JPS64931B2 - - 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 Factor
The fraction containing Hemagglutinin) was collected, and the HA
A method for producing a pertussis vaccine by detoxifying the fraction in the presence of amino acids, more specifically, a method for producing a pertussis vaccine by cultivating Bordetella pertussis in a liquid medium containing cyclodextrin or its derivatives with aeration and stirring. By controlling the amount of oxygen within a specific range and performing under antifoaming conditions, the infection-protective antigen HA fraction of Bacillus pertussis is collected, and this is detoxified in the presence of amino acids to produce a pertussis vaccine on an industrial scale. Regarding the method. 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 to this, and further treated with aluminum adjuvant if necessary, gelatin,
Stabilizers such as glucose are added to create a precipitated and purified pertussis, diphtheria, and 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, 1979
). 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 method 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 solution. (Hiroki et al., Proceedings of the 29th Toxin Symposium, 1-5,
(see 1982). However, when this method was applied to the production of pertussis vaccine on an industrial scale using 10 or more scale fermenters, results were obtained that could not be inferred from the knowledge based on conventional agitation culture. In other words, although it is known that the number of bacteria increases in shaking culture or agitation culture systems in which stirring conditions are kept constant, 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,
We found that even in large-scale culture, especially in aerated agitation culture using a conventional fermenter, B. pertussis can significantly proliferate and the B. pertussis HA fraction can be significantly increased. It was found that detoxification was achieved by adding formalin, and no reversal of toxicity occurred even after long-term heating at 37°C.
In this detoxification treatment, the amino acid addition system does not produce aggregate precipitates compared to the amino acid non-addition system, and there is no need for steps such as sonication, and the detoxified HA fraction is directly passed through sterile filtration using a membrane filter. They discovered what they could do and completed 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. Aerated agitation culture is carried out at a pH of 6.0 to 9.0, preferably at a pH of 6.0 to 9.0, and an infection-protecting antigen HA fraction is collected during the logarithmic growth phase or stationary phase of bacterial growth. The vaccine is then detoxified with formalin and used to economically produce a desired purified pertussis vaccine, precipitated purified pertussis vaccine, precipitated purified pertussis/diphtheria/tetanus combined vaccine, etc. in large quantities. 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 preferably, the Steiner-Scholte medium is used as the basis, and this contains casamino acids in an amount of 0.1 to 20 g/
A Steiner-Schorte improved medium (hereinafter simply referred to as improved medium) is used, in which ascorbic acid and glutathione are adjusted to a range of 0.01 to 1 g/0.1 to 5 g/. Cyclodextrin (hereinafter referred to as
(abbreviated as CD) or its derivatives, α
-isomers such as CD, β-CD, γ-CD, methylated α-CD, methylated β-CD (listed above), methylated γ
In addition to etherified derivatives such as -CD, aminated derivatives and esterified derivatives may be mentioned, and these 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 0.001/5 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),
Culture temperature ranged from 17℃ to 42℃ with shaking speed 60 times/
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 amount and stirring speed, and the aeration amount and stirring speed are not particularly limited, but when a normal aeration stirring tank is used, the amount of air aeration 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. In addition, in order to obtain the desired HA fraction with a high yield, it is important to check the culture state of the bacteria, and it is important to collect the bacteria at the growth stage from the logarithmic growth phase through the conversion phase to the stationary phase. Desirably, it 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. A sedimentation-purified pertussis vaccine is prepared from the HA-containing culture solution produced as described above. That is, after directly or continuously centrifuging the obtained culture solution, ammonium sulfate is added to approximately 1/3 saturation, and the resulting precipitate is collected by centrifugation or filtration, and then added with 1M sodium chloride. Dissolve in phosphate buffer, PH7.2. Add ammonium sulfate to this solution until it is about 1/2 saturated,
The resulting precipitate is collected by centrifugation or filtration, placed in a dialysis tube, and dialyzed against 1M sodium chloride-added phosphate buffer, pH 7.2, to dissolve. This is subjected to ultracentrifugation, and the resulting supernatant is further subjected to sucrose density gradient centrifugation to obtain the supernatant (B. pertussis HA fraction). These series of purification steps are desirably carried out at 4°C or lower. The obtained supernatant contains a large amount of LPF-HA and F-HA. When this was investigated by electrophoresis, it was found that F-HA and LPF-derived from the conventional static culture method.
It has the same molecular weight and charge as HA, and also has the same morphology (electron microscopy findings), antigenicity (in-gel precipitation reaction findings), and mouse LPF toxin activity. This HA fraction is diluted appropriately, and formalin is added to it at 0.1 to 1.2 v/v%, preferably 0.4 to 0.8 v/v%.
Add to a concentration of 20-43℃, preferably 37-40℃
Process for 3 to 60 days. This formalin detoxification treatment reduces the toxicity of LPF activity, HSF activity (Histamine-sensitizing factor), etc. in the HA fraction. The present inventors have found that if amino acids are added in this detoxification treatment, the time required for detoxification is significantly shortened, and no aggregate precipitation occurs and toxicity reversion phenomenon (reverse) does not occur. At this time, Tween 80 and gelatin may be added as stabilizers along with the amino acids as appropriate. As the amino acid, one or more amino acids are selected and used from glycine, methionine, cysteine, monosodium glutamate, aspartic acid, serine, alanine, leucine, isoleucine, valine, threonine, γ-aminobutyric acid, lysine, and the like. When the HA fraction purified from a culture solution obtained using a fermenter in a liquid medium containing cyclodextrin or its derivatives is detoxified with formalin after adding the above amino acids, aggregate precipitates are generated. Therefore, it is possible to perform sterilization using a membrane filter. On the other hand, applying formalin detoxification treatment in the substantial absence of amino acids is similar to the case of detoxifying the HA fraction purified from the culture medium obtained by static culture in a liquid medium without the addition of cyclodextrin. In this case, an agglomerate precipitate is formed, and it is necessary to crush this precipitate by sonication in a subsequent step. In this case, it is difficult to carry out sterilization in the process after detoxification. After the above detoxification treatment, adjust to an appropriate protein concentration (usually final protein nitrogen concentration 8-20 μg TCAPN/ml)
Then, if desired, diphtheria toxoid and tetanus toxoid are further added, and aluminum hydroxide or aluminum phosphate is added as is or as an adjuvant if necessary to a final concentration of 0.15 to 0.3 μg/ml.
Treat in addition to the degree. Finally, appropriate amounts of gelatin and glucose as stabilizers and thimerosal as a preservative are added to prepare the vaccine. EXAMPLES Next, the present invention will be explained in more detail with reference to experimental examples and examples, but the present invention is not limited thereto. Experimental 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.).
to a final concentration of 1.0 g/medium
35 and inoculated with pertussis bacteria at an amount of 1.0 IOU/ml. The control range of DO was varied in an aerated culture system with a sparger from the bottom of the tank, and the temperature was increased to 35.
℃ 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.Infect.Immun. 7 , 929-999, 1973)
In vitro, LPF−HA was measured by Hp−
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 (Hyoshiki et al., Proceedings of the 29th Toxin Symposium, 1-5,
1982). The second result is
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.
~60ppm, and particularly good results were obtained for DO1.0-5.5ppm. Comparative Examples 1 to 10 and Experimental Examples 2 to 5 The production amount of the Bordetella pertussis HA fraction was comparatively studied by changing various 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 Experimental 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 Experimental Examples (hereinafter simply referred to as 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, although a certain degree of logarithmic growth (approximately 10 IOU/ml) was shown up to 10 hours of culture, the growth rate decreased rapidly after that and remained at approximately 15 IOU/ml even after 36 hours, indicating that the production amount of the HA fraction F-HA is 16HA/ml,
LPF-HA was extremely low at 100 IOU/ml, and hardly increased even when 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 over 6.0 ppm, which is inappropriate for HA fraction production, and as the culture progressed, the DO
The concentration continued to decrease and reached a level of 0.7 ppm or less, which is inappropriate for bacterial growth and HA production, within 36 hours. In Comparative Example 9, in which chemical defoaming treatment was carried out in part by the method of the present invention, F-HA reached 80 IOU/ml after 36 hours of culture, but F-HA was 128 HA/ml.
ml, LPF-HA was approximately 500 LPEU/ml.

[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 the amount of F-HA and LPF-HA 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 HA produced could be significantly increased. Comparative Experimental Example 11 and Experimental Examples 6 and 7 As described above, the target Bordetella pertussis HA is cultivated under specific controlled 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: Lou 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) (Experimental Examples 6 and 7) Culture container: 300 volume fermenter (manufactured by Marubishi Rika) Medium: Same improved medium as used in Experimental Example 1 ,
200; Methylated β-CD (Experimental Example 6) or Methylated α-CD (Experimental 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 Experimental 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. Example 1 The HA fraction obtained in Experimental Example 1 was
Dilute to 30 μg TCAPN/ml, add formalin to a concentration of 0.6 or 1.0 v/v%,
Process at 37°C or 39°C for 5-21 days. During this detoxification treatment with formalin, various amino acids shown in Table 4 are added at the indicated concentrations. The presence or absence of agglomerate precipitation during this detoxification treatment was examined, and after the detoxification treatment, the samples were dialyzed using a dialysis tube to remove formalin, and then toxicity to mice was examined. Furthermore, the samples were heated at 37°C for 3 weeks and the presence or absence of toxicity recovery was examined. The titer of the obtained vaccine was also measured. The results are shown in Table 4.

[Table] Example 2 The pertussis bacteria Higashihama strain was subcultured in Bordet-Jeyang medium and used as a seed strain, and the methylated β
- Inoculated into a 2-volume triangular container containing 0.4 of the same improved medium used in Experimental Example 1, supplemented with 10 mg/ml of CD, at a final bacterial concentration of 1.0 IOU/ml, and cultured at 35°C for 18 hours. The original cultured bacteria was obtained. Add 1.0 IOU/ml of the above original culture to a 300-capacity fermenter (manufactured by Marubishi Rika) containing 200 of the same medium as above.
Inoculate and culture with aeration and agitation. In this culture, both the aeration amount and stirring speed were automatically controlled so that the DO was 1.8 to 2.7 ppm. In addition, the culture temperature was automatically controlled to 35°C and pH to 7.2, and mechanical defoaming (rotating disk method) was also performed. After culturing for 35 hours, collect the culture medium. When the number of bacteria and the amount of HA fraction were measured for the culture solution obtained in this way, the number of bacteria was 210 IOU/ml, F-
HA amount 1.024HA/ml, LPF-HA amount 2400LPEU/
It was hot in ml. Centrifuge the culture solution obtained above and collect the supernatant.
Add ammonium sulfate to the supernatant to 1/3 saturation, collect the resulting precipitate by centrifugation at 10,000 rpm for 30 minutes, dissolve it in phosphate buffer (PH7.2) containing 1M sodium chloride, and remove the undissolved precipitate. of
Removed by centrifugation at 10,000 rpm for 30 minutes. Ammonium sulfate was added to this supernatant to approximately 1/2 saturation, and the resulting precipitate was similarly centrifuged and collected, dissolved again in the same buffer as above, placed in a dialysis tube, and dialyzed at 4°C to remove the undissolved precipitate. It was removed by centrifugation in the same manner. This was subjected to ultracentrifugation, and the resulting supernatant was further centrifuged with 10-30% sucrose density gradient (39,000 rpm, 20
time), and the supernatant (HA fraction) was collected. This was sterilized using a membrane filter.
The protein concentration of this HA fraction was adjusted to 30 μg TCAPN/ml using the same buffer as above. Incidentally, the treatment process of the collected culture solution and the purification process of the HA fraction were mainly performed at 2 to 4°C. 0.6v/v% formalin was added to the obtained HA fraction.
Tween 80 at 0.05v/v%, gelatin at 0.02w/v
0.25M of glycine was added to the mixture, and the mixture was heated at 39°C for 7 days. Place in a dialysis tube and dialyze against 0.7w/v% sodium chloride-added phosphate buffer (PH7.2) to remove formalin, and dilute this with the above buffer to a protein concentration of 8μgTCAPN/ml. Then, a diluted detoxified HA fraction was obtained. Add 0.20% aluminum hydroxide gel to this diluted solution.
mg/ml (aluminum equivalent)
The HA fraction was adsorbed. A precipitated and purified pertussis vaccine was prepared by adding 0.01 w/v % of thimerosal as a preservative. This vaccine was tested in accordance with the national testing standards (see Biological Product Standards, Yakuhin No. 287, 1971) and was found to comply with all the requirements as shown in Table 5.

[Table] Example 3 To the detoxified fraction diluted solution obtained in Example 1,
Diphtheria toxoid (33 Lf/ml) and tetanus toxoid (5 Lf/ml) were added thereto, and aluminum hydroxide (0.20 mg/ml (in terms of aluminum)) was added thereto for gel adsorption. Gelatin is used as a stabilizer
0.02w/w, glucose 0.1w/v%, and thimerosal 0.01w/v% as a preservative were added to prepare a precipitated and purified pertussis/diphtheria/tetanus combined vaccine. This combination vaccine was tested in accordance with the national testing standards (see Biological Product Standards, Yakuhin No. 287, 1981) and was found to comply with all the requirements as shown in Table 6.

【table】

【table】

【table】 [Brief explanation of the drawing]

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.
Graphs showing the F-HA amount and LPF-HA amount after 24-hour culture, Figures 3 and 4 show the number of bacteria, F-HA amount, and It is a graph showing the change in LPF-HA amount 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 a pertussis vaccine, which comprises collecting a protective antigen HA fraction during the bacterial growth stage of logarithmic growth phase or stationary phase, and then detoxifying the HA fraction in the presence of amino acids. 2 Amino acids are glycine, methionine, cysteine, monosodium glutamate, aspartic acid,
1. The method according to item 1, wherein one or more selected from serine, alanine, leucine, isoleucine, valine, threonine, γ-aminobutyric acid, and lysine. 3. 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. 4 Cyclodextrin or its derivative is one or two selected from methylated α-cyclodextrin, methylated β-cyclodextrin, methylated γ-cyclodextrin, α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin The method of item 1 above. 5. The method of item 1 above, wherein the culture time is 7 to 40 hours. 6. 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. 7. The method of item 1 above, wherein the pH is in the range of 6.0 to 9.0.