examples 2 to 5 The production amount of the Bordetella pertussis HA fraction was compared and studied by varying the culture conditions. That is, a comparison was made between a conventional method in which aeration and agitation are kept constant and a method in which aeration and agitation are continuously varied based on the present invention. An improved medium 10 containing the same methylated β-CD as used in Example 1 at a final concentration of 1.0 g was added to a 14 aerated stirring culture device (manufactured by NBS).
Inoculate the pertussis bacteria at an amount of 1.0 IOU/ml,
All cultures were cultured at 35°C for 36 hours under the conditions shown in Table 2 in a culture system with aeration and agitation from the bottom of the tank using a sparger. Comparative Examples 1 to 5, 7, and 8 in Table 2 are cultures in which aeration and agitation are constant and no defoaming treatment is performed.
Among them, the conditions of Comparative Example 5 of 100 rpm and 0.5 rrm produced a good amount of HA fraction. However, even in this case, a certain degree of logarithmic growth (approximately 10 IOU/ml) was shown up to 10 hours of culture, but the growth rate decreased rapidly after that, and even after 36 hours, approximately
The production amount of HA fraction remained at 15 IOU/ml, and the amount of F-
HA was 16 HA/ml and LPF-HA was extremely low at 100 IOU/ml, with almost no increase even when the culture was continued for 48 hours. In addition, Comparative Examples 7 and 8 are culture systems with aeration at 0.2 vvm, constant stirring at 500 rpm or 600 rpm, and no antifoaming treatment, but in both cases, after 5 to 10 hours of culture, intense foaming occurred. If the bacterial cells adhere to the upper part of the tank wall or the culture solution flows out of the tank,
HA even when all collected and mixed after hours
The fraction amount was extremely low. In Comparative Example 10, a culture system in which no antifoam treatment for controlling DO was performed, similar flow-off of the culture solution and adhesion of bacterial cells to the tank wall occurred, and the amount of HA fraction was low. Comparative Examples 6 and 9 are culture systems in which aeration and agitation are constant and defoaming treatment is performed. In either case, the DO at the initial stage of culture was above 6.0 ppm, which is inappropriate for HA fraction production, and as the culture progressed, the DO
Before 36 hours, the concentration continued to decrease and reached a level of 0.7 ppm or less, which is inappropriate for bacterial growth and HA production. In Comparative Example 9, in which chemical defoaming treatment was carried out in part by the method of the present invention, the concentration of F-HA reached 80 IUO/ml after 36 hours of culture, but F-HA reached 128 HA/ml.
ml, LPF-HA was approximately 500 LPEU/ml.

【table】

[Table] On the other hand, the DO controller (manufactured by NBS) was used to automatically and continuously change the aeration rate or stirring speed to control the DO in the culture solution to 1.6 to 3.5 ppm and defoaming. Examples 2 to 5 were cultured while being treated, and they maintained logarithmic growth even after 10 hours of culture, and finally reached extremely high bacterial counts and F-HA amounts within 36 hours. and LPF-HA amount could be obtained. In addition, in the method using surface ventilation from above,
No production of HA fraction was observed. As is clear from these results, in aerated agitation culture using a fermenter, bacterial growth is observed when antifoaming is applied in the absence of DO control, but in these examples, F-HA and LPF-HA It was found that the production amount was low in both cases. On the other hand, when the experiment was carried out under DO control, not only bacterial growth but also F-HA and LPF-HA levels were significantly increased. In this way, in the aerated agitation culture using a fermenter, DO control not only allows for bacterial growth but also for the target pertussis bacteria.
It was found that the amount of hyaluronan produced could be significantly increased. Comparative Example 11 and Examples 6 and 7 As described above, the desired Bordetella pertussis HA was obtained by culturing under specific control conditions according to the present invention.
A large amount of fractions can be produced, as shown in Table 3, when compared with conventional static culture. The conditions for each culture shown in the table are as follows. However, the bacterial inoculation amount and culture temperature were 1.0 IOU/ml and 35°C, respectively. (A) Static culture (Comparative Example 11) Culture container: Roux bottle, 1.5 volume Medium: Same improved medium as used in Example 1
0.2 Culture time: 120 hours (B) Controlled culture (method of the present invention) (Examples 6 and 7) Culture container: 300 capacity fermenter (manufactured by Marubishi Rika) Medium: Same improved medium as used in Example 1 ,
200; Methylated β-CD (Example 6) or Methylated α-CD (Example 7) 1.0
DO control: 2.2-2.4 ppm Defoaming: Mechanical defoaming means (by rotating disk method) PH control: PH7.3 Culture time: 35 hours The above results are shown in Table 3.

[Table] As is clear from the results in Table 3, the method of the present invention reduces bacterial growth and
Both LPF and HA amount increased significantly, and F-
The amount of HA is the same or higher, for example, the number of bacteria is 2 to 3.
times, the amount of LPF-HA increased more than 10 times, and the culture time
The time has been significantly reduced from 120 hours to 35 hours. In addition, the number of bacteria, amount of F-HA, and LPF- in the case of culture under DO control in Examples 6 and 7
Figures 3 and 4 show the changes in HA amount over time.
The figures are shown in Figs. As is clear from these figures, when cultured under DO control according to the present invention, as the number of bacteria increases, the amounts of F-HA and LPF-HA also increase significantly.

[Brief explanation of drawings]

Figure 1 is a graph showing the relationship between the growth of Bordetella pertussis and culture temperature, and Figure 2 is a graph showing the DO control range in the culture solution.
Bacterial count, F-HA amount and LPF after 24-hour culture
Graphs showing the amount of HA, Figures 3 and 4, show the number of bacteria, F--HA amount, and LPF-- when cultured under the control of DO2.2 to 2.4 ppm.
2 is a graph showing changes in the amount of HA over time.

Claims (1)

[Claims] 1. Bordetella pertussis is inoculated into a liquid medium supplemented with cyclodextrin or its derivatives, and cultured at a temperature of 20°C.
Cultivate with aeration at ~37°C while maintaining the amount of dissolved oxygen in the medium within the range of 0.7 to 6.0 ppm and performing antifoaming treatment.
1. A method for producing an infection-protective antigen HA fraction of Bordetella pertussis, which comprises collecting the infection-protection antigen HA fraction at a bacterial growth stage of logarithmic growth phase or stationary phase. 2. The liquid medium contains 0.1 to 20 g of casamino acid, 0.01 to 1 g of ascorbic acid, and glutathione.
The method according to the above item 1, containing 0.1 to 50 g/and 0.001 to 5 g/of cyclodextrin or its derivative. 3 Cyclodextrin or its derivative is one or two selected from methylated α-cyclodextrin, methylated α-cyclodextrin, methylated γ-cyclodextrin, α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin The method of item 1 above. 4. The method of item 1 above, wherein the culture time is 7 to 40 hours. 5. The method according to item 1 above, wherein the defoaming treatment is performed by adding a mechanical defoaming means, a chemical defoaming agent, or a combination thereof. 6. The method of item 1 above, wherein the pH is in the range of 6.0 to 9.0.