KR100344703B1 - Method for preparation of diphtheria toxin and toxoid - Google Patents

Abstract

The present invention relates to a method for mass-producing high-purity diphtheria toxins and toxoids. Specifically, high-purity toxins are produced in large quantities by optimizing the culture conditions of diphtheria, and such diphtheria toxins are purified through double filtration and ion exchange chromatography. The present invention relates to a method for producing a stable high purity toxoid by detoxification, and the method of the present invention can be usefully used for preparing a high purity diphtheria vaccine having improved safety.

Description

Method for preparation of diphtheria toxin and toxoid

The present invention relates to a method for mass-producing high-purity diphtheria toxins and toxoids. Specifically, high-purity toxins are produced in large quantities by optimizing the culture conditions of diphtheria, and such diphtheria toxins are subjected to double ultrafiltration and ion exchange chromatography. The present invention relates to a method for preparing a stable high purity toxoid by purification after detoxification, and the method of the present invention can be usefully used for preparing a high purity diphtheria vaccine having improved safety.

Diphtheria is a bacterial respiratory disease caused by the Gram-positive bacillus Corynebacterium diphtheria. As a method of active immunity against this disease for a long time, a toxoid vaccine inactivated exo-toxin produced by C. diphtheria has been used, and a medium for developing and culturing medium for obtaining diphtheria toxin more efficiently is used. Many studies have been conducted.

In general, in order to mass-produce diphtheria toxin, it is known that hydrolyzing bovine muscle with enzyme is the most suitable medium. That is, it is reported that toxin production of 200 Lf (Limit of Flocculation) or more can be produced in 48 hours of culture using this medium, but there is a fatal weakness that is expensive and contains a bee-sensitizing antigen. On the other hand, when a casein is hydrolyzed by an enzyme as a medium, there is no risk of impurities acting as an antigen, and it is suitable for mass production of toxins.

Steiner also reported that high concentrations of toxins could be produced by the addition of casein-free inorganic phosphates and CaCl ₂ to produce calcium-phosphate precipitates in the medium (Stainer DW, Can. J. Microbiol . 13, 1967, 963-968). Since C. diphtheria is known to secrete diphtheria toxin when iron is depleted in the medium, iron is fixed in the precipitate when calcium-phosphate precipitation occurs in the medium, and then it is released slowly during the incubation period and iron in the medium. By allowing the concentration of ions to be properly maintained throughout the incubation period, it is possible to produce high concentrations of toxins.

Maltose is generally used as a carbon source for the culture medium of C. diphtheria because it prolongs the toxin production period by allowing growth constraints by the carbon source to be maintained for a long time. Therefore, maltose is more advantageous for toxin production than glucose. As such, growth restriction by carbon source is an important factor in the production of high yield of toxin, and in fact, Rigelato et al. Shows that toxin production is 0.3 toxin / bacterial protein (g / g) in the growth of glucose restriction conditions, which is more toxin than in glucose excess condition. The production was reported to increase fivefold (Righelato RC, Gen Microbiol. 58, 1965, 403-410).

During the incubation period of C. diphtheria, pH is known to be determined by the metabolites and aeration conditions. Maltose present in the medium is broken down into glucose and converted into organic acids such as propionic acid, formic acid and acetic acid by metabolism, so the pH drops at the beginning of the culture and the organic acid is carboxylic acid. The pH is increased again by oxidative metabolism which converts carbonic acid and water. In addition, the pH value during the culture is sensitive to the change in the rate of oxygen transfer in the medium, and when the oxygen supply is excessive, the pH is also increased to lower the yield of toxin production.

Diphtheria toxin can generally be produced in both culture and fermenters using culture bottles. The advantage of the political culture is that it is simple and inexpensive to purchase, and incubation in a Roux bottle for about 7 days yields a toxin of 50-100 Lf / mL. However, in order to produce high concentration and purity of toxin, the production by fermenter is essential. In this case, 150 to 300 Lf / mL of toxin can be produced within 48 hours. However, until now, domestic production of diphtheria toxin is still under way.

Diphtheria vaccines use a toxoid detoxified diphtheria toxin, where the quality of the diphtheria vaccine is closely related to the purity of the diphtheria toxoid. This is because higher purity contains fewer heterologous proteins that cause side effects.

Purity of such toxoid is determined by a purification method, and in the purification method, detoxification of toxin and purification of toxoid (hereinafter referred to as 'toxoid purification') and purification of toxin and detoxification by detoxification (hereinafter referred to as Called toxin tablets). Currently, both of the above purification methods are used to manufacture diphtheria vaccine, and each purification method has advantages and disadvantages.

The advantage of toxoid tablets is that they do not return to toxin. However, the toxoid purified in this way is of low purity. The reason is that when formaldehyde is added to crude-toxin contained in the medium component, methyl bridge is formed between the toxin and the amino acids and peptides derived from the medium, resulting in a large number of peptides. Is attached to the toxin to become a toxoid. Low purity toxoid can cause side effects due to media components. That is, when beef meat is used as a medium component, toxoid binds to numerous peptides derived from cows, and these cow-derived peptides can be covalently bound to toxoid to act as an antigen, causing side effects. In particular, adults who have been continuously exposed to bovine antigens can cause more side effects than children.

Prior to the 1950s, there was only a method of purifying toxoid after detoxifying zotoxin, but in 1958, a method for purifying toxin and detoxification was developed by Pop and Steven by developing a method for purifying toxin in large quantities. (Pope, CG and MF Steven, Brit. J. Exp. Path. 39, 1958, 139-149). In other words, the toxin purification was performed by removing the peptide from the medium component from the crude toxin, purifying only diphtheria toxin and then detoxifying the toxin by using methods such as ultrafiltration (hereinafter abbreviated as 'UF') and column chromatography. To prepare. As such, toxoid production through toxin purification has a risk of toxoid returning to toxin, but high purity toxoid can be obtained and side effects due to the medium-derived material can be reduced. In particular, adult diphtheria vaccines require purity of more than 2,500 Lf / mg protein nitrogen (PN) in Korea's biologics standards, because adults have been exposed to bovine antigens for long periods of time to reduce side effects due to bovine-derived substances. Therefore, the production of diphtheria vaccine through toxin purification is a purification method necessary for the production of high purity diphtheria vaccine and adult diphtheria vaccine.

Therefore, the inventors of the present invention, in order to prepare a large amount of high-purity diphtheria toxin and toxoid, first conduct a study to optimize the culture conditions of C. diphtheria in a fermenter and to search for a medium having high toxin productivity, and to prepare a high-purity toxoid vaccine. As a result of repeated studies to establish a toxin purification method, high-purity diphtheria toxin can be obtained by using casein enzymetic digest as a nitrogen source and culturing with fermenter by properly adjusting pH and aeration of the medium. Produced in large quantities, this toxin is a double UF The present invention was completed by confirming that, after purification through ion exchange chromatography, detoxification can yield a high purity diphtheria toxoid.

It is an object of the present invention to provide a method for producing a high purity diphtheria toxin and a method for producing a high purity diphtheria toxoid by purifying diphtheria toxin.

Figure 1 is a graph showing the growth and toxin production of cells over time when Corynebacterium diphtheriae ( Corynebacterium diphtheriae ) was cultured using the fermentor culture method of the present invention.

Figure 2 shows the results of performing the Ouchterlony double immunodifusion method to compare the antigenicity of the toxin purified toxoid and the conventional toxoid purified toxoid according to the present invention.

1: diphtheria antitoxin; 2, 3: toxin tablet toxoid

4, 5: Toxoid Tablet Toxoid

Figure 3 is the result of performing SDS-PASE to confirm the molecular weight of the toxin purified toxoid and the conventional toxoid purified toxoid according to the present invention.

One; Markers; 2, 3, 4: Toxoid Tablet Toxoid

5, 6, 7: Toxin Tablet Toxoid

In order to achieve the above object, the present invention comprises the steps of: a) culturing Corynebacterium diphtheria in a fermenter containing a medium using a casein enzymatic degradation product as a nitrogen source; b) it provides a method for producing diphtheria toxin comprising the step of recovering diphtheria toxin from the culture.

The casein enzymatic degradation product of the present invention preferably has an average molecular weight of at least 400 Daltons of amino acids.

At this time, the initial pH of the medium is preferably within the range of 6.5-7.5, more preferably about pH 7.0. In addition, the iron ion concentration of the medium is preferably added to 0.7 mg / L or less, more preferably 0.5 mg / L or less.

In addition, the present invention a) toxins produced by diphtheria strains are double U.F. Making; b) performing ion exchange chromatography with filtered toxin; And c) detoxifying the purified toxin.

Wherein the double U.F. Is the molecular weight cut off (hereinafter abbreviated as 'M.W.C.O.') 100,000 modules and M.W.C.O. Using 30.000 modules, step b) is accomplished by adding lysine to the purified toxin and then detoxifying by adding formalin.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of: a) culturing Corynebacterium diphtheria in a fermentor containing a medium using a casein enzymatic degradation product as a nitrogen source; b) it provides a method for producing diphtheria toxin comprising the step of recovering diphtheria toxin from the culture.

Specifically, the present invention provides culture conditions for optimized diphtheria strains for toxin production. First, the present invention searches for a medium optimized for toxin production in the culture of diphtheria strains using a fermentor.

In the present invention, Corynebacterium dihptheriae Park-Willium No. 8 strain was used as the diphtheria strain.

First, in order to determine the nitrogen source of the medium best suited for cell growth and toxin production, casamino acid, toxiprotone and casein enzymatic degradation products (NZ-Case, NZ-Amine) are selected as the nitrogen source. And cultured C. diphtheria strains. Toxiproton is a medium for diphtheria culture made by processing meat of poultry.

As a result, NZ-Case and oxyproton were superior to other media in the growth and toxin production ability of bacteria and the fermentation time was short. However, in the case of oxyprotone, there is an additional burden of verifying the effect of meat-derived protein components as antigens, whereas casein-derived media has a low risk of side effects and a high toxin-producing ability, which is a nitrogen source for diphtheria strain culture. It was confirmed as preferable. In particular, the case of the casein enzymatic degradation products showed a more excellent result in the strain culture capacity and toxin production capacity when the average molecular weight of the amino acid is more than 400 Daltons.

In the present invention, in order to determine the pH of the medium most suitable for cell growth and toxin production, C. diphtheria strains were cultured by adjusting the initial pH of the medium to 6.6-7.8. As a result, it was found that the pH is in the range of 6.5-7.5, and it is more preferable to adjust the pH to around 7.0.

This is a different result than the initial pH reported for the medium for diphtheria toxin production is generally 7.5-7.8. These results indicate that the initial pH 7.0 medium, which can provide a relatively low pH at the end of the cultivation, showed the best cell growth and toxin production since the pH continuously increased under good aeration conditions.

Iron ion concentration in the medium is a very important factor in the production of diphtheria toxin. Murphy et al. Reported that iron ions act as corepressors in the regulation of tox genes that synthesize toxins through studies using mutants (Murphy, JR et al., J. Virol. 18) . , 1976, 23-44). That is, only when the concentration of iron ions in the cell is low, the tox operon is derepressed to allow toxin production. Therefore, it is necessary for the high yield of diphtheria toxin to be maintained in the iron ion concentration in the medium during the culture period.

In the present invention, in order to determine the iron ion concentration in the medium that is most suitable for cell growth and toxin production, C. diphtheria strains were cultured in a medium to which the iron ion concentration was added at 0.2 to 0.8 mg / L using FeSO ₄ solution. As a result, the iron ion concentration is preferably added at 0.6 mg / L or less, more preferably 0.5 mg / L or less. However, in the early stage of incubation with low cell concentration, it is preferable that a certain amount of iron ion exists for growth of the cell.

In the present invention, in order to determine the dissolved oxygen amount of the medium most suitable for cell growth and toxin production, the aeration amount is fixed at 0.22 vvm and the agitation speed is changed to 200 ~ 500rpm to incubate C. diphtheria strains and stirred at 380 rpm or more. It was found that toxin production at the speed reached the highest, particularly at about 400-430 rpm.

In order to obtain a high yield of toxin, it is essential that an appropriate concentration of calcium-phosphate precipitate is formed in the medium. Therefore, in the present invention, in order to determine the optimal calcium-phosphate precipitation in the medium, phosphate solution and CaCl ₂ solution were added at various concentrations so that the ratio of calcium-phosphate was 5.7-8.8, and C. diphtheria strains were cultured. -The amount of precipitation of phosphate did not significantly affect cell growth, but when it was above or below the proper amount, it affected the concentration of iron ions in the medium and changed the yield of toxin production.

In order to confirm the effectiveness of the fermenter culture method optimized in the present invention, the amount and quality of each of the diphtheria toxin produced through the culture of the fermenter and the conventional political culture to which the culture conditions such as the above-described medium composition was applied were compared.

Existing political culture was inoculated with C. diphtheria in Mueller, Miller (Ju, J. Immunol. 37, 1980, 103-112) using casamino acid as a nitrogen source. As a result of comparing the toxin produced in both cultures, the toxin produced in the culture using the fermenter showed more than two times higher toxin concentration and toxin purity than the toxin produced by the political culture. In addition, the reaction time with the diphtheria antitoxin also appeared faster in the toxin obtained in the fermentor culture, it was confirmed that the quality of the toxin produced by the fermentor culture is excellent.

In addition, the toxins produced by the two culture methods were purified to prepare a final stock solution for vaccine production, and a potency test comparing the immunogenicity of the two toxoids was carried out through animal experiments using guinea-pigs. It was. As a result, the toxin obtained through the culture of the fermenter of the present invention was found to have a superior titer than the political culture toxin.

Through the above experiments, it was confirmed that when the toxin is produced in the fermentation broth, it is possible to produce diphtheria toxoid having excellent yield, productivity, and immunogenicity.

The present invention provides a method for producing high purity diphtheria toxoid by purifying and detoxifying the toxin produced by the diphtheria strain.

Specifically, the method for preparing toxoid of the present invention comprises the steps of: a) dual U.F. toxins; b) performing ion exchange chromatography with filtered toxin; And c) detoxifying the purified toxin.

In the present invention, C. diphtheria strains were cultivated by the above-mentioned static culture method or fermentation tank culture method, and then the bacteria were removed from the culture medium by filter paper, followed by bacterial filtration with membrane filter paper to obtain Jotoxin.

Jotoxins are composed of toxins and other proteins. Cox contains 15% of diphtheria toxins, 12% more molecular weight than toxins, and less of toxins. (Cox, JC, Appl. Microbiol. 29, 1975, 464-468).

In the past, U.F. The process was used, and the salting out method using ammonium sulfate (hereinafter abbreviated as 'A / S') was mainly used to remove proteins having a molecular weight greater than that of toxin. This is because toxins do not precipitate at low concentrations of A / S, while those with higher molecular weight do precipitate. However, this method requires higher concentrations of A / S, resulting in higher conductivity, which requires buffer exchange to reduce conductivity prior to DEAE ion exchange chromatography. In the present invention, a double U.F. was performed to overcome this inconvenience.

Specifically, in the present invention, M.W.C.O. The module was used and the M.W.C.O. was determined in consideration of the molecular weight of diphtheria toxin of 58,500. 100,000 modules were used to remove proteins of high molecular weight and to recover the filtrate, followed by M.W.C.O. 30,000 modules were used to remove low molecular weight proteins. As a result, the purity was improved about 41% compared to that of zotoxin, which was similar to the result of low concentration A / S treatment.

Therefore, in the present invention, the double U.F method was found to be a useful method for removing other proteins of higher molecular weight than diphtheria toxin without using A / S in toxin purification.

The main feature of the U.F concentrate is the removal of the dark brown pigment of zotoxin. This dye is not a protein, and when DEAE-ion exchange chromatography was performed, the DEAE-gel was more strongly adsorbed than toxin and eluted at 0.5N NaOH. Therefore, it is thought that such a pigment component is adsorbed to aluminum hydroxide gel adjuvant prior to toxoid in the preparation of a vaccine, thereby inhibiting the adsorption rate. Therefore, the U.F process has great significance not only for the concentration and purity of the sample but also for the removal of the pigment.

Toxin in zotoxin can be purified by ion exchange chromatography using the difference in charge amount because it has one kind of charge, and in the case of ion exchange chromatography, it is a purification method suitable for mass production system.

In the present invention, the toxin was purified using an ion exchange chromatography method because of the above advantages.

Specifically, in the present invention, the toxin concentrated in double U.F. was further purified by ion exchange chromatography using DEAE-sepharose column.

As a result, four peaks were observed in the dissolution profile, and Lf was measured only at the peak eluted at a double conductivity of 10.7 mS / cm, confirming that this was a toxin peak. As a result of the recovery, the toxin recovery solution was a transparent toxin solution having almost no pigment component.

Dual U.F. according to the invention. As a result of measuring the purity of toxin purified through ion exchange chromatography, Cox showed similar values to that of detoxified toxoid after toxin purification. Therefore, the toxin purification method of the present invention was confirmed as a very effective method.

In the present invention, diphtheria toxoid is prepared by detoxifying the toxin purified by the above process. Toxin detoxification is known to be important detoxification factors such as toxin content, type and concentration of amino acids used, concentration of formalin used, detoxification time, temperature, pH.

Detoxification with only 0.5% formalin added to the toxin results in incomplete detoxification and a decrease in Lf because the toxin does not form sufficient methyl crosslinks due to a decrease in pH during detoxification. The addition of 0.5% sodium bicarbonate to prevent pH drop during detoxification can prevent the pH drop but a return to toxin is found. To prevent this return, Neumula used 0.1M glycine, which had no stability and immunogenicity, although no return to toxin was found after detoxification (Neumuller, C., Nature 174, 1954, 405-412). Later, et al. Experimented with several amino acids and found that lysine was the most suitable amino acid (Linggood, FV, Brit. J. Exp. Path. 55, 1962, 177-188).

In the present invention, a difference in the properties of the toxin solution was observed according to the order of lysine and formalin addition during the detoxification process, but when formalin was first added to the toxin solution, aggregates were formed, which did not disappear during the detoxification period. On the other hand, when formalin was added after lysine addition, clear appearance was maintained throughout the detoxification period. When formalin is added without lysine, an agglomerate is formed by forming a methyl bridge between toxin and toxin by formalin, whereas when lysine is present, the soluble form is prevented by binding of toxin and toxin due to binding of lysine and toxin. It is judged to be maintained.

Therefore, in the present invention, the addition of lysine first, followed by the addition of formalin was judged as a stable detoxification method for detoxification without loss of toxoid.

Specifically, in the present invention, 0.5% sodium bicarbonate and 0.05 M lysine were added to the toxin solution, 0.5% formalin was added, and the pH was adjusted to 7.5%.

Toxin-purified toxin prepared according to the present invention, to confirm whether the return to toxin, a detoxification test was performed. As a result of observing the guinea-pig to which the toxoid of the present invention was administered for 30 days, no poisoning symptoms such as toxin poisoning, necrosis, paralysis, remarkable weight loss, and other abnormalities were observed in the animal.

In addition, as a result of comparing the quality of the toxin tablet toxoid and the conventional toxoid tablet toxoid according to the present invention, it was confirmed that the toxin tablet toxoid of the present invention was excellent in purity and titer.

Therefore, the production of diphtheria toxoid with high purity and excellent titer and production of adult diphtheria vaccine using the toxoid production method of the present invention is possible.

The present invention will be described in more detail with reference to the following examples. The following examples illustrate the invention but are not intended to limit the scope thereof.

I. Production of toxin

<Example 1> Strain and medium composition

In the present invention, Corynebacterium diphtheria park-William No. 8 strain, which has been generally used to produce toxoid necessary for active immunity against diphtheria, was distributed and used by the Korea Food and Drug Administration (KFDA).

In order to perform the culture using a fermentation tank, sterilized 7 L jar fermentor (KFC. Korea), filtered and sterilized 4.5L of the composition of the composition shown in Table 1, and the phosphate solution and before inoculation of the strain Precipitation was generated by adding 40% CaCl ₂ solution, and then inoculated with 0.4% (v / v) of the strain and incubated at 35 ° C., and the initial pH of the medium was adjusted to 7.0˜7.4. The effect of dissolved oxygen was carried out while stirring in the range of 200 ~ 500rpm under aeration conditions of 0.22 vvm.

Furtherance density 10% NZ-Case stock solution 33.3% (v / v) Maltose 25.0 g / L 69% sodium lactate solution 0.17% (v / v) Mueller growth factor solution 0.8% (v / v) 10% L-Cystine HCI solution 0.2% (v / v) 0.1 mg / mL Fe ^2- solution 0.2-0.5 mg / L

The 10% NZ-Case stock solution of Table 1 was prepared as follows. NZ-Case (Sheffield, USA) 500.0g, Na ₂ HPO ₄ 19.5g, KH ₂ PO ₄ 6.5g was dissolved in 4.2L of distilled water to adjust the pH to 9.3 with 10N NaOH and heated to 79 ℃ CaCl ₂ 20.46 g was added to form a precipitate. The supernatant from which the precipitate was removed by centrifugation was used as stock solution.

Example 2 Cell Growth and Toxin Generation

In order to examine the relationship between cell growth and toxin production, the concentration of cells and the concentration of toxin were determined according to the progress of culture in NZ-Case medium using a fermentor (see FIG. 1).

The concentration of cells was measured by optical density using a spectrophotometer at 650 nm. The concentration of toxin was used by Ramon's floculation method, and the measured value was expressed as Lf / mL. In order to determine the Lf value, 100 IU / mL antitoxin (WHO diphtheria antitoxin) was prepared by diluting in multiple steps from 30 IU to 70 IU at 5 IU intervals, and the sample was prepared by diluting with saline to 50 Lf / mL. . 1 mL of the prepared antitoxin and 1 mL of the sample were placed in a 5 mL tube and reacted at 50 ° C. in an incubator.

As shown in FIG. 1, toxins began to be produced after 12 hours, and toxins were also actively generated in the exponential growth phase, thereby rapidly increasing the concentration of toxins. After 30 hours at the end of the exponential growth phase, the toxin concentration continued to increase, but the overall growth of the toxin was also observed.

After 42 hours of incubation, the concentration of toxins began to decrease gradually, because the toxins produced were denatured by proteases released during cell death and were not measured in the determination of toxins through antigen-antibody reactions. .

The concentration of the bacterial cells corresponds to 34-38% of the maximum concentration of the bacterial cells until the first 24 hours of incubation, but the toxin production amount is only 24-28%, so the amount of toxin produced per unit weight of the bacterial cells is gradually increased. Therefore, cell growth and toxin production could not be explained by simple linear proportional function, and the trend could be changed according to factors of various culture conditions.

<Example 3> Influence of nitrogen source

To examine the effects on cell growth and toxin production according to the type of nitrogen source of the medium, as a nitrogen source, casamino acid, NZ-Case (Sheffield, USA), NZ-Amine (Sheffield, USA), and oxyproton (Solavia, France) The experiment was carried out using the fermenter as in Example 1 by selecting the results are shown in Table 2 below.

badge Incubation time (hours) OD (650 nm) Toxin Concentration (Lf / mL) Cassamino 48 15.3 20 Oxyproton 48 45.2 220 NZ-Amine 40 33.7 180 NZ-Case 39 43.0 225

In the above NZ-case and NZ-Amine both show a difference as shown in Table 3 and Table 4 in the amino acid distribution and the molecular weight distribution of each of the casein-enzyme-mediated medium.

NZ-Amine (mg / g) NZ-Case (mg / g) Aspartic acid 66.1 63.2 Threonine 38.8 38.0 Serine 50.4 49.4 Glutamic acid 181.0 178.0 Proline 89.6 88.5 Glycine 17.6 16.5 Alanine 28.4 26.8 Valine 60.4 56.2 Methionine 24.6 24.6 Isoleucine 46.4 45.0 Leucine 75.8 78.6 Tyrosine 29.7 30.8 Phenylalanine 40.7 42.4 Histidine 23.3 23.4 Lysine 39.8 69.7 Arginine 27.6 31.3 Tryptophan 10.4 10.2

100-200 200-500 500-1000 Average molecular weight (daltons) NZ-Amine 58% 32% 10% 283 NZ-Case 30% 20% 10% 410

As shown in Table 2, the casein enzymatic degradation product and oxyproton were excellent in the growth and toxin production capacity of the bacteria and the fermentation time was short. However, in the case of oxyprotone, there is an additional burden of verifying the effect of the protein-derived protein as an antigen, whereas casein-enzyme degradation products are casein-derived media such as conventional culture culture media, and there is no risk of side effects. It is small and is excellent in toxin generating ability and is preferable as nitrogen source for fermenter culture.

In particular, when the casein enzymatic degradation product having an average molecular weight of 400 or more Daltons was used, the toxin producing ability was excellent.

Example 4 Initial pH Effect of Medium

In order to examine the effect on the cell growth and toxin production according to the pH of the medium, the experiment was carried out using the fermenter as in Example 1 by adjusting the initial pH of the medium to 6.6 ~ 7.8, the results are shown in Table 5 Indicated.

Initial pH of the medium Incubation time (hours) OD (650 nm) Highest toxin concentration (Lf / mL) 6.6 42 32.5 205 7.0 42 38.6 223 7.4 44 30.4 197 7.8 41 36.3 160

As shown in Table 5, optimal cell growth and toxin production were at pH 7.0.

<Example 5> Influence of dissolved oxygen amount

In order to examine the effect on the cell growth and toxin production according to the dissolved oxygen content of the medium, the aeration was fixed at 0.22 vvm and the agitation speed was changed to 200, 300, 400, 500 rpm, and cultured using the fermenter as in Example 1. The results are shown in Table 6 below.

At this time, the protein content was measured by the Bradford method (bovine serum albumin) as a standard material. Quantification of maltose and glucose in the medium was measured by HPLC (Bionex, USA) using a sugar analysis column (Bionex PA1, USA), and the detector was a pulsed electrochemical detector (Pulsed Amperometric Detecter).

Aeration rate (rpm) Incubation time (hours) OD (650 nm) Highest toxin concentration (Lf / mL) Remaining maltose (g / L) 200 42 8.2 0 16.4 300 39 16.8 50 7.95 400 39 43.0 228 0.00 500 38 29.3 130 0.19

When the stirring speed was 200 ~ 300rpm almost did not occur during the incubation period, no toxin was produced or produced at a low concentration and there was a significant amount of maltose unused in the medium even after 45 hours incubation. On the other hand, in the culture of 400rpm or more, all carbon sources were used for metabolism, and the cell concentration and toxin concentration showed the maximum value at 400rpm. However, at 500 rpm or higher, dissolved oxygen is supplied higher than the appropriate concentration, and thus, it does not provide optimal conditions for the metabolic process in the early stage of culture in which sugar is converted into organic acid. Therefore, in order to obtain the maximum cell growth and toxin yield, the optimum dissolved oxygen amount was found to be one of the important factors.

<Example 6> Influence of iron ion concentration

In order to examine the effect on the cell growth and toxin production according to the iron ion concentration of the medium, the iron ion concentration was added at 0.2, 0.3, 0.4, 0.6, 0.8 mg / L using a 0.1 mg / l FeSO ₄ solution. The culture was carried out using a fermenter as in Example 1 in the medium, and the results are shown in Table 7 below. Iron ions were measured by instrumental analysis through ICF-AES.

Iron Ion Concentration (mg / L) Incubation time (hours) OD (650 nm) Highest toxin concentration (Lf / mL) 0.2 38 38.1 215 0.3 42 40.7 230 0.4 42 37.9 210 0.6 42 38.5 160 0.8 41 37.2 95

As a result, in the range of 0.2 ~ 0.4mg / L, all the toxins were produced more than 200Lf / mL, but it was found that the toxin production rapidly decreased at the 0.6mg / L or more. In addition, the iron ion concentration did not appear to have a significant effect on cell growth, which is consistent with the report that excessive iron ion concentration did not significantly affect the cell growth rate.

Example 7 Influence of Calcium-Phosphate Precipitation

To determine the optimal calcium-phosphate precipitation in the medium, the phosphate solution (K ₂ HPO ₄ 255 g / L, KH ₂ PO ₄ 85 g / L) and CaCl ₂ solution (40%) were used to achieve a calcium-phosphate ratio of 5.7-8.8. CaCl ₂ 2H ₂ O) was added at various concentrations, and the results of the incubation by the method of Example 1 are shown in Table 8 below.

Addition of Phosphate and CaCl ₂ Solution (ml) OD (650 nm) Highest toxin concentration (Lf / mL) Phosphate solution CaCl ₂ solution 4.3 13.0 30.3 140 6.5 20.0 34.3 190 13.0 41.0 43.0 228 19.5 61.0 34.2 180

The experimental results showed that the precipitation of calcium-phosphate did not significantly affect the cell growth, but if it was above or below the proper amount, the concentration of iron ions in the medium changed the production yield of toxin.

Example 8 Comparison between Political Culture and Fermenter Culture

In order to compare the quality of C. diphtheria toxin produced by culturing in a fermentor and the toxin produced by conventional political culture, the culture medium was first prepared by adding a Muller-Enmir medium using cassamino acid as a nitrogen source in 250 mL of 2L Lux bottles. It was incubated for 7 days at 36 ℃. Cultivation of the inoculation strains was inoculated to 0.4% (v / v) in a 1L shielded flask containing 200 mL of culture cultured in a Loeffler slant for 2 days to 2% at 35 ℃ Incubated daily. Fermenter culture was performed by the method of Example 1 above.

The results of the two cultures were compared and the results are shown in Table 9 below.

Culture method Political Culture Fermentation tank culture Incubation time (hours) 168 48 Toxin Concentration (Lf / mL) 90-110 200-230 Kf (min) 4-5 1-2 Purity (Lf / mg PN) 875-1020 1720-1825 Titer (iu / mL) 2> 4>

First, in the case of fermentation tank culture, a culture condition capable of producing more than 200 Lf / mL of toxin was reproducibly established, which was more than two times higher than that of stationary culture when compared to the maximum toxin concentration.

Purity naturally increased as the concentration of toxin increased. Generally, the purity of political culture is 800 ~ 1000 Lf / mg PN (Protein Nitrogen), and in case of fermenter culture, toxins of 1000 ~ 2000 Lf / mg PN can be produced. Known. In the present invention, the toxin having a maximum purity of 1825 Lf / mg PN was produced in the fermentation broth, which is more than two times higher than the conventional political culture method.

The reaction time with the diphtheria antitoxin (Kf) also appeared faster in the toxin obtained from the fermentor culture, which means that the toxin produced through the fermentor has a better quality than the conventional political culture toxin.

In addition, the toxins produced by the two culture methods were purified to prepare a final stock solution for vaccine production, and a potency test comparing the immunogenicity of the two toxoids was performed through animal experiments.

First, the final stock solution of purified toxoid was adsorbed onto aluminum hydroxide gel adjuvant (Superfos Biosector A / S), and injected subcutaneously by injection of 0.75 mL into 300-400 g of guinea-pig and immunized for 4 weeks. After immunization, serum was collected and neutralized with standard toxin 1L + (green cross). Subcutaneous injection of 2 mL in 230-280 g of guinea-pig was performed for 12 hours, followed by 120 hours observation. It is shown in Table 10 below.

mix Dilution rate Observation time (dead pigs / experimental pigs) 24 48 72 96 120 Standard Antitoxin + Standard Toxin 1 IU / mL 0/2 2/2 2/2 Final solution of political culture × 2 0/2 0/2 2/2 × 4 0/2 1/2 2/2 Final solution of fermenter culture × 2 0/2 0/2 0/2 0/2 0/2 × 4 0/2 0/2 0/2 0/2 0/2

As a result, the toxoid obtained from the culture of the fermenter was not only surviving all the guinea pigs in 2 units, which is the national standard, but also all of the 4 units. Through the above experiments, it was confirmed that when the toxin is produced in the fermentation broth, it is possible to produce diphtheria toxoid having excellent yield, productivity, and immunogenicity.

In addition, in the case of fermenter culture, the high titer may lower the content of toxoid during vaccine production, and thus, the possibility of other proteins causing side effects is also lowered.

II. Preparation of Toxoid Vaccine

Example 9 Production of Toxin

Lyophilized seeds of Corynebacterium diphtheria park-William strain 8 were inoculated into a tube containing seed media (Wads Worth Broth) and then incubated at 36 ° C. for 48 hours. After incubation, diphtheria cells grown on the surface of the medium were gram-stained to determine whether they were contaminated, and then Muller En contained 2.5% (w / v) maltose (Difco) and 0.29% (w / v) casamino acid (Difco). It was inoculated in Miller medium and left to incubate for 7 days at 36 degreeC using Lux disease. After the cells were removed from the culture by Toyo # 2 filter paper, bacteria were filtered through membrane filter paper (Sartorious, 0.2 μm). This was called jotoxin.

Toxin production was confirmed through the Limit of flocculation test. Nitrogen content of the protein was measured by the Kjeldahl method.

As a result of measuring Lf and protein of jotoxin, Lf content was 100Lf / mg, protein concentration was 702µg / mL and purity was 890 Lf / mg PN. This was the same as the results of the diphtheria politic culture published by Mueller N. Miller.

Example 10 Toxin Tablets

In the present invention, Zotoxin was U.F. using Sartocon-mini U.F system (Sartorious). Two types of U.F modules are used. M.W.C.O. The filtrate was recovered using 100,000 modules and then returned to the M.W.C.O. Toxins were recovered by concentration 10 × with 30.000 modules. As a buffer, 25 mM phosphoric acid solution (pH 7.5) was used, and the purification degree of U.F purification step was analyzed by gel filtration (gel filtration; superdex 200), and the results are shown in Table 11 below.

process Purity of Jotoxin Purity of Purified Toxin (Lf / mg of Protein Nitrogen) Zotoxin → UF (MWCO ² 30.000) 870 Lf / mg PN 927 Lf / mg PN Zotoxin → U.F (M.W.C.O 30.000) → A / S treatment 870 Lf / mg PN 1.447 Lf / mg PN Zotoxin → U.F (M.W.C.O 100,000) → U.F. (M.W.C.O 30.000) 890Lf / mg PN 1.300 Lf / mg PN

As a result, the purity was 1,230 Lf / mg PN, which is about 41% improvement compared to that of jotoxin, which is similar to the low concentration of A / S treatment.

Example 11 Ion Chromatography

Toxins concentrated in U.F. were subjected to ion exchange chromatography using DEAE-Sepharose FF (Pharmacia). The column was a 5 cm diameter Pharmacia column with a packing height of 5 cm and a packing volume of 100 mL.

50 mM Tris-Cl (pH 8.5) was used as a buffer, and other proteins and pigments were removed with NaCl and 0.5 N NaOH in the buffer. First, as a result of experiment with NaCl gradient to confirm the conductivity of toxin to be eluted for the first time, as shown in Table 12, toxin was first eluted at the conductivity of 7.7 mS / cm, and the protein was detected at the conductivity of 10.9 mS / cm. Lf was not detected. Thus, the conductivity of the loading and washing buffer was 6.70 mS cm, the flow rate was 8 mL / min, the elution buffer was 10.7 mS / cm and the flow rate was 6 mL / min. The conductivity of the 0.5 M NaCl wash buffer was 16.0 mS / cm and the flow rate was 8 mL / min.

As a result of performing the experiment under the above conditions, four peaks were observed as shown in Table 12 below, and the protein eluted without being adsorbed to DEAE-Sepharose FF during sample loading (Peak 1), and eluted at a conductivity of 10.7 mS / cm. Toxins (peak 2), and proteins eluted at 0.5 M NaCl (peak 3) and proteins eluted at 0.5 N NaOH (peak 4).

Fraction Conductivity (mS / cm) Lf / ml Protein concentration (µg / ml) Purity (Lf / mg of protein nitrogen) 6 5.35 - - - 7 6.46 - 18 - 8 7.70 320 850 2353 9 8.88 86 228 2357 10 10.90 - 78 - 11 11.46 - 11 -

Lf was not measured at peaks 1, 3 and 4 except for the diphtheria toxin of peak 2 among the four peaks. The peak 2 was recovered and measured for purity. The purity was 2560 Lf / mg PN. This is a result similar to the purity of 1050-2500 Lf / mg PN of Cox detoxified after detoxification.

In particular, the toxin recovery solution of peak 2 was a transparent toxin solution with little pigment component, and the pigment component was eluted with 0.5 M NaCl and 0.5 N NaOH. Purity step by step purification is shown in Table 13.

Purification stage Purity (Lf / mg of protein nitrogen) Zotoxin 870 U.F (M.W.C.O. 100,000-M.W.C.O. 30,000) 1,300 DEAE-Sepharose FF 2,560

Example 12 Toxin Detoxification and Diphtheria Toxoid Production

After measuring the Lf of the toxin purified by the method in Example 10, the toxin purification liquid was adjusted to 500 Lf / mL. 0.5% sodium bicarbonate, 0.05 M lysine, 0.5% formalin was added to the L-adjusted toxin solution, and the pH was adjusted to 7.5%.

After detoxification, A / S was added to allow the saturation to be 60% and left at room temperature overnight, followed by centrifugation at 7.000 rpm for 30 minutes to recover the precipitate. The precipitate was dissolved in water for injection, put in a dialysis membrane (M.W.C.O.1,000), dialyzed in distilled water for 4 days in sodium chloride solution for 2 days, and filtered through a membrane filter (sartorious, 0.2 μm) to produce diphtheria toxoid.

<Example 13> Molecular weight confirmation through SDS-PAGE

After purifying the toxin, SDS-PAGE was performed to confirm the degree of purification of the detoxified toxoid.

That is, after loading the sample on a 10% SDS-polyacrylamide gel, the electric field was energized to 12.5 V / cm and then electrophoresed until bromophenyl blue reached the end of the gel. After the electrophoresis, the gel was fixed, stained with Coomassie Brilliant Blue and Silver staining, and then decolorized to observe a band pattern.

As a result, a toxoid band was observed at a molecular weight of 58,500, which is the same size as the known diphtheria toxin molecular weight.

<Example 14> Detoxification test

In the present invention, a detoxification test was performed to confirm that the toxin tablet toxoid is returned to toxin.

That is, the detoxified sample was diluted with 0.017M phosphate-buffered sodium chloride solution to contain 200 Lf of toxoid in 1 mL and the same concentration as the final stock solution, and left at 37 ° C. for 20 days, followed by 300-400 g of guinea-pig 4 5 mL each was used for subcutaneous injection and observed for 30 days.

In the meantime, no toxin poisoning, necrosis, paralysis and other poisoning symptoms, significant weight loss, and other abnormalities were observed. Therefore, the detoxification method of the present invention using formalin and lysine is considered a suitable method.

<Example 15> Confirmation of antigenicity

Toxin confirmation of the toxin and detoxification of the toxin after detoxification of the toxin, an oxterlony double immunodifusion method (weakly referred to as 'I.D') was performed.

First, agarose (Sigma) was dissolved in 0.07M barbital buffer (pH 8.6) to 1% and dissolved in Petri dishes. Holes were punched at 2 cm intervals to add anti-toxin (WHO diphtheria antitoxin) 4 IU and diphtheria toxoid 2 Lf, and then placed in a humidity chamber for 36 hours at 37 ° C. Agar gel was stained with Coomassie brilliant blue and decolorized to observe sedimentation lines reacted with toxoid and diphtheria antitoxin.

As a result, both the toxin tablet toxoid and the toxoid tablet toxoid responded to diphtheria antitoxin, and a sedimentation line was observed at the same position (see FIG. 2).

<Example 16> Activity test

However, the diphtheria titer test was performed because it was not confirmed by I.D which of the two toxoids had better antigenicity.

After detoxifying zotoxin and purified toxoid after detoxification, the detoxified toxoid was adsorbed onto aluminum hydroxide gel adjuvant, and subcutaneously injected with 0.75 mL of 300-400 g of guinea-pig and immunized for 4 weeks. After immunization, the serum was collected, neutralized with standard toxin 1L + (green cross), and subcutaneously injected into 230-280 g of guinea-pig, 2 ml each time, and the survival was observed for 12 hours. The results are shown in Table 14 below. Indicated.

process Purity (Protein Nitrogen Lf / mg) Titer (IU / mL) return Toxoid Tablets Toxoid 2000 3 0 Toxin Tablet Toxoid 1560 3 0

In the case of the toxin tablet toxoid, it survived at 2,3 units, whereas the purified toxoid after detoxification of the toxin was survived at 2 units, but one of them died at 3 hours and 72 hours. . Therefore, the titer of the toxin tablet toxoid showed better results than the purified toxoid after detoxifying the toxin. That is, after detoxifying the toxin, the toxin was recovered from the toxoid through UF concentration, A / S treatment, and gel filtration. It was.

mix Dilution rate Observation time (dead pigs / experimental pigs) 24 48 72 96 120 Standard Antitoxin + Standard Toxin 1 IU / mL 0/2 2/2 Antitoxin + Standard Toxin of Toxin Tablet × 2 0/2 0/2 0/2 0/2 0/2 × 3 0/2 0/2 0/2 0/2 0/2 Antitoxin + Standard Toxin of Toxoid Tablet × 2 0/2 0/2 0/2 0/2 0/2 × 3 0/2 0/2 1/2 1/2 1/2

As described above, by culturing the diphtheria strain by the cultivation method of diphtheria according to the present invention, it is possible to prepare a high purity toxin in large quantities, and further, such a diphtheria toxin according to the present invention is a double U.F. And detoxification after purification with high purity through ion exchange chromatography, it is possible to produce a stable high purity toxoid.

Therefore, the method of the present invention can be usefully used for the preparation of high purity diphtheria vaccines and adult diphtheria vaccines with improved safety.

Claims (7)

a) culturing Corynebacterium diphtheria in a fermenter at a stirring rate of 200-500 rpm using a casein enzymatic product as a nitrogen source and adding a medium with iron ion concentration of 0.7 mg / L or less; b) a method for producing diphtheria toxin, which comprises recovering diphtheria toxin after removing the cells from the culture medium with a filter paper. The method according to claim 1, wherein the casein enzymatic product of step a) has an average molecular weight of at least 400 Daltons of amino acids. The method of claim 1, wherein the initial pH of the medium of step a) is in the range of 6.5-7.5. delete a) double ultrafiltration of the toxin produced by the diphtheria strain; b) purifying by performing ion exchange chromatography with filtered toxin; And c) a method for preparing detoxified diphtheria toxoid comprising detoxifying the purified toxin. 6. The method of claim 5, wherein the dual ultrafiltration of step a) uses a molecular weight cutoff of 100,000 modules and a molecular weight cutoff of 30.000 modules. 6. The method according to claim 5, wherein the detoxification of the toxin of step c) is followed by formalin treatment after lysine treatment.