KR20060121507A - Method for the preparation of diphtheria toxoid vaccine - Google Patents

Abstract

A method for the preparation of diphtheria toxoid vaccine is provided to improve preparation yield of diphtheria toxin by optimizing the cultivation condition, and reduce purification costs and improve purity of diphtheria toxin by using ammonium sulfate treatment and ion exchange chromatography. The method for the preparation of diphtheria toxoid vaccine comprises the steps of: culturing Corynebacterium diphtheriae in medium for 24-72 hours by supplying 0.1-1 vvm of a mixture containing air and oxygen into the fermenter to maintain dissolved oxygen amount of the medium at 10-50%, adding 1-30g/l of maltose into the medium after 10-30 hours of the cultivation, and further culturing it; centrifuging the cultured medium, separating the supernatant and concentrating the supernatant with ultrafiltration and diafiltration; precipitating the concentrated diphtheria toxin with ammonium sulfate and dialyzing the precipitates with 5-8 mS/cm of phosphate buffer; purifying the crude purified diphtheria toxin with column chromatography using 5-8 mS/cm of phosphate buffer; and removing toxicity of diphtheria toxin with formalin.

Description

Method for the preparation of diphtheria toxoid vaccine

Figure 1 shows the results of the SDS-PAGE to confirm the purified diphtheria toxin and diphtheria toxoid after detoxification.

[Explanation of symbols in the drawings]

1: molecular weight marker 2: purified diphtheria toxin

3: detoxified diphtheria toxoid

The present invention relates to a method for preparing a diphtheria toxoid vaccine, and more specifically, to optimize the culture conditions of the diphtheria strain ( Corynebacterium diphtheriae ) to produce diphtheria toxin (diphtheria toxin) and then using ammonium sulfate treatment and ion exchange chromatography By purifying and detoxification, a large amount of high-purity diphtheria toxin can be easily produced and purified, thereby providing a method for producing an improved diphtheria toxoid vaccine which can easily obtain a high-purity diphtheria toxoid vaccine with excellent stability and immunity in large quantities. It is about.

Diphtheria disease is a local disease of the respiratory mucosa and skin caused by infection with the aerobic Gram-positive bacillus, Corynebacterium diphtheriae , and is most common in children aged 2 to 4 years in winter.

To prevent this disease, diphtheria toxoid vaccines have been used for decades, either alone or in combination with pertussis vaccines and tetanus vaccines. Since the late 1950s, the introduction of diphtheria vaccines has significantly reduced the incidence of infections, and the use of antibiotics has reduced the mortality rate.

Since 1987, there have been no reports of domestic outbreaks, but in the 1990s, diphtheria re-emerged in countries that had become independent from the former Soviet Union, resulting in more than 4,000 deaths (Emerg. Infect. Dis., Vol. 4, pp. 539-550 , 1998). Therefore, there is a risk of foreign influx in Korea, and in recent years, adults are also recommended to be inoculated with diphtheria vaccine because of low titer of antibody in the blood due to less chance of additional immunity due to natural infection.

The strain used to obtain diphtheria toxin used as a vaccine is Corynebacterium diphtheriae Park-Williams No. 8 or a mutant strain thereof, and the cultivation method of this strain can be largely divided into surface static culture and submerged culture. The surface cultivation method was developed by Muller and Miller in 1941. The liquid synthetic medium containing acid hydrolysis casein was added to a Roux bottle to grow the cells on the surface of the liquid medium, about 70 to 90 Lf / ml. It has been used until recently to produce toxins (Mueller and Miller, J. Immun., 40, 21-32, 1941). However, the Muller and Miller method described above has the advantage of low production cost but is not suitable for mass production of high purity toxin.

In order to overcome the above problems, Ringard and Fenton (Br. J. Exp. Path. No. 28, pages 354-364, 1947) in 1947 developed a shake culture method using popped medium containing enzyme hydrolysis casein. In addition, Edward (Journal of general microbiology, Vol. 22, pp. 698-704, 1960) used a papain digest of beef broth to surface air in a 20 liter stirred tank to provide 200 in 48 hours. Techniques for producing toxins up to Lf / ml have been developed. Since then, the introduction of a fermentor with a stirrer has led to the development of a method for mass production of high purity diphtheria toxin.

Toxin obtained by culturing the diphtheria cells as described above is to improve the purity through the purification process, there are two ways to purify this toxin. One is a toxoid purification method that removes the cells from the culture medium in which the diphtheria cells have grown and converts them to toxoid by detoxifying the supernatant containing the toxin, and the other is toxin purification of the toxin contained in the supernatant first. Toxin purification after detoxification.

The toxoid purification method has the advantage that it is not converted to toxin again after vaccine preparation, but there is a problem in that the content of impurities is increased because several impure proteins including the media component included in the culture medium are detoxified during the detoxification. In addition, once denatured with toxoid, the purification method using ion exchange chromatography cannot be applied. Therefore, the toxoid purification method uses only a salting-out method by treating a high concentration of ammonium sulfate, and for this reason, there is a disadvantage in that the final purity is low in the purification through the toxoid purification method.

On the other hand, the toxin purification method toxins are purified through ion exchange chromatography to high purity and then detoxified using formalin, which has the advantage of being able to be purified with higher purity than the toxoid purification method. Therefore, the toxin purification method in addition to the culture method using a fermenter is a trend that is currently used by each vaccine manufacturer as a method for producing a diphtheria vaccine.

In other words, in order to secure immunity, safety, and commerciality as the vaccine, the toxin released from the bacteria as the diphtheria grows can be produced in high purity in high purity through the easy culture and purification methods. Should be

Therefore, it is required to secure the production yield by studying the optimum culture conditions using fermenters suitable for mass production, and also to develop a technology capable of purifying toxins obtained in large quantities through simple and low cost methods. This is necessary.

Therefore, the inventors of the present invention have been researched to provide a method for producing industrially useful diphtheria toxoid vaccine with high safety. As a result, the growth of the diphtheria cells by adding a carbon source during the cultivation while maintaining the dissolved oxygen amount of the fermenter is above a certain level. And toxin production can be maximized, and the resulting diphtheria toxin is concentrated and treated with ammonium sulfate and dialyzed with a buffer with controlled conductivity, followed by ion exchange column chromatography using a support material equilibrated with a buffer with controlled conductivity. By carrying out, it has been found that diphtheria toxin can be purified without adsorption on a support packed in a column, and thus it is possible to mass produce a high-purity diphtheria toxin in a simple manner, thereby completing the present invention.

Therefore, the present invention can easily isolate and purify the diphtheria toxin obtained by culturing the diphtheria strain under optimal conditions, thereby obtaining a high-purity diphtheria toxin and using it to prepare a diphtheria toxoid vaccine which can effectively prevent diphtheria infection. The purpose is to provide a method.

In the present invention, a diphtheria toxoid vaccine is prepared by dispensing a diphtheria strain ( Corynebacterium diphtheriae ) into a medium and supplying a mixture of air and oxygen to the fermenter at 0.1-1 vvm to obtain a dissolved oxygen content of 10-50% air saturation. Culturing for 24 to 72 hours while maintaining, culturing while adding maltose at a concentration of 1 to 30 g / l at 10 to 30 hours after the start of the culture to obtain a diphtheria toxin culture solution; Concentrating the supernatant obtained by centrifuging the diphtheria toxin culture solution, followed by ultrafiltration followed by diafiltration. Treating the concentrated diphtheria toxin with ammonium sulfate, followed by dialysis with a phosphate buffer having a conductivity of 5 to 8 mS / cm to adjust the modifier; Purifying the purified diphtheria toxin using column chromatography packed with a support material equilibrated with phosphate buffer having a conductivity of 5-8 mS / cm; And a method for preparing a diphtheria toxoid vaccine comprising the detoxification of the purified diphtheria toxin.

Hereinafter, the present invention will be described in detail.

The present invention is to optimize the culture conditions of the diphtheria strain ( Coynebacterium diphtheriae ) to produce diphtheria toxin (diphtheria toxin) and then purified and detoxified using ammonium sulfate treatment and ion exchange chromatography, high purity of diphtheria toxin The present invention relates to a method for preparing an improved diphtheria toxoid vaccine which can be easily produced and purified, and thus easily obtains a large amount of high purity diphtheria toxoid vaccine having excellent stability and immunity.

Hereinafter, the method for preparing the diphtheria toxoid vaccine of the present invention will be described in detail step by step.

In the step of producing toxin by culturing diphtheria strains, the cultivation method can be both a surface politics culture using a Lux disease and a fermentation culture using a fermentation tank, but a fermentation tank is more suitable for mass production and obtaining a homogenous culture solution. have. Therefore, the present invention provides a culture method using a fermenter.

Firstly, Corynebacterium diphtheriae was dispensed into the medium, and a mixture of air and oxygen was supplied to the fermenter at 0.1 to 1 vvm, followed by incubation for 24 to 72 hours while maintaining the dissolved oxygen content of the fermenter at 10 to 50%. However, the step of obtaining a diphtheria toxin culture solution by culturing while adding maltose at a concentration of 1 to 30 g / ℓ 10 to 30 hours after the start of the culture.

First, as a medium for fermenter culture, a medium using papain digested beef juice or a semi-synthetic medium containing enzyme hydrolyzed casein may be used. However, the medium using beef juice is expensive and the latter may be caused by susceptibility to beef antigen. More appropriate.

Therefore, in the present invention, a stainer modified medium in which yeast extract is added to a semisynthetic medium (Canadian journal of microbiology, 14, pages 1155-1160, 1968) is used.

Specifically, NZ-Amine (Sheffield, USA) or NZ-Case (Sheffield, USA), which is an enzyme hydrolysis casein, is used as the nitrogen source, and the yeast extract (Difco, USA) is 1 to 7.5 g / L, preferably 4-6 g / L is added.

Inoculate 0.1 to 5 (v / v)%, preferably 1 to 3 (v / v)%, of the spawned broth pre-cultured to the fermenter containing the above-mentioned statin modified medium, preferably for 24 to 72 hours. Incubate for 36 to 60 hours.

After 20 hours of incubation of the diphtheria strains, the dissolved oxygen level drops rapidly to 10% or less. If sufficient dissolved oxygen is not maintained, cell growth stops and toxin production stops. At this time, as a method for sufficiently maintaining the dissolved oxygen amount, there is a method of increasing the air flow rate, a method of increasing the speed of the stirrer, and a method of supplying oxygen. However, the method of increasing the air flow rate can cause the generation of bubbles to suppress cell growth, and the increase in the stirrer speed has a limit of the stirrer rotation speed according to the performance of the fermentor.

Therefore, in the present invention, while maintaining the stirrer speed of the fermenter at 200 rpm during the culture of diphtheria cells, the mixed air of air and oxygen is supplied, but the aeration amount is supplied at 0.1 to 1 vvm, preferably 0.2 to 0.5 vvm. By supplying the oxygen (purity of 98% or more), the amount of dissolved oxygen in the fermenter is maintained in the range of 10 to 50%, preferably 20 to 40% air saturation.

In addition, the antifoaming agent (Antifoam A, Sigma) is automatically supplied to suppress the foam generated during the culture.

When culturing the diphtheria strain as described above, 1 to 30 g / l of maltose, which is a carbon source, is added within 10 to 30 hours after the start of the culture, and preferably 10 to 20 g / l within 24 hours after the start of the culture. The use of a modified medium and the addition of maltose, a carbon source, were more effective in cell growth and toxin production.

Secondly, the supernatant obtained by centrifuging the diphtheria toxin culture solution is concentrated by ultrafiltration and then diafiltration.

The obtained diphtheria culture solution was continuously centrifuged to separate the cells and the supernatant, the cells were removed, and finally the supernatant was ultrafiltered and subjected to diafiltration. The ultrafiltration is performed using a 30 kDa ultrafiltration module, and diafiltration is performed using physiological saline.

In the culture supernatant from which the diphtheria cells are removed, pigment components, medium components, impurity proteins, and inorganic ions that are consumed during the culture are present. Therefore, the supernatant was concentrated by ultrafiltration and diafiltration to remove impurities.

That is, the ultrafiltration was concentrated using a ultrafiltration concentrator (Sartorius) equipped with a 30 kDa ultrafiltration module (Sartorius) to concentrate the supernatant to 1/10 to 1/5 times the initial volume of the culture. Use physiological saline, which is 5 to 20 times the volume of the concentrate, and perform diafiltration in an ultrafiltration concentrator.

After dialysis filtration, physiological saline is added to the final toxin content of 200 to 800 Lf / ml, preferably diluted to 400 to 600 Lf / ml, and NaHCO ₃ is added to 0.5 (w / v)%. To dissolve.

By the above method, it was possible to obtain a concentrated diphtheria toxin, while reducing the capacity of the supernatant and at the same time removing the impurities remaining in the supernatant.

Third, the concentrated diphtheria toxin is treated with ammonium sulfate, followed by dialysis with a phosphate buffer having a conductivity of 5 to 8 mS / cm to adjust the modifier.

In the present invention, the supernatant (diphtheria toxin) concentrated by the ultrafiltration and diafiltration is adjusted by applying a salt-out method using ammonium sulfate. The salting-out method, which is generally used to purify proteins or enzymes, is a method of recovering when the salt in the solution containing the protein is increased and the solubility of the protein is reduced and separated from the solution in the form of precipitate.

The precipitate obtained by treating ammonium sulfate at low concentration in the diafiltration was finished by centrifugation, and only the supernatant was recovered, and again, high concentration of ammonium sulfate was added to precipitate the toxin. The resulting precipitate is recovered by centrifugation, redissolved in physiological saline and placed in a dialysis membrane (MW 12,000) for dialysis to remove residual ammonium sulfate with physiological saline.

That is, in order to remove impure protein in the diafiltration coarse diphtheria toxin concentrate, ammonium sulfate concentration is first added to dissolve 22 to 27 (w / v)% to dissolve other impurity proteins not containing diphtheria toxin. The formed precipitate is removed by centrifugation (9,000 rpm) and only the supernatant is recovered.

In addition to the ammonium sulphate treated firstly to the recovered supernatant, ammonium sulphate was further added so that the final ammonium sulphate concentration was 38 to 43 (w / v)% to dissolve to form a precipitate. Since the precipitate formed at this concentration contains diphtheria toxin, centrifugation is performed at 9,000 rpm to discard the supernatant and recover only the precipitate.

By treating ammonium sulfate at low and high concentrations as described above, the diphtheria toxin was precipitated by the salting-out method to adjust the crude agent. At this time, if the concentration in the first ammonium sulfate treatment is less than 22 (w / v)% purification purity is lowered, if it exceeds 27 (w / v)% toxin recovery is reduced. In addition, when the concentration of secondary ammonium sulfate is less than 38 (w / v)%, the recovery rate is lowered, and when it exceeds 43 (w / v)%, the purity is lowered.

The recovered precipitate is redissolved again to 1500 to 2000 Lf / ml with saline solution, and the redissolved diphtheria toxin solution is placed in a sterile dialysis membrane (MW 12,000) and dialyzed until the remaining ammonium sulfate is removed with saline solution. .

The diaphragm toxin solution containing physiological chlorine is again dialyzed with a buffer having a conductivity of 5 to 8 mS / cm, preferably a phosphate buffer of 6 to 7 mS / cm, by dialysis until the ammonium sulfate is removed using saline solution. Replace with saline solution. The diphtheria toxin containing solution substituted with the phosphate buffer solution was purified by column chromatography using a membrane filter (0.2 μm, Gelman) to purify the diphtheria toxin adjusting agent solution by column chromatography to increase purity.

Fourth, purifying the purified diphtheria toxin using column chromatography packed with a support material equilibrated with phosphate buffer having a conductivity of 5 to 8 mS / cm.

In the purification method using column chromatography, the material to be purified is adsorbed and then eluted.

In the present invention, however, the support material is equilibrated with a buffer having a specific conductivity, so that impurities are adsorbed to the support material in the column and diphtheria toxin is passed without adsorption. At this time, the method used is ion exchange chromatography, and the functional group (functional group) is characterized in that using a diethylaminoethyl (DEAE, diethylaminoethyl) group. The above-described method was possible by adjusting the conductivity of the buffer, and it can be said to be an industrially useful method because no additional elution step is required in addition to the purification process using column chromatography.

Therefore, in the present invention, the adjusted diphtheria toxin solution previously substituted in the above step with a buffer having a conductivity of 5 to 8 mS / cm, and an ion exchange resin, DEAE-sepharose FF (Pharmacia) support material, are applied to the column. After filling, washing and activating, equilibrating with a phosphate buffer having a conductivity of 5 to 8 mS / cm, and then passing through at a constant flow rate, the high-purity diphtheria toxin can be recovered.

In the present invention, DEAE-Sepharose FF (Pharmacia) is used as a support for use in the column chromatography process for purifying diphtheria toxin.

As described above, the diphtheria toxin contained in the buffer having a conductivity of 5 to 8 mS / cm is inferior in adsorption to the DEAE-Sepharose FF support material. Therefore, the impure protein including the pigment component is adsorbed on the support material, but the diphtheria toxin is It can be passed without adsorption, allowing for simple purification of diphtheria toxin without the need for further elution.

Fifth, detoxify the purified diphtheria toxin.

The purified diphtheria toxin has inherent toxicity and therefore must be detoxified to remove it. In general, formalin, phenol, glutaraldehyde, and the like are used as a drug for detoxifying toxin. Formalin was used in the present invention. First, L-lysine is added to the diphtheria toxin purified solution to dissolve it, and formalin is added at 0.9 to 1.0 (w / v)% to detoxify at 36 to 37 ° C for 30 days.

After completion of the detoxification, dialysis water was used to remove residual formalin, and chimerosal 0.01 (w / v)% and NaCl 0.85 (w / v)% were added to the stock solution from which formalin was removed, and a membrane filter (0.2 μm, Aseptic filtration using Gelman) prepares diphtheria toxoid stock solution.

The prepared diphtheria toxoid can be used by preparing an adsorbent diphtheria toxoid vaccine having excellent stability by adsorbing aluminum salt in a conventional manner.

The adsorbed diphtheria toxoid vaccine of the present invention thus prepared is prepared as a final stock solution so that the toxoid content does not exceed 50 Lf, 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 detail with reference to Examples, but the present invention is not limited by the following Examples.

1. Production of Diphtheria Toxin

Preparation Example: Preparation of Stainer Variation Medium

1) Preparation of Enzymatic Hydrolysis Casein Solution

To prepare the enzyme hydrolysis casein solution, 2,700 g of NZ-Amine or NZ-Case were added together with 400 g of yeast extract in 23 L of distilled water and heated to 60 ° C to dissolve. 105.3 g of Na ₂ HPO ₄ and 35.1 g of KH ₂ PO ₄ were dissolved therein, and the pH was adjusted to 9.3 with 10N NaOH. The mixture was heated to 79 ° C., followed by 83.7 g of CaCl ₂ , and further heated to 85 ° C. Cooled to room temperature and stored in the refrigerator overnight. Overnight solution was stored in Toyo No. The supernatant was collected by filtration with 2 filter paper and the volume of the supernatant was corrected to 27 L using distilled water.

2) Preparation of Stainer Variation Medium

After modifying 27 L of the enzyme hydrolysis casein solution to pH 8.1 with acetic acid, 136 ml of 60% sodium lactate solution and stock and modified Muller growth factor were prepared and stored [MgSO ₄ · 7H ₂ O per 1000 ml]. 22 5 g, β-alanine 2.3 g, pimelic acid 0.15 g, nicotinic acid 4.6 g, CuSO ₄ · 5H ₂ O 0.5 g, ZnSO ₄ · 7H ₂ O 0.8 g, MnCl ₂ 0.24 g of 4H ₂ O and conc. 30 ml of HCl] was added to adjust the volume to 60 l. Add 160 ml of 10% cystine-HCl sol. And 240 ml of 0.05% FeSO ₄ · 7H ₂ O solution, adjust the final volume to 75 ℓ, and adjust the pH to 7.5 with 5N HCl. It was.

Example 1 Culture of Diphtheria Strains

1) Master Seed & Walking Seed Manufacturer

The strain used in the present invention is a known strain for producing diphtheria vaccine, Corynebacterium diphtheriae Park-Williams No. 8, which is used to prepare a master seed (master seed), from which the working seed (working seed) is made again and then rapidly Stored in a deep freezer was used for the experiment.

2) Cultivation of diphtheria strains: spawn and main culture

The spawn culture consists of two stages, the first spawn culture and the second spawn culture. In the primary seed culture, 45 ml of the medium prepared in Preparation Example was dispensed into a 250 ml Erlenmeyer flask and sterilized at 121 ° C. for 30 minutes, and 2.5 ml of the sterilized 50% maltose solution was added thereto, followed by 1 vial of the working seed. (vial) was added and incubated for 24 hours in a shaker at a shaking speed of 150 rpm at 35 ℃.

In the secondary seed culture, 190 ml of the medium prepared in the above Preparation Example was dispensed into four 1 L Erlenmeyer flasks and sterilized at 121 ° C. for 30 minutes, and 10 ml of the sterilized 50% maltose solution was added thereto. 10 ml of the spawn culture was added and incubated in a shaker for 24 hours at a shaking speed of 150 rpm at 35 ° C.

In this culture, 75 l of the medium prepared in Preparation Example was added to a 200 l fermenter (KFC, Korea) and sterilized at 121 ° C. for 30 minutes, and 4,000 ml of sterile 50% maltose solution and phosphate solution (25.5) were added thereto. 228 ml of% K ₂ HPO ₄ , 8.5% KH ₂ PO ₄ ), 734 ml of 40% CaCl ₂ solution, and 320 ml of 25 (v / v)% antifoam (Antifoam A, Sigma) were added and inoculated with 800 ml of secondary seed culture. Incubated for 48 hours.

In the culture conditions, the aeration amount was supplied at 0.3 vvm, and the oxygen (purity 98% or more) was supplied while maintaining the speed of the stirrer at 200 rpm to maintain the dissolved oxygen amount at 20% air saturation. In addition, 50 (v / v)% antifoam (Antifoam A, Sigma) was automatically supplied to suppress the foaming during the culture.

3) Cell growth and toxin production with and without yeast extract

When yeast extract was added to the stainer medium, the effect on cell growth and toxin production was examined, and the results are shown in Table 1 below.

The cell concentration according to the growth of the cells was checked by optical density using a spectrophotometer (Kontron) at 650 nm, and the production of diphtheria toxin was measured using a known Ramon aggregation method. Values are expressed in Lf / ml.

Culture medium and culture conditions are the same as in Example 1, yeast extract in the stainer medium to which maltose is added at 25 g / l 0 g / L, 5 g / l and 10 g, respectively, as shown in Table 1 below. Comparative tests were made by adding / l.

Remarks Stainer Badge Lawful stainer badge ¹⁾ Lawful stainer badge ²⁾  Maximum cell growth (O.D650nm) 38.4 45.2 36.2  Maximum Toxin Concentration (Lf / mL) 140 160 120  Maximum Toxin Purity (Lf / mgPN) 664 688 529 1) Add 5 g / l yeast extract to stainer medium 2) Add 10 g / l yeast extract to stainer medium

As shown in Table 1, the addition of 5 g / L yeast extract to the stainer medium was found to increase cell growth and toxin production. This result may be due to the increase of cell growth because diphtheria toxin is produced with cell growth. However, when more than 10 g / ℓ of yeast extract was added, cell growth was inhibited and toxin production was decreased. This may be due to the result of inhibiting cell growth by high yeast extract of 10 g / L or more. According to the above experimental results, it was found that it was effective to use the modified strainer medium in which yeast extract was added in the range of 1.0 to 7.5 g / L to the stainer medium.

4) Cell growth and production of diphtheria toxin by addition of carbon source

In order to increase cell growth and toxin production of diphtheria, a sufficient carbon source is required, but maltose, a carbon source, is known to be limited to 25 g / l (Journal of general microbiology, 48, 37-44, 1967). .

Therefore, when 25 g / l or more is added at a time, cell growth and toxin production are inhibited. In addition, in the present invention, in addition to 10 g / l maltose added to the stainer modified medium, maltose is each 0 g / l while the culture is performed. 1, 15 g / L, 20 g / L was further added to the experiment was shown in Table 2 the results. Culture medium was a stainer modified medium and the culture conditions are the same as in Example 1.

division Steiner Variant Badge Maltose added amount (g / ℓ) 0  15 20 Maltose Extra Time - 24 hours after incubation 30 hours after incubation  Maximum cell growth (O.D650nm) 45.2 57.2 52.4  Max Toxin Concentration (Lf / mL) 160 225 200  Maximum Toxin Purity (Lf / mgPN) 688 703 695

As shown in the results of Table 2 above, after incubating 24 g / l of maltose, a carbon source, after 24 hours from the start of the culture in the stainer modified medium, the maximum toxin purity and cell growth were increased, in particular, the amount of toxin production was 40 % Increased.

These results suggest that the addition of maltose promoted cell growth, thereby increasing the amount of toxin production. However, in the experiment in which maltose was additionally added to 20 g / l after 30 hours of incubation, toxin production and toxin purity were lower than those of maltose added to 15 g / l. Therefore, the addition of 15 g / l yeast extract 24 hours after the start of the culture was found to be the most effective.

5) Dissolved oxygen content in fermenter culture

20 hours after the start of the culture, the dissolved oxygen content of the fermenter dropped below 10%, oxygen (more than 98% purity) and air were mixed and supplied at 0.3 vvm to maintain the dissolved oxygen content of the fermenter above 20% air saturation.

Example 2 Diphtheria Culture Recovery and Concentration

1) Diphtheria culture solution recovery

A continuous centrifuge (Westfelia) was used to separate the cells and the culture supernatant from the culture solution obtained through the fermentor culture of Example 1. The supernatant containing diphtheria toxin could be separated from the cells by passing the culture solution through a continuous centrifuge rotating at a rotational speed of 9,600 rpm. The recovered diphtheria culture supernatant was subjected to primary filtration with K300 (Seitz) filter paper, and secondary filtration with KS80 (Seitz) filter paper, and stored in the refrigerating chamber and used for the toxin purification process.

2) Diphtheria culture concentrate

The culture supernatant from which the diphtheria cells were removed was concentrated using an UF concentrator (Sartorius) equipped with a 30 kDa UF module. At this time, while maintaining the inlet pressure of the concentrator at 1 kg / cm 2 or less and the discharge pressure at 0.5 kg / cm 2 or less, it was concentrated until the toxin concentration of the recovered liquid became about 1000 Lf / ml.

After concentration, diafiltration was performed using physiological saline 10 times the concentration of the concentrate to remove the remaining minerals. After diafiltration, the toxin content was diluted to a final 500 Lf / mL, and NaHCO ₃ was added to 0.5 (w / v)% to dissolve it.

Example 3 Modulators of Diphtheria Toxin

1) Primary Ammonium Sulfate Treatment

The primary ammonium sulfate treatment process removes impurities protein except diphtheria toxin, and takes samples in diafiltered diphtheria toxin solution, and samples are taken at 1% intervals so that the concentration of ammonium sulfate is 22-27 (w / v)%, respectively. Treated and dissolved. At this time, the precipitate formed was removed by centrifugation at 3,000 rpm, and only the supernatant of each sample was collected to measure protein content and toxin content to determine the concentration of ammonium sulfate in the highest purity sample.

Ammonium sulfate corresponding to the concentration was weighed and added to the diafiltered diphtheria toxin main solution, completely dissolved, and reacted overnight in a refrigerating chamber. The primary ammonium sulfate main treatment solution reacted overnight was centrifuged at 9000 rpm for 30 minutes to discard the precipitate and recover only the supernatant.

2) secondary ammonium sulfate treatment

Secondary ammonium sulfate treatment processes induce precipitation formation to recover diphtheria toxin. Take a sample from the supernatant obtained by the primary ammonium sulfate treatment, and add ammonium sulfate at 1% intervals such that the final concentration is 38 to 43 (w / v)%, including the concentration of the primary ammonium sulfate main treatment. It was 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 by measuring the protein content and toxin content, respectively. 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.

3) Conductivity control by buffer dialysis

The precipitate recovered after the second ammonium sulfate treatment was dissolved in physiological saline to a content of toxin of 1500-2000 Lf / ml, which was placed in a dialysis membrane (MW 12,000) and physiological saline until the remaining ammonium sulfate was removed. Dialysis was performed.

After dialysis by physiological saline, the dialysis was performed with 1 / 15M phosphate buffer (pH 7.0) having a conductivity of 6.8 mS / cm, thereby replacing the toxin solution. The diphtheria toxin solution thus prepared was aseptically filtered using a membrane filter (0.2 μm, Gelman) and used as a column chromatography process as a diphtheria toxin adjusting solution.

Example 4 Purification of Diphtheria Toxin

1) Activation of column chromatograph

The column chromatography support used in the diphtheria toxin purification process is DEAE-Sepharos FF (Pharmacia), and 300 ml of this support is filled in a 50/30 XK column (Pharmacia), and the column is subjected to the chromatography system GradiFrac (Pharmacia). ) And the diphtheria toxin purification process was carried out in a refrigerating chamber at 2 to 8 ℃.

The support material is washed by passing a 0.5N NaOH solution, which is 3 to 5 times the capacity of the support material charged to the column mounted in the chromatography system, at a flow rate of 8 ml / min, and becomes 3 to 5 times the capacity of the support material. The support was activated by passing 1 / 15M phosphate buffer (pH 7.0, 2M NaCl, 125 mS / cm) at a flow rate of 8 ml / min. The 1 / 15M phosphate buffer (pH 7.0, 6.8 mS / cm), which is 3 to 5 times the capacity of the support material, is passed through the activated material column at a flow rate of 8 ml / min. The column was equilibrated by removing phosphate buffer.

2) Purification of Diphtheria Toxin by Column Chromatography

The diphtheria toxin modifier obtained in Example 3 was passed through a column filled with the equilibrated support material at a flow rate of 8 ml / min.

Under column chromatography conditions equilibrated with 1 / 15M phosphate buffer solution having a conductivity of 6.8 mS / cm, DEAE-Sepharose FF adsorbs the pigment component and impure protein contained in the diphtheria toxin adjusting agent and passes through the diphtheria toxin without binding. Therefore, all toxin solutions coming out of the support material were recovered.

This method is advantageous for the industrial purification of diphtheria toxin since no additional elution step is required. The final purified diphtheria toxin could be recovered at a concentration of 800 to 1,300 Lf / ml, and at the same time, high purity diphtheria toxin of 2000 Lf / mgPN or higher could be obtained.

Example 5 Preparation of Diphtheria Toxoid Stock Solution by Detoxification of Diphtheria Toxin

In order to remove the toxicity of the diphtheria toxin finally purified by Example 4, a detoxification method using formalin was used.

L-lysine was added to the purified diphtheria toxin solution at 50 mM to completely dissolve it, and aseptic filtration was performed using a membrane filter (0.2 μm, Gelman). Formalin was added thereto at 0.9 to 1.0 (w / v)%. Formalin-added diphtheria toxin solution was stored in a constant temperature room of 36 to 37 ℃ detoxification for 30 days.

Dialysis was performed using distilled water to remove formalin and L-lysine remaining in the diphtheria toxoid solution obtained by the detoxification process. After dialysis, NaCl 0.85 (w / v)% and chimerosal 0.01 (w / v)% were added to the diphtheria toxoid solution, followed by aseptic filtration using a membrane filter (0.2 μm, Gelman) and refrigerated as a diphtheria toxoid stock solution. It was used for diphtheria vaccine related tests while storing.

SDS-PAGE was performed to confirm the diphtheria toxoid obtained by detoxification of the diphtheria toxin and formalin purified by the above method, and the results are shown in FIG. 1. As shown in FIG. 1, diphtheria toxin and toxoid were identified at a molecular weight of 58 kDa.

Experimental Example 1 Test of Diphtheria Toxoid Stock Solution

The prepared stock solution of diphtheria toxoi was tested according to the Biological Standards and Test Methods published by the Korea Food and Drug Administration. The main test items consisted of purity test, sterility test, detoxification test and toxic recovery denial test, and the results are shown in Table 3 below.

Test Items  standard  Test result Purity Test  Over 1,500 Lf of toxoid in 1 mg of protein 2,000-2,600 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 Table 3, the diphtheria toxoid stock solution prepared according to the present invention showed that the diphtheria toxoid stock solution was commercially prepared as a diphtheria vaccine and could be used as a vaccine for preventing diphtheria infection.

Example 6 Preparation of Adsorption Diphtheria Toxoid Stock Solution

To carry out the test according to biological standards and test methods, diphtheria toxoid stock solution was diluted with 0.01 M phosphate buffer (pH 6.8, 0.7% NaCl) to a final 125 Lf / mL, and aluminum hydroxide was added to the final aluminum content. The diphtheria toxoid stock solution was adsorbed to aluminum to 0.5 mg / ml and the test of the final stock solution was performed by diluting 5 times the adsorption diphtheria toxoid stock solution.

Experimental Example 2 Test of Adsorption Diphtheria Toxoid Stock Solution

The adsorption diphtheria toxoid was tested according to the Biological Standards and Test Methods published by the Korea Food and Drug Administration. The main test items consisted of detoxification test, abnormal toxicity test, and potency test. The results are shown in Table 4 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 Table 4, the adsorbed diphtheria toxoid stock prepared by the present invention showed satisfactory efficacy as a vaccine for preventing diphtheria infection.

As described above, the diphtheria toxoid vaccine prepared by the present invention is not only non-toxic and can effectively prevent diphtheria disease, and can be used as a DTaP vaccine by using diphtheria toxoid vaccine alone or in combination with purified pertussis vaccine and tetanus vaccine. In addition, it can be applied to adult diphtheria and tetanus prevention vaccine.

In addition, high-purity diphtheria toxoid vaccines can be prepared and purified in large quantities in an easy manner, making them more commercially useful.

Claims (6)

In the method for producing a diphtheria toxoid vaccine, Diphtheria strain ( Corynebacterium diphtheriae ) was dispensed in the medium, and a mixture of air and oxygen was supplied to the fermenter at 0.1-1 vvm to incubate for 24 to 72 hours while maintaining the dissolved oxygen content of the fermenter at 10-50% air saturation. Culturing while adding maltose at a concentration of 1 to 30 g / l at 10 to 30 hours thereafter to obtain a diphtheria toxin culture solution; Concentrating the supernatant obtained by centrifuging the diphtheria toxin culture solution, followed by ultrafiltration followed by diafiltration.  Treating the concentrated diphtheria toxin with ammonium sulfate, followed by dialysis with a phosphate buffer having a conductivity of 5 to 8 mS / cm to adjust the modifier; Purifying the modulated diphtheria toxin using column chromatography equilibrated with phosphate buffer having a conductivity of 5 to 8 mS / cm; And Detoxifying the purified diphtheria toxin Method for producing a diphtheria toxoid vaccine comprising a. According to claim 1, wherein the medium is a method for producing a diphtheria toxoid vaccine, characterized in that the yeast extract 1 to 7.5 g / L was added to the stainer semi-synthetic medium. The method of claim 1, wherein the ultrafiltration is performed using a 30 kDa UF module. The diphtheria toxoid vaccine according to claim 1, wherein the ammonium sulfate precipitation treatment is performed first at a concentration of 22 to 27 (w / v)% and second at a concentration of 38 to 43 (w / v)%. Manufacturing method. The method of claim 1, wherein the column chromatography is a method for producing a diphtheria toxoid vaccine, characterized in that using a diethylaminoethyl (DEAE) group as a reactor. The method of claim 1, wherein the detoxification is performed using formalin.

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