Method for refining tetanus toxoid

Abstract

The invention discloses a tetanus toxoid refining method which comprises the following steps: S1, centrifuging tetanus fermentation harvest liquid by adopting a butterfly centrifuge to remove thalli, and then performing ultrafiltration concentration to obtain a concentrated solution; s2, treating the concentrated solution obtained in the step S1 with a D390 ion exchange chromatographic column to obtain a chromatographic solution; s3, treating the chromatographic solution obtained in the step S2 by virtue of a Superdex 200 ion exchange chromatographic column, eluting by virtue of a sodium carbonate buffer solution, and collecting an elution peak, namely target protein, so as to obtain an eluent; and S4, performing formaldehyde detoxification treatment on the eluent obtained in the step S3, performing ultrafiltration to remove formaldehyde, and finally performing sterilization through a filter membrane to obtain the toxoid stock solution. In the refining process, the loss of the target protein is less, and the prepared toxoid is relatively excellent in immune effect and relatively high in purity.

Description

Technical Field

[0001] The present application relates to the technical field of vaccine preparation, and in particular to a method for refining tetanus toxoid. Background Art

[0002] Tetanus is an acute disease with a high mortality rate caused by infection with toxin-producing Clostridium tetani. Vaccination with vaccines containing tetanus toxoid is currently the most economical and effective way to prevent tetanus, and tetanus toxoid vaccines have been included in the routine immunization program for children in all countries around the world. Tetanus toxoid is usually a toxin produced by the cultivation of toxin-producing Clostridium tetani, which is made after formaldehyde detoxification. It can not only be used to prepare tetanus toxoid vaccines, but also as a carrier protein to prepare polysaccharide protein conjugate vaccines such as Haemophilus influenzae type b conjugate vaccine, meningococcal conjugate vaccine, and pneumococcal conjugate vaccine.

[0003] The existing tetanus vaccine is prepared by first fermenting and culturing the toxin-producing Clostridium tetani, and then detoxifying and purifying the toxin for preparing the vaccine through purification. The most widely used purification process is the ammonium sulfate salting-out process. This process first removes the bacteria by centrifugation or plate and frame filtration, then concentrates the toxin stock solution, purifies the protein by two stages of ammonium sulfate salting-out, removes the ammonium sulfate by ultrafiltration, and finally detoxifies to obtain the toxoid.

[0004] For example, the Chinese invention patent with publication number CN102961741A and invention name "A method for preparing a tetanus toxoid vaccine" discloses a method for refining tetanus toxoid using two salting-out steps. Although the protein yield of this method is relatively high, the purity of the toxoid obtained is insufficient, and the consumption of ammonium sulfate during preparation is relatively high. Summary of the invention

[0005] In order to solve at least one of the above technical problems, a refining process is developed in which the target protein loss is less during the refining process, the obtained toxoid has a better immune effect and a higher purity. The present application provides a method for refining tetanus toxoid.

[0006] The present application provides a method for refining tetanus toxoid, comprising the following steps: S1, centrifuging the tetanus fermentation harvest liquid to remove the bacteria by butterfly centrifuge, and then concentrating it by ultrafiltration to obtain a concentrated liquid; S2, treating the concentrated solution obtained in step S1 through a D390 ion exchange chromatography column to obtain a chromatographic solution; S3, treating the chromatographic solution obtained in step S2 with a Superdex 200 ion exchange chromatography column, eluting with a sodium carbonate buffer, collecting the elution peak as the target protein, and obtaining an eluate; S4. The eluate obtained in step S3 is subjected to formaldehyde detoxification treatment, and then formaldehyde is removed by ultrafiltration, and finally sterilized by a filter membrane to obtain a toxoid stock solution.

[0007] By adopting the above technical scheme, the present application adopts a completely salting-free tetanus toxoid refining process, with relatively less protein loss and relatively high protein recovery rate; the refining method of the present application adopts the steps of first removing impurities by D390 ion exchange chromatography column treatment and then purifying the target protein by Superdex 200 ion exchange chromatography column treatment, which can greatly remove impurities and obtain a toxoid with high purity; the process steps of the present application are relatively simple, the refining process is relatively mild, the damage to the target protein is small, the quality of the toxoid obtained is high, and the immune effect is relatively excellent.

[0008] Optionally, in step S1, the bacteria are removed by centrifugation at 4°C, 10,000-12,000g, and for more than 30 minutes.

[0009] Optionally, in step S1, ultrafiltration and concentration are performed to concentrate the solution to 1 / 12 to 1 / 8 of the original volume.

[0010] Optionally, in step S2, D390 ion exchange chromatography column treatment is performed at a flow rate of 15-20 mL / min.

[0011] Optionally, in step S3, the chromatographic fluid needs to be filtered through a 0.45 μm filter membrane before being treated with the Superdex 200 ion exchange chromatography column.

[0012] Optionally, in step S3, Superdex 200 ion exchange chromatography column treatment is performed at a flow rate of 2.5-5 mL / min.

[0013] Optionally, in step S3, the pH of the sodium carbonate buffer is 9.5-10, and the concentration of sodium carbonate is 20-25 mM.

[0014] Optionally, in step S4, the final concentration of formaldehyde after detoxification treatment is controlled at 0.1-0.25%.

[0015] Optionally, in steps S1 and S4, ultrafiltration uses a 25~35KD ultrafiltration membrane.

[0016] Optionally, in step S4, a 0.25 μm filter membrane is used for filter membrane sterilization.

[0017] In summary, the present invention includes at least one of the following beneficial technical effects: 1. This application adopts a completely salt-free tetanus toxoid refining process, with relatively less protein loss and relatively high protein recovery rate.

[0018] 2. The purification method of the present application adopts the steps of first removing impurities by D390 ion exchange chromatography column treatment and then purifying the target protein by Superdex200 ion exchange chromatography column treatment, which can greatly remove impurities and the obtained toxoid has a higher purity.

[0019] 3. The process steps of this application are relatively simple, the refining process is relatively mild, the damage to the target protein is small, the toxoid obtained is of high quality, and the immune effect is relatively excellent. DETAILED DESCRIPTION

[0020] The present application is further described in detail below in conjunction with embodiments.

[0021] The present application designs a method for refining tetanus toxoid, comprising the following steps: S1, centrifuging the tetanus fermentation harvest liquid to remove the bacteria by butterfly centrifuge, and then concentrating it by ultrafiltration to obtain a concentrated liquid; S2, treating the concentrated solution obtained in step S1 through a D390 ion exchange chromatography column to obtain a chromatographic solution; S3, treating the chromatographic solution obtained in step S2 with a Superdex 200 ion exchange chromatography column, eluting with a sodium carbonate buffer, collecting the elution peak as the target protein, and obtaining an eluate; S4. The eluate obtained in step S3 is subjected to formaldehyde detoxification treatment, and then formaldehyde is removed by ultrafiltration, and finally sterilized by a filter membrane to obtain a toxoid stock solution.

[0022] The main differences between the present application and the prior art are as follows. First, the present application does not adopt the traditional salting-out process, nor does it adopt the process combining salting-out and ion exchange chromatography, but adopts the process combining filtration and ion exchange chromatography. Secondly, the present application adopts a specific ion exchange chromatography step design.

[0023] The following are preparation examples and embodiments of the present application.

[0024] The following are examples of the present application: Example 1 The method for refining tetanus toxoid of the present embodiment comprises the following steps: S1. The tetanus fermentation harvest liquid was centrifuged at 8500g for 45 min at room temperature using a butterfly centrifuge, the supernatant was taken, and then concentrated to 1 / 6 of the original volume by 30KD ultrafiltration to obtain a concentrate; S2. The concentrated solution obtained in step S1 is treated with a D390 ion exchange chromatography column at a flow rate of 5 mL / min to obtain a chromatographic solution; S3, the chromatographic solution obtained in step S2 was treated with a Superdex 200 ion exchange chromatography column at a flow rate of 5 mL / min; then eluted with a sodium carbonate buffer with a pH of 8.5, and the elution peak was collected as the target protein to obtain an eluate; S4. The eluate obtained in step S3 is detoxified with formaldehyde at a final concentration of 0.1%, then the formaldehyde is removed by 30KD ultrafiltration, and finally sterilized by a 0.22μm filter membrane to obtain a toxoid stock solution.

[0025] Example 2 The difference between this embodiment and embodiment 1 is that in step S1, the centrifugation is performed at 4° C., 10,000 g, and for 30 min.

[0026] Example 3 The difference between this embodiment and embodiment 1 is that in step S1, the centrifugation is performed at 4° C., 12000 g, and for 30 min.

[0027] Example 4 The difference between this embodiment and embodiment 2 is that in step S1, ultrafiltration and concentration are performed to 1 / 12 of the original volume.

[0028] Example 5 The difference between this embodiment and embodiment 2 is that in step S1, ultrafiltration and concentration are performed to 1 / 8 of the original volume.

[0029] Example 6 The difference between this embodiment and embodiment 2 is that in step S1, ultrafiltration and concentration are performed to 1 / 10 of the original volume.

[0030] Example 7 The difference between this embodiment and embodiment 6 is that in step S2, D390 ion exchange chromatography column treatment is performed at a flow rate of 15 mL / min; in step S3, Superdex 200 ion exchange chromatography column treatment is performed at a flow rate of 2.5 mL / min.

[0031] Example 8 The difference between this embodiment and embodiment 7 is that in step S3, the chromatographic solution needs to be filtered through a 0.45 μm filter membrane before being treated with the Superdex 200 ion exchange chromatography column.

[0032] Example 9 The difference between this embodiment and embodiment 8 is that in step S3, the pH of the sodium carbonate buffer is 10 and the concentration of sodium carbonate is 20 mM.

[0033] Example 10 The difference between this embodiment and embodiment 9 is that in step S4, a 0.25 μm filter membrane is used for filter membrane sterilization.

[0034] Comparative Example 1 Example 1 with publication number CN114805504A and invention name "A method for preparing oligomeric tetanus toxoid" is adopted as comparative example 1 of the present application.

[0035] Comparative Example 2 Example 4 with publication number CN108003225A and invention name "A method for refining tetanus toxoid" was adopted as comparative example 2 of the present application.

[0036] Detection The fermentation harvest liquid with a known target toxin protein content of 102 Lf / ml was taken as the refining object, and the refining methods of Examples 1 to 10 and Comparative Examples 1 to 2 were used to purify it.

[0037] 1) Purity The HPLC method described in the Chinese Pharmacopoeia was used to detect the purity of the toxin-like monomers after purification in Examples 1 to 10 of the present application and Comparative Examples 1 to 2.

[0038] 2) Protein recovery rate The HPLC method recorded in the Chinese Pharmacopoeia was used to detect the content of toxin protein in the tetanus fermentation harvest liquid. The protein recovery rate was calculated according to protein recovery rate = protein monomer content after purification / protein content before purification × 100%.

[0039] The results are shown in Table 1 below.

[0040] Table 1 Test results of Examples 1 to 10 and Comparative Examples Monomer purity (%) Protein recovery rate (%) Example 1 96.8 56.4 Example 2 97.1 57.6 Example 3 97.2 57.3 Example 4 97.1 57.5 Example 5 97.3 57.7 Example 6 97.4 58.3 Example 7 97.6 58.9 Example 8 97.9 59.1 Example 9 97.9 59.2 Example 10 98.0 59.3 Comparative Example 1 92.2 49.8 Comparative Example 2 97.4 41.7 It can be seen from the data in Table 1 that the purification methods of Examples 1 to 10 of the present application, compared with the salting-out exchange chromatography method of Comparative Example 1 and the single exchange chromatography method of Comparative Example 2, have no significant difference in the results of toxicity identification, and the toxicity of the toxin culture is very close. It can be seen from this that the method of the present application, through a specific step design, can effectively remove impurities, and the purity of the obtained monomer protein is extremely high, with a purity of more than 96%. In addition, the protein recovery rate of Examples 1 to 10 of the present application is close to 60%, far exceeding Comparative Examples 1 and 2.

[0041] From the data comparison of Examples 1 to 10 in Table 1, it can be seen that after further optimization of the process parameters of the present application, the purity of the toxin and the protein recovery rate can be further improved.

[0042] Animal experiments The toxoid stock solutions of Examples 1 to 10 of the present application and Comparative Examples 1 to 2 were prepared into vaccines with a concentration of 700 Lf / ml.

[0043] Twelve groups of mice were taken, with 6 mice in each group; the vaccines prepared in Examples 1 to 10 of the present application and Comparative Examples 1 to 2 were used for immunization injection, respectively. Each mouse was injected once every 5 days, for a total of 3 injections.

[0044] After the third injection, 7 days later, one mouse was randomly selected from each group, its tail was cut off and blood was taken, and the antibody level of the mouse was detected by ELISA.

[0045] The results are shown in Table 2 below.

[0046] Table 2 Immunoassay results of Examples 1 to 10 and Comparative Examples Antibody level (GMU) Example 1 94.24 Example 2 98.69 Example 3 96.42 Example 4 95.81 Example 5 101.47 Example 6 102.63 Example 7 97.92 Example 8 92.18 Example 9 104.46 Example 10 102.12 Comparative Example 1 48.92 About Billy 2 55.26 It can be seen from the data in Table 2 that the toxoid vaccines prepared in Examples 1 to 10 of the present application have extremely excellent immunogenicity, and the immune effect is far superior to the toxoid vaccines of Comparative Examples 1 and 2. This fully demonstrates that the purification method designed in the present application has minimal damage to the target protein and can effectively ensure the quality of the target protein.

[0047] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for refining tetanus toxoid, characterized in that: The following steps are involved: S1, centrifuging the tetanus fermentation harvest liquid to remove the bacteria by butterfly centrifuge, and then concentrating it by ultrafiltration to obtain a concentrated liquid; S2, treating the concentrated solution obtained in step S1 through a D390 ion exchange chromatography column to obtain a chromatographic solution; S3, treating the chromatographic solution obtained in step S2 with a Superdex 200 ion exchange chromatography column, eluting with a sodium carbonate buffer, collecting the elution peak as the target protein, and obtaining an eluate; S4. The eluate obtained in step S3 is subjected to formaldehyde detoxification treatment, and then formaldehyde is removed by ultrafiltration, and finally sterilized by a filter membrane to obtain a toxoid stock solution.

2. The method for refining tetanus toxoid according to claim 1, characterized in that: In step S1, the bacteria are removed by centrifugation at 4°C, 10,000-12,000 g, and for more than 30 min.

3. The method for refining tetanus toxoid according to claim 1, characterized in that: In the step S1, ultrafiltration and concentration are performed to 1 / 12 to 1 / 8 of the original volume.

4. The method for refining tetanus toxoid according to claim 1, characterized in that: In the step S2, the D390 ion exchange chromatography column is used for treatment at a flow rate of 15-20 mL / min.

5. The method for refining tetanus toxoid according to claim 1, characterized in that: In step S3, the chromatographic solution needs to be filtered through a 0.45 μm filter membrane before being treated with the Superdex 200 ion exchange chromatography column.

6. The method for purifying tetanus toxoid according to claim 1, characterized in that: In the step S3, the Superdex 200 ion exchange chromatography column is used for treatment at a flow rate of 2.5-5 mL / min.

7. The method for purifying tetanus toxoid according to claim 1, characterized in that: In step S3, the pH of the sodium carbonate buffer is 9.5-10, and the concentration of sodium carbonate is 20-25 mM.

8. The method for purifying tetanus toxoid according to claim 1, characterized in that: In step S4, the final concentration of formaldehyde after detoxification treatment is controlled at 0.1-0.25%.

9. The method for purifying tetanus toxoid according to claim 1, characterized in that: In the steps S1 and S4, the ultrafiltration uses a 25-35KD ultrafiltration membrane.

10. The method for purifying tetanus toxoid according to claim 1, characterized in that: In step S4, a 0.25 μm filter membrane is used for filter membrane sterilization.