KR20060121501A - Method for the preparation of tetanus toxoid vaccine - Google Patents

Abstract

A method for the preparation of tetanus toxoid vaccine is provided to improve safety and immunity by removing toxicity of tetanus toxin, so that tetanus in young and adult people is prevented. The method for the preparation of tetanus toxoid vaccine comprises the steps of: culturing Clostridium tetani in the medium for 24-72 hours by supplying 1-10 l/min of nitrogen gas, and further culturing it for 3-5 days by supplying 1-10 l/min of nitrogen gas or compressed air; concentrating the tetanus toxin by filtering it with ultrafiltration and diafiltration; precipitating the concentrated tetanus toxin with ammonium sulfate solution; purifying the crude purified tetanus toxin with column chromatography using 5-8 mS/cm of phosphate buffer; and removing toxicity of tetanus toxin with formalin.

Description

Method for the preparation of the adsorption tetanus toxoid vaccine {Method for the preparation of tetanus toxoid vaccine}

Figure 1 shows the results of the SDS-PAGE to confirm the tetanus toxin obtained through the culture and purification process.

[Explanation of symbols in the drawings]

1: molecular weight marker 2: tetanus culture

3: ultrafiltration and diafiltration treatment solution 4: primary ammonium sulfate treatment solution

5: Secondary Ammonium Sulfate Treatment Solution 6: Purified Tetanus Toxin (DEAE-Sepharose)

The present invention relates to a method for preparing an adsorption tetanus toxoid vaccine, and more particularly, to optimize the culture conditions of fermentation tanks of tetanus strain ( Clostridium tetani ), and to produce a mass produced tetanus toxin by ammonium sulfate treatment and ion exchange chromatography. The present invention relates to a method for preparing an adsorption tetanus toxoid vaccine which can effectively prevent tetanus diseases that may occur in infants and adults by improving and purifying high purity tetanus toxin.

Tetanus disease is an anaerobic Gram-positive bacillus and Clostridium tetani that forms resistant spores. Toxins produced when spores of these pathogens attach to wounds in humans or livestock and germinate multiply from the wound to the body. Absorbed and invaded the peripheral nervous system and spinal cord cells, causing stiffness in the muscles of the whole body with a high mortality rate of 30-50%.

These tetanus bacteria are widely distributed in the environment, and their spores are commonly found in soil, animal stools and the like, and therefore everyone is at risk of tetanus infections from traumatic types such as cuts and wounds.

Tetanus toxoid vaccines have been used alone or in combination with pertussis vaccines and diphtheria vaccines for decades to prevent the disease. Since the use of the tetanus toxoid vaccine, tetanus infectious disease has been shown to decrease significantly. Tetanus toxoid is immunized by adsorbing on aluminum salt to increase antigenicity. This toxoid induces antitoxin production, which neutralizes toxins, and when active immunity is given to the mother, antitoxins are delivered through the placenta to prevent tetanus in newborns.

In Korea, tetanus was designated as a type 2 epidemic in 1976 and started receiving reports, but its reporting rate is very low. Neonatal tetanus rarely occurred as the inoculation rate of diphtheria, tetanus and pertussis vaccines (DTaP) in the 1980s exceeded 90%. Since the 1990s, fewer than 10 cases have been reported. However, 16 cases in 2000 and 8 cases in 2001 are required to maintain a protective defensive immunity against tetanus even after pediatric age, and adult tetanus and diphtheria vaccine (Td) is recommended.

These tetanus antitoxin levels are generally thought to be the minimum level of defense of 0.01 IU / ml in serum by neutralization reaction tests. Like other vaccines, the disease sometimes occurs after vaccination, but the tetanus toxoid vaccine is more than 95% It is a vaccine that has a prophylactic effect.

Tetanus spores were first discovered in the soil by Nikolaier in 1884, and were specifically identified in 1889 by Kitasato's successful cultivation of the bacteria.

The toxin produced by the tetanus is a powerful neurotoxin and is composed of A, B, and C fragments as a single-chain protein having a size of 150 kDa. Among the fragments, the B fragment is a basic skeleton necessary for showing toxicity, and the A-B fragment also exhibits relatively strong toxicity, and the C fragment is a receptor for toxin binding to nerve tissue.

The strain for tetanus toxin production has been using Clostridium tetani Harvard strain or its strain since 1945. Since tetanus is an anaerobic bacterium for the culture of tetanus, liver-bouillon medium has been used in the past. Until recently, it was designed by Muller (J. Immunol., 50, 377-384, 1945). Medium to which enzyme hydrolysis casein, bee heart infusion, glucose and other salts are added is widely used.

Stone (Appl. Microbiol., Vol. 1, pp. 166-168, 1953) developed toxins by developing a strain (Massachusetts strain) that grows even in medium without bee heart infusion, , Vol. 10, pp. 146-152, 1962) used this strain to produce high concentrations of toxins of 70-90 Lf / ml in medium without beef heart infusion.

The method of culturing tetanus bacteria has traditionally been applied to the political culture method using a small volume of culture bottles, J. Appl. Bacteriol., Vol. 28, pp. 52-55, 1965 and Nielsen (Appl) in the 1960s. Microbiol., Vol. 15, pp. 453-454, 1967) produced tetanus toxin using fermenters, and Zacharias (Appl. Microbiol., Vol. 16, pp. 69-72, 1968) produced toxins in a continuous culture. Produced. That is, tetanus strains can be cultured in a small volume of culture bottles as well as mass production using a large-scale fermenter.

Since tetanus toxin is a powerful neurotoxin, tetanus toxin is generally detoxified by the addition of formalin, but toxin is purified, but both toxin and toxoid purification are possible. There are largely a purification method by a chemical aggregation method, a column chromatography method, and a combination of two methods.

First, in 1948, filler (J. Exp. Med., Vol. 88, pp. 205 ~ 221, 1948) isolated tetanus toxin crystallized by precipitation method using cold methanol. Biochim. Biophys. Acta, Vol. 21, pages 433-438, 1956) purified toxins using ammonium sulfate precipitation.

Purification by column chromatography was carried out by terpine (Ann. Inst. Pasteur (Paris), vol. 97, pp. 718, 1959) using calcium phosphate chromatography, and Aust. J. Sci., 23. Vol., Pp. 131-132, 1960) purified toxins precipitated with ammonium sulfate and potassium phosphate by DEAE-cellulose chromatography. In addition, Latam (J. Immunol., Vol. 95, pages 487-493, 1965) purified toxoid through Sephadex G-100 gel filtration, and Murphy (J. Bacteriol., Vol. 94, 580). 585, 1967) purified toxoids using ion exchange cellulose chromatography. And Huggis (J. Appl. Bacteriol., Vol. 37, p. 603, 1974) purified the toxoid by affinity chromatography.

The main antigenic component of tetanus vaccine is detoxified tetanus toxin. This toxin, produced by tetanus, must be able to be mass-produced in high purity through easy culture and purification methods to ensure immunity, safety and commercial viability as a vaccine.

Therefore, it is necessary to study the culture conditions using fermenters suitable for mass production and to develop a purification method that requires low cost.

Therefore, the inventors of the present invention carried out the present study to provide a method for producing a large amount of high-safety tetanus toxoid vaccine in an industrially useful method, when the absolute anaerobic bacteria cultured in consideration of the physiological characteristics of tetanus bacteria Incubate while injecting nitrogen at a constant rate, and incubating the nitrogen or compressed air at a constant rate after incubation is sufficient to induce self-fusion of tetanus bacteria to obtain a large amount of toxins. By using column chromatography packed with Sepharose support material equilibrated with a range of phosphate buffers, high-purity tetanus toxin can be obtained without a separate toxin elution process, and detoxification produces a tetanus toxoid vaccine with excellent immunity and stability. I know I can And it completed the invention.

Therefore, the present invention can be isolated and purified in the optimum culture conditions and mass production of tetanus toxin, which can produce a large amount of tetanus strains to secure a large amount of high purity tetanus toxin and at the same time can effectively prevent tetanus. An object of the present invention is to provide a method for preparing an improved adsorption tetanus toxoid vaccine.

In the present invention, a method for producing an adsorption tetanus toxoid vaccine using a fermenter, after dispensing a tetanus strain ( Clostridium tetani ) into the medium and incubated for 24 to 72 hours while supplying nitrogen to the medium surface at 1 to 10 l / min, Re-incubating for 3 to 5 days while supplying nitrogen or compressed air at 1 to 10 l / min to the surface of the medium to obtain tetanus toxin; Concentrating the tetanus toxin by ultrafiltration and then diafiltration; Precipitating the concentrated tetanus toxin in an ammonium sulfate solution to adjust it; Purifying the adjusted tetanus toxin using column chromatography equilibrated with phosphate buffer with a conductivity of 5 to 8 mS / cm; And characterized in that the method for producing the adsorption tetanus toxoid vaccine comprising the detoxification of the purified tetanus toxin.

Hereinafter, the present invention will be described in detail.

The present invention is to optimize the fermentation tank culture conditions of tetanus strain ( Clostridium tetani ) to concentrate the mass produced tetanus toxin by ultrafiltration (UF) and diafiltration method, ammonium sulfate treatment and ion exchange The present invention relates to a method for preparing an adsorption tetanus toxoid vaccine which can effectively prevent tetanus diseases that may occur in infants and adults by improving chromatographic purification and recovering high purity tetanus toxin.

Hereinafter will be described in detail step by step the preparation method of the adsorption tetanus toxoid vaccine of the present invention.

As a method of culturing the strain to produce tetanus toxin by culturing tetanus bacteria, both culturing and culture using a fermentation tank are possible, but a method using a fermentation tank may be more suitable for mass production and obtaining a homogenous culture solution. . Therefore, the present invention provides a method for preparing an adsorption tetanus toxoid vaccine using a fermenter.

First, after dispensing the tetanus strain ( Clostridium tetani ) to the medium and incubated for 24 to 72 hours while supplying nitrogen to the surface of the medium at 1 to 10 l / min, and then to the medium surface 1 to 10 of nitrogen or compressed air It is a step of obtaining tetanus toxin by culturing again for 3-5 days while supplying in l / min.

The medium used to cultivate tetanus bacteria in fermenter includes the addition of enzyme hydrolysis casein, beef heart infusion, glucose and other salts developed by Muller, and the modified Mueller medium without beat heart infusion devised by Latham. (Latham, 1961) can be used in all cases, but when beep heart infusion is added, it may be said that Latam medium is more suitable than Muller's medium because it may reduce the purity and contain sensitive antigens. As a result of culturing tetanus bacteria by fermenter for each medium, it was found that the growth of the cells was more active than when yeast extract (yeast extract) was properly added than in the muller and latam medium.

Therefore, in the present invention, it is more preferable to use a latam modified medium in which yeast extract is further added in the range of 0.1 to 1 g / L, more preferably 0.1 g / L to the semisynthetic medium of latam.

That is, a medium prepared by mixing NZ-Amine or NZ-Case and yeast extract (Difco, USA) with a hydrolase casein solution added with a lactam semisynthetic medium is used as the Latam semisynthetic medium. 25 mg Thiamine, 25 mg Riboflavin, 25 mg Pyridoxin, 100 mg Ca Panantothenate, 0.25 mg Biothin, 0.005 mg per 1000 ml Cyanocobalamin], Muller solution IV [component: 125 mg of uracil per 1000 ml], 25 mg nicotinic acid], Muller solution V [component: 12.5 g of L per 1000 ml -L-cystine], Mueller solution VI [component: 3.2 g of FeCl ₃ .6H ₂ O per 1000 ml] and the like.

Generally, since tetanus is an absolute anaerobic bacterium, cells are grown while injecting nitrogen during incubation, and then cultured while injecting nitrogen and compressed air during the toxin secretion by autolysis.

That is, a method of culturing by supplying compressed air to the medium surface of the fermentation tank has been used for a long time (WHO Manual, BLG / UN / 77.1), and the compressed air supplied at this time is used for toxic H ₂ S gas produced during the culturing process. By removing it, it prevents cell growth inhibition. In 1967, Nielsen found that more toxins were produced by surface supply of nitrogen than in compressed air in tetanus fermenters.In 1996, Lucca used a nitrogen gas bubbling culture (Nitrogen-) to supply nitrogen using a sparger. The gas bubbling culture also produced tetanus toxin. The method of supplying nitrogen to the fermenter may be supplied to the medium using a sparger or directly to the medium surface.

However, in the case of cultured by supplying compressed air from the beginning of culture, the amount of dissolved oxygen in the medium increased and the cells did not grow at all, and after the maximum growth of the cells, the sparger was used during autolysis for toxin secretion. In case of supplying nitrogen to the culture medium, the aggregation of the cells occurred and the autolysis did not occur completely, resulting in less toxin production.

In the present invention, it was possible to promote the growth of the cells by culturing the tetanus fungi while supplying nitrogen to the medium surface at a constant rate, and incubated while injecting nitrogen or compressed air to the medium surface at a constant rate after sufficient growth of the cells. In the case of promoting the autolysis of the strain was able to obtain a large amount of tetanus toxin.

In other words, inoculated with 1 to 10 (v / v)% of the spawn culture medium incubated for 24 hours in the Latam transformation medium prepared by adding the yeast extract, most preferably 4 (v / v)% after inoculating the fermenter (Korean fermenter) Incubate while maintaining the speed of the stirrer at about 60 rpm.

At this time, nitrogen is incubated for 24 to 72 hours, preferably 48 to 60 hours, feeding the surface of the medium at a rate of 1 to 10 L / min, preferably 2 to 6 L / min. If the infusion rate of the nitrogen is out of the above range and the incubation time is less than 24 hours, the growth of the cells is not sufficient, and if the time exceeds 72 hours, the cells are killed. After culturing the cells, nitrogen or compressed air is further incubated for 3 to 5 days while injecting at a rate of 1 to 10 L / min, preferably 3 to 5 L / min. At this time, when the infusion rate of the nitrogen or compressed air is out of the range and the incubation time is less than 3 days, the yield of tetanus toxin is low because the cells do not sufficiently melt, and when the exceeding time exceeds 5 days, the number of toxins is increased. There is no profit. After the incubation is completed, the filter is filtered with K 300 (Seitz) filter paper to remove tetanus cells from the culture.

According to the culturing method of the present invention described above, the recovery amount of toxin in the culture solution is 70 Lf / ml or more, and therefore, it is increased by 70% or more than the recovery amount of 40 Lf / ml obtained by the conventional static culture. That is, according to the present invention, a large amount of tetanus toxin can be obtained.

Secondly, the tetanus toxin is concentrated by diafiltration after ultrafiltration.

In order to effectively purify the tetanus toxin produced by the culture, diafiltration with a phosphate buffer solution of 5 to 10 volume ratio of the concentrate volume obtained by first concentrating with 30 kDa ultra filtration (Sartorius) module. Do this.

Thirdly, the concentrated tetanus toxin is precipitated with ammonium sulfate solution to adjust it.

In another aspect, the present invention is characterized in that the purification process is performed in two stages: a salting-out method using a ammonium sulfate and a column chromatography process to purify tetanus toxin with high purity.

First, in order to remove the impurity protein contained in the tetanus toxin concentrate after the diafiltration process, ammonium sulfate is first added to 10 to 15 (w / v)% to sufficiently dissolve to precipitate the impurity proteins except for the tetanus toxin. The formed precipitate is removed by centrifugation (Sorvall) at 9,000 rpm to recover only the supernatant. Ammonium sulfate is added to the recovered supernatant so that the final concentration is 20-26 (w / v)% including ammonium sulfate used in the first treatment, and then dissolved to form a precipitate. Since the precipitate contains tetanus toxin, the supernatant is discarded by centrifugation at 9,000 rpm and only the precipitate is recovered. The recovered precipitate is redissolved again to 1,000 to 1,500 Lf / ml using phosphate buffer and placed in sterile dialysis membrane (MW 12,000) for dialysis until the remaining ammonium sulfate is removed with phosphate buffer.

Fourth, the adjusted tetanus toxin is purified using column chromatography equilibrated with phosphate buffer having a conductivity of 5 to 8 mS / cm.

In the present invention, in order to purify the adjusted tetanus toxin with high purity, the support material is washed and activated, and the conductivity is 5-8 mS / cm, preferably 6-7 mS / cm, 1 / 15M phosphate buffer (pH 7.0 Purification process is performed using a column chromatograph equilibrated with), wherein the support material filled in the column is preferably DEAE-Sepharose FF (Sepharose FF, Pharmacia).

Tetanus toxin in the buffer conditions having a conductivity in the above range is poor adsorption to the DEAE-Sepharose FF support. Therefore, the impure protein containing the pigment component is adsorbed to the support material, but since tetanus toxin can pass through without adsorption, it is a simpler purification method that does not require an additional tetanus toxin elution step, and it is easy to apply it industrially.

Fifth, detoxify the purified tetanus toxin.

Purified tetanus toxin has inherent neurotoxicity and must be detoxified to remove it. In general, formalin, phenol, glutaraldehyde, and the like are used as a method of detoxifying toxin. In the present invention, formalin is used.

That is, after dissolving L-lysine to 0.1M in tetanus toxin purified liquid, formalin is added so as to be 0.7-0.9 (w / v)%, and detoxified at 36-37 ° C for 30 days. After completion of detoxification, dialysis is carried out with distilled water to remove residual formalin. A tetanus toxoid stock solution was prepared by aseptic filtration with a membrane filter (0.2 μm, Gelman) by adding chimerosal 0.01 (w / v)% and NaCl 0.85 (w / v)% as a preservative to the formalin-free stock solution, followed by tetanus toxoid vaccine. Manufacture.

The adsorbed tetanus toxoid vaccine of the present invention thus prepared is prepared as a final stock solution so that the protein content does not exceed 80 µg, and 0.75 ml per head is injected once per 4 or more guinea pigs weighing 300 to 400 g. When blood is collected from each animal after 4-6 weeks, the anti-toxin level in blood should be at least 2 IU / ml.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited by the following Examples.

1. Production of Tetanus Toxin

Preparation Example: Preparation of Latam Modification Medium

1) Preparation of Enzymatic Hydrolysis Casein Solution

NZ-Amine [Sheffield, USA] or NZ-Case [Sheffield, USA) is used and a 20% enzymatic hydrolysis casein solution with 0.1 g / l yeast extract (Difco, USA) is used.

That is, 216.7 g of NZ-Amine (or NZ-Case) and 0.668 g of yeast extract are put in 1000 ml of distilled water, and then boiled for 15 minutes, and 6.7 g of K ₂ HPO ₄ and 3.0 g of CaCl ₂ · 2H ₂ are dissolved therein. O was dissolved, and after cooling to 20 ° C., it was adjusted to pH 9.0 using 10N NaOH and filtered through a Toyo No. 2 filter paper.

The filtrate was stored in a refrigerator for one day and then filtered again with Toyo No. 2 filter paper, and modified to pH 7.7 using 5N HCl to prepare a culture medium.

2) Preparation of Latam Modification Medium

8.8 g of glucose, 2.5 g of salt, and 0.125 g of MgSO ₄ · 7H ₂ O were added and dissolved in 500 ml of distilled water. Muller Solution III [Component: 25 mg Thiamine, 25 mg Riboflavin, 25 mg Pyridoxin, 100 mg Ca Panantothenate, 0.25 mg per 1000 ml Biothin, 10 ml of 0.005 mg of cyanocoballamin], Muller solution IV [component: 125 mg of uracil per 1000 ml of component, 25 mg of nicotinic acid], 10 ml of muller Solution V [component: 10 ml of 12.5 g L-cystine per 1000 ml], Muller solution VI [component: 3.2 g of FeCl ₃ .6H ₂ O per 1000 ml]] and 20 prepared above 150 ml of% enzymatic hydrolysis casein solution was added and mixed, the final volume of the medium was adjusted to 1000 ml, and then adjusted to pH 7.4 using 5N NaOH.

Example 1 Culture of Tetanus Strains and Preparation of Tetanus Toxin

1) Master Seed & Walking Seed Manufacturer

The strain used in the present invention is Clostridium tetani Harvard, a known strain for the production of tetanus vaccine. First, a master seed is prepared by using a strain distributed by the Korea National Institute of Health, and then again on the master seed. Working seeds were made and stored in a deep freezer and used for testing.

2) Culture of tetanus strains: spawn passage, spawn culture and main culture

The spawn passage was performed twice. First, 1 vial of the working seed was added to 30 ml of fluid thioglycollate [Difco, USA] sterilized at 121 ° C. for 20 minutes, and the culture solution obtained by incubating for 24 hours at 35 ° C. It was inoculated again in fluid thioglycolate medium and incubated at 35 ° C for 24 hours.

For the spawn culture, 3000 ml of the Latam transformation medium prepared in Preparation Example was placed in a 5 ° C. fermenter (KFC, Korea) and sterilized at 121 ° C. for 30 minutes, followed by quenching, and the spawn subculture was added to 1 (v / v). Inoculated to%. It was incubated for 24 hours with surface feeding of 0.5 L / min while stirring at 35 ° C. at 60 rpm.

The culture was put into a 200 L fermenter (KFC, Korea) 80 L of the Latham modified medium prepared in Preparation Example, sterilized for 30 minutes at 121 ℃ and then quenched to inoculate 4 (v / v)% of the spawn culture solution therein It was. The mixture was incubated for 48 hours while supplying nitrogen at 2 L / min while stirring at 35 ° C. at 60 rpm. After 48 hours of incubation, the mixture was further incubated for 3 days while supplying compressed air at 3 L / min.

3) Tetanus toxin production and culture solution recovery

The cell concentration according to cell growth was measured by optical density at 650 nm using a spectrophotometer (Kontron), and the production of tetanus toxin was determined using Ramon's flocculation method. The measured value is expressed in Lf / ml.

As a result of incubation with the present culture conditions in a 200 L fermenter using the Latam-modified medium prepared in Preparation Example, the maximum cell growth was from 1.8 to 2.3 at 20 to 24 hours after the start of culture. After incubation for a total of 5 to 7 days, all the tetanus bacteria self-thawed, and the maximum toxin production was 70 to 90 Lf / ml.

Depth filtration was performed using K300 (Seitz) filter and KS80 (Seitz) filter paper to separate the cells and culture supernatant from the culture medium. The filtrate was membrane filter (0.2 μm, Gelman). Aseptic filtration using.

Example 2 Concentration of Tetanus Toxin

The culture solution of the tetanus strain recovered by the fermenter culture and filtration was concentrated in a UF concentrator (Sartorius) equipped with a 30 kDa UF module. The inlet pressure of the UF concentrator was concentrated at 1 kg / cm 2 and the discharge pressure was 0.5 kg / cm 2 until the toxin concentration was about 500 Lf / ml.

The UF concentrate of tetanus toxin was subjected to diafiltration using the same UF module. After concentration, impurities such as residual medium components and pigment components were removed using a 1 / 15M phosphate buffer solution corresponding to 10 times the concentration. After diafiltration, 1 / 15M phosphate buffer was added to a final toxin content of 500 Lf / ml, and NaHCO ₃ was added to 0.5 (w / v)% to dissolve.

Example 3: Purification of Tetanus Toxin

1) Primary Ammonium Sulfate Treatment: Elimination of Impurity Proteins except Tetanus Toxin

Samples were taken in the concentrated tetanus toxin solution, and samples were prepared at 1% intervals so that the concentration of ammonium sulfate was 10-15 (w / v)%, respectively, and the samples were dissolved. At this time, the precipitate formed was removed by centrifugation at 3,000 rpm, and only the supernatant of each sample was recovered to determine the concentration of ammonium sulfate in the highest purity sample by measuring protein content and toxin content. Was added to the tetanus present solution concentrated by the diafiltration and completely dissolved and reacted overnight in a refrigerating chamber. Primary ammonium sulfate reacted overnight was centrifuged at 9000 rpm for 30 minutes to discard the precipitate and recover only the supernatant.

2) Secondary Ammonium Sulfate Treatment: Induction of Precipitation for Tetanus Toxin Recovery

Take a sample from the supernatant obtained by the first ammonium sulphate treatment, and add ammonium sulphate at 1% intervals so that the final concentration is 20 to 26 (w / v)%, including the concentration of the first ammonium sulphate. It was added and dissolved.

When protein precipitate is formed, the supernatant is removed by centrifugation at 3,000 rpm, and only the precipitate is recovered, and redissolved by adding the same amount of physiological saline, and the concentration of ammonium sulfate in the highest purity sample is measured. It was confirmed. Ammonium sulfate corresponding to this concentration was weighed (including the primary ammonium sulfate main treatment concentration), added to the primary ammonium sulfate main treatment solution, completely dissolved, and reacted overnight in the refrigerating chamber. The reacted secondary ammonium sulfate treatment solution was centrifuged at 9,000 rpm for 30 minutes to discard the supernatant and recover only the precipitate.

The recovered precipitate was dissolved in 1 / 15M phosphate buffer (pH 7.0) to 1000-1500 Lf / mL. The dissolved tetanus toxin containing solution was placed in a dialysis membrane (MW 12,000) and dialyzed with 1 / 15M phosphate buffer (pH 7.0) with a conductivity of 6.8 mS / cm until the remaining ammonium sulfate was removed. The tetanus toxin solution thus prepared was aseptically filtered using a membrane filter (0.2 μm, Gelman) and used as a column chromatography treatment as a tetanus toxin adjusting solution.

3) column chromatography treatment:

The column chromatography support used in the tetanus toxin purification process is DEAE-Sepharos FF (Pharmacia), and 300 ml of this support is filled in a 50/30 XK column (Pharmacia), followed by column chromatography system. (GradiFrac, Pharmacia).

The DEAE-Sepharose FF column mounted in the chromatography system was washed with support by passing a 0.5 N NaOH solution at a flow rate of 8 ml / min, which was 3-5 times the capacity of the filled support. In order to activate the column again, 1 / 15M phosphate buffer (pH 7.0, 2M NaCl, 125 mS / cm), three to five times the volume of the support, was passed through the column at a flow rate of 8 mL / min to activate the support. 8 ml / min of 1 / 15M phosphate buffer (pH 7.0, 6.8 mS / cm), 3 to 5 times the capacity of the support, to remove the high ionic strength phosphate buffer contained in the activated support and equilibrate the column. The column was equilibrated by washing at a flow rate of.

The following tetanus toxin purification process was advanced in the refrigerator of 2-8 degreeC.

The tetanus toxin modifier solution was passed through the equilibrated DEAE-Sepharose FF column at a flow rate of 8 ml / min to recover all the toxin solution passing through the support material. That is, in the column chromatography conditions equilibrated with 1 / 15M phosphate buffer solution having a conductivity of 6.8 mS / cm, the DEAE-Sepharose FF adsorbs the pigment component and the impure protein contained in the tetanus toxin control agent, and the tetanus toxin is adsorbed. Since it does not require additional elution step, it can be said to be an advantageous method for the industrial purification of tetanus toxin.

The final purified tetanus toxin could be recovered at a concentration of 700-1,200 Lf / ml and at the same time, high purity of 2000 Lf / mgPN or higher could be obtained.

Example 4 Preparation of Tetanus Toxoid Stock Solution by Tetanus Toxin Detoxification

L-lysine was added 0.1 M to the purified tetanus toxin solution to completely dissolve it, and aseptic filtration was performed using a membrane filter (0.2 μm, Gelman). To this formalin was added to 0.8 (w / v)%. Tetanus toxin solution to which formalin was added was stored in a constant temperature room of 36 to 37 ° C. and detoxified for 30 days.

Dialysis was performed using distilled water to remove formalin and L-lysine remaining in the tetanus toxoid solution obtained by the detoxification process. After dialysis, 0.85 (w / v)% NaCl and 0.01 (w / v)% of chimerosal were added to the tetanus toxoid solution, followed by aseptic filtration using a membrane filter (0.2 μm, Gelman). Cold storage was used to test for tetanus toxoid vaccine.

SDS-PAGE was performed to confirm the tetanus toxoid obtained by detoxifying the toxin with formalin and tetanus toxin purified by the above method, and the results are shown in the accompanying drawings in FIG. 1, and as shown in FIG. Tetanus toxin and toxoid were identified at the kDa position.

Experimental Example 1 Test of Tetanus Toxoid Stock Solution

The tetanus toxoid stock solution prepared above was tested according to the biological standards and test methods published by the Korea Food and Drug Administration. The main items consisted of purity test, sterility test, detoxification test and toxic recovery denial test. The results are shown in the following Table 1.

Test Items  standard  Test result Purity Test  Over 1,500 Lf of toxoid in 1 mg of protein 2,000-2,500 Lf Sterility Test  Bacteria, fungi should not be identified fitness Detoxification Test  No laboratory animals should be abnormal fitness Toxic recovery negative test  No specific reactions and other abnormalities should occur by injection into rabbit skin after 6 weeks at 4 ° C and 37 ° C. fitness

According to the test results of Table 1, the tetanus toxoid stock solution prepared according to the present invention showed that the tetanus toxoid stock solution was commercially prepared as a tetanus vaccine and can be used as a vaccine for preventing tetanus infection.

Example 5: Preparation of adsorption tetanus toxoid stock solution

To carry out the test according to the biological standards and test methods, the tetanus toxoid stock solution was diluted with 0.01 M phosphate buffer (pH 6.8, 0.7% NaCl) to a final 50 Lf / mL, and aluminum hydroxide was added to the final aluminum content. The tetanus toxoid stock solution was adsorbed with aluminum to make 0.5 / ml, thereby preparing the adsorption tetanus toxoid stock solution. The tetanus toxoid stock solution was diluted 5 times to carry out the following experiment.

Experimental Example 2: Test of adsorption tetanus toxoid stock solution

The adsorption tetanus toxoid was tested in accordance with the Biological Standards and Test Methods published by the Korea Food and Drug Administration. The main items consist of the detoxification test, the abnormal toxicity test, and the potency test. The results are shown in Table 2 below.

Test Items  standard  Test result Sterility Test  Bacteria, fungi should not be identified fitness Detoxification Test (GP)  No laboratory animals should be abnormal fitness Adverse Toxicity Negative Test Guinea pig  Laboratory animals should not have abnormalities fitness mouse Station test  2 units (IU) or more in 1 ml based on the final stock solution 2 units or more

According to the test results of Table 2, the adsorption tetanus toxoid stock solution prepared by the present invention showed satisfactory efficacy as a vaccine for preventing tetanus infection.

As described above, the tetanus toxoid vaccine prepared by the present invention showed not only non-toxicity but also effective prevention of tetanus disease.

In addition, tetanus toxoid vaccine prepared by the production method of the present invention can be used as a DTaP vaccine by mixing with purified pertussis vaccine and diphtheria toxoid vaccine, as well as applicable to adult diphtheria and tetanus prevention vaccine.

Claims (5)

In the manufacturing method of the adsorption tetanus toxoid vaccine using a fermentation tank, After dispensing the tetanus strain ( Clostridium tetani ) into the medium and incubated for 24 to 72 hours while supplying nitrogen to the surface of the medium at 1 to 10 l / min, and supplying nitrogen or compressed air to the medium surface at 1 to 10 l / min. While incubating again for 3 to 5 days to obtain tetanus toxin; Concentrating the tetanus toxin by diafiltration after ultrafiltration; Precipitating the concentrated tetanus toxin in an ammonium sulfate solution to adjust it; Purifying the adjusted tetanus toxin using column chromatography equilibrated with phosphate buffer with a conductivity of 5 to 8 mS / cm; And Detoxifying the purified tetanus toxin Method for producing an adsorption tetanus toxoid vaccine comprising a. The method of claim 1, wherein the medium is a method for producing an adsorption tetanus toxoid vaccine, characterized in that 0.1 to 1.0 g / l of yeast extract is added to a latam semisynthetic medium. The method of claim 1, wherein the ultrafiltration is performed using a 30 kDa ultrafiltration membrane (UF) module. The method of claim 1, wherein the column chromatography is filled with DEAE-Sepharose FF support material. The method of claim 1, wherein the detoxification is performed using formalin.

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