Tetanus vaccine and application thereof

Abstract

The invention provides tetanus toxoid as well as a preparation method and application thereof. The flocculent purity of the tetanus toxoid provided by the invention is greater than 2000Lf / mg PN. The preparation method disclosed by the invention comprises the step of adding chromatography treatment between the step of refining the toxoid and the step of preparing the preparation, impurities in the system are further removed, the purity and monomer content of the finally produced refined tetanus toxoid stock solution after chromatography are obviously improved, and the adsorption tetanus vaccine prepared by using the refined tetanus toxoid stock solution has the advantages that the adsorption effect is good, and the production cost is low. In the preparation process, an aluminum adjuvant with the level within 1 micron and high uniformity is selected for preparation, and the obtained product has the advantages of good titer and better quality standard, and has a good application prospect.

Description

Technical Field

[0001] The present disclosure relates to the field of biopharmaceutical technology, and in particular, to a tetanus vaccine preparation and application thereof. Background Art

[0002] Tetanus toxin is a neurotoxin produced by Clostridium tetani under hypoxic or anaerobic conditions, which can cause spasmodic contraction of skeletal muscles, causing illness or even death. In 1923, Roman et al. treated tetanus toxin with formaldehyde to make it lose its immunoreactivity, thus producing a refined tetanus toxoid stock solution.

[0003] The 2020 edition of the "Chinese Pharmacopoeia" stipulates the inspection standards for refined tetanus toxoid concentrate, including pH value, flocculent unit, purity, sterility test, specific toxicity test, toxicity reversal test and other inspection contents.

[0004] In recent years, with the rapid development of vaccine production technology and industry, the allergenicity of vaccines has received widespread attention. Studies have shown that the increase in the content of impurities and tetanus toxoid polymers in vaccines may be an important cause of allergic reactions. The main reason for the formation of tetanus toxoid polymers is the addition reaction between formaldehyde and tetanus toxin in the detoxification process. Therefore, many vaccine manufacturers prefer to use refined tetanus toxoid stock solutions with higher purity and tetanus toxoid monomer content for research and vaccine production. How to control the content of impurities and tetanus toxoid polymers in refined tetanus toxoid stock solutions has received more and more attention, and the common problem of low purity and monomer component content in refined tetanus toxoid stock solutions needs to be solved urgently. Summary of the invention

[0005] In order to solve the above-mentioned problems existing in the prior art, the present disclosure provides a preparation method of an adsorbed tetanus vaccine, which is mainly based on the production of the original refined tetanus toxoid stock solution, and a chromatography treatment is added between the refined toxoid and the preparation process steps to further remove impurities in the system. The purity and monomer content of the refined tetanus toxoid stock solution finally produced after chromatography are significantly improved. In addition, the adsorbed tetanus vaccine prepared using the refined tetanus toxoid stock solution is prepared by using an aluminum adjuvant with a grade of less than 1 micron and a higher uniformity. The obtained product has the advantages of good potency and better quality standards, and has good application prospects.

[0006] In a first aspect, the present disclosure provides a tetanus toxoid, wherein the flocculent purity of the tetanus toxoid is greater than 2000 Lf / mg PN. The adsorbed tetanus vaccine containing the tetanus toxoid has high potency and better stability.

[0007] In some embodiments, the flocculent purity of the tetanus toxoid is greater than 2200 Lf / mg PN, more preferably greater than 2300 Lf / mg PN.

[0008] In some embodiments, the HPLC purity of the tetanus toxoid is greater than 70%, preferably greater than 85%, more preferably greater than 90%, and further preferably greater than 95%.

[0009] In some embodiments, the SDS-PAGE purity of the tetanus toxoid is greater than 80%, preferably greater than 85%, more preferably greater than 90%, and further preferably greater than 93%.

[0010] In a second aspect, the present disclosure provides a method for preparing the above-mentioned tetanus toxoid, which includes subjecting the crude tetanus toxoid solution after detoxifying the toxin to chromatography.

[0011] In some embodiments, the chromatography is gel filtration chromatography.

[0012] In some embodiments, the chromatography medium for the chromatography is selected from one or more of Superdex 200 and / or S-300 and / or S-200.

[0013] In some embodiments, the equilibration buffer and / or elution buffer for the chromatography is selected from PBS solutions, preferably the concentration of the PBS solution is 4-6 mM, and / or the pH is 7.0-7.2.

[0014] In some embodiments, the preparation method further includes the following steps before the chromatography:

[0015] (1) Inactivating the cells in the fermentation broth of Clostridium tetani with formaldehyde, followed by solid-liquid separation to obtain a filtrate containing tetanus toxin;

[0016] (2) After concentrating and ultrafiltering the filtrate containing tetanus toxin, salting out with ammonium sulfate, and then ultrafiltering to remove salt, a diluted stock solution of the toxin is obtained;

[0017] (3) Detoxifying the diluted stock solution of the toxin with formaldehyde to obtain a detoxified solution of tetanus toxin;

[0018] (4) Concentrating and ultrafiltering the detoxified solution of tetanus toxin to remove formaldehyde, and then performing sterile filtration to obtain a crude tetanus toxoid solution.

[0019] In some embodiments, in step (1), the conditions for cell inactivation include: the final concentration of formaldehyde is 0.2-0.4% (w / w), preferably 0.25-0.35% (w / w), and / or the time for cell inactivation is 0.5-2 h, preferably 45-75 min.

[0020] In some embodiments, in step (1), the solid-liquid separation is carried out by centrifugation. Preferably, it is carried out by at least one of large-capacity centrifugation, disc centrifugation or depth filtration.

[0021] In some embodiments, in step (2), the salting out is carried out in a two-stage precipitation manner; preferably, the conditions for the first-stage precipitation include: the final concentration of ammonium sulfate is 10%-18% (w / w), preferably 12%-18% (w / w); and / or the temperature < 15°C, the time is 20-60 min; and / or the centrifugation conditions are 6500-7000 rpm, 8-12°C, 30-50 min; preferably, the conditions for the second-stage precipitation include: the final concentration of ammonium sulfate is 20%-28% (w / w), preferably 24%-28% (w / w); and / or the temperature < 15°C, the time is 20-60 min; and / or the centrifugation conditions are 6500-7000 rpm, 8-12°C, 30-50 min.

[0022] In some embodiments, in step (2), the ultrafiltration membrane package used for the concentration ultrafiltration and / or the ultrafiltration desalting has a pore size of 30-70 KD, for example 50 KD.

[0023] In some embodiments, in step (2), the buffer solution for the concentration ultrafiltration and / or the ultrafiltration desalting is selected from PBS solutions. Preferably, the concentration of the PBS solution is 4-6 mM, and / or the pH is 7.0-7.2.

[0024] In some embodiments, in step (3), the toxin detoxification is carried out in the presence of L-lysine. Preferably, the conditions for the toxin detoxification include: the toxin concentration is 300-800 Lf / ml, preferably 400-600 Lf / ml; and / or the final concentration of formaldehyde is 0.050%-0.080% (w / w), preferably 0.060%-0.075%; and / or the final concentration of L-lysine is 0.02-0.05 mol / L, preferably 0.02-0.03 mol / L; and / or the buffer solution is selected from PBS solutions. Preferably, the concentration of the PBS solution is 4-6 mM, and / or the pH is 7.0-7.2; and / or the pH value for the toxin detoxification is 7.0-7.5, preferably 7.3-7.5; and / or the temperature for the toxin detoxification is 35-40°C, preferably 36-38°C; and / or the time is 21-40 days, preferably 28-32 days.

[0025] In some embodiments, in step (4), the ultrafiltration membrane package used for the concentration ultrafiltration has a pore size of 30-70 KD, for example 50 KD; and / or the buffer solution is selected from PBS solutions. Preferably, the concentration of the PBS solution is 4-6 mM, and / or the pH is 7.0-7.2.

[0026] In some embodiments, in step (4), the pore size of the filter element for sterilization filtration is 0.2 μm.

[0027] In some embodiments, the preparation method further includes the step of preparing a fermentation broth of Clostridium tetani.

[0028] In some embodiments, the preparation of the fermentation broth of Clostridium tetani includes the following specific steps:

[0029] Inoculating Clostridium tetani into a strain culture medium for strain culture to obtain a seed solution; and

[0030] Inoculating the seed solution into a fermentation medium for fermentation culture to obtain a fermentation broth of Clostridium tetani.

[0031] In some embodiments, the Clostridium tetani is purchased from the National Institutes for Food and Drug Control, China, and the strain number is: CMCC 64008.

[0032] In some embodiments, the strain culture conditions include: 33 - 37 °C, 44 - 52 h, culturing for 2 - 3 generations, preferably 34 - 36 °C, 47 - 49 h, culturing for 3 generations.

[0033] In some embodiments, the fermentation medium is a tetanus double peptone toxin - producing medium.

[0034] In some embodiments, the fermentation culture conditions include: 33 - 37 °C, preferably 33.5 - 36.5, not exceeding 144 h, preferably the culture time is 120 h; and / or the pH value is 7.0 ± 0.6. In some specific embodiments, intermittent stirring is performed every 2 h in the first 48 h of the fermentation culture, and every 4 h after 48 h.

[0035] In a third aspect, the present disclosure provides a vaccine composition, comprising the above - mentioned tetanus toxoid or the tetanus toxoid obtained by the above - mentioned preparation method, and an adjuvant.

[0036] In some embodiments, the particle size of the adjuvant is 1 μm or less, preferably 100 - 400 nm.

[0037] In some embodiments, the adjuvant is an aluminum adjuvant, preferably an aluminum hydroxide adjuvant.

[0038] In some embodiments, the tetanus vaccine composition further includes a sodium chloride solution.

[0039] In some embodiments, in each 1 mL of the vaccine composition, the content of the tetanus toxoid is 7-10 Lf, preferably 8-9 Lf.

[0040] In some embodiments, in each 1 mL of the vaccine composition, the content of the adjuvant is 1.0-1.5 mg, preferably 1.3-1.4 mg.

[0041] In some embodiments, in each 1 mL of the vaccine composition, the content of sodium chloride is 7.5-9.5 mg, preferably 8-9 mg.

[0042] In some embodiments, the pH value of the vaccine composition is 6.0-7.0, preferably 6.30-6.50.

[0043] In some embodiments, during the preparation of the vaccine composition, the selected adjuvant is an aluminum adjuvant with a particle size distribution within 1 micron, which has the typical advantages of smaller particles and more uniform distribution.

[0044] In some embodiments, during the preparation of the vaccine composition, the refined tetanus toxoid stock solution separated by chromatography is selected, which has the remarkable effect of higher purity and lower impurity content. By adding molecular sieve chromatography to further purify the refined tetanus toxoid after the detoxification process, the content of impurity proteins is reduced, the presence of dimers and polymers is decreased, and it is beneficial to improve the purity of the refined tetanus toxoid stock solution, making it more suitable for adsorbed tetanus vaccine.

[0045] In a fourth aspect, the present invention provides the use of the above-mentioned tetanus toxoid or the tetanus toxoid obtained by the above-mentioned preparation method or the above-mentioned vaccine composition in the preparation of a vaccine for preventing diseases caused by tetanus toxin.

[0046] On the basis of the traditional production of refined tetanus toxoid stock solution, the present invention adds molecular sieve chromatography suitable for protein purification to further purify the tetanus toxoid, so as to further improve the purity of the refined tetanus toxoid stock solution for vaccine preparation. And the design of this process step can further reduce the content of heterologous proteins in the detoxification system. The adsorbed tetanus vaccine prepared by this method has good immunogenicity, excellent safety, clearer quality standards and good economic benefits. At present, there are many manufacturers that can be selected for molecular sieve chromatography equipment and medium fillers, and the equipment can meet the GMP requirements. This method is suitable for commercial-scale production. In addition, the present invention selects an aluminum adjuvant with a particle size distribution at the micron level, which has the typical advantages of smaller particles and more uniform distribution, and the safety of the obtained product is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1Shows the Sephacryl S-300 chromatography profile of the purified tetanus toxoid prepared in Example 1.

[0048] Figure 2 Shows the SEC-HPLC purity inspection profile of the molecular sieve chromatography solution of the purified tetanus toxoid prepared in Example 1.

[0049] Figure 3 Shows the SDS-PAGE purity inspection profile of the molecular sieve chromatography solution of the purified tetanus toxoid prepared in Example 1.

[0050] Figure 4 Shows the Superdex 200 chromatography profile of the purified tetanus toxoid prepared in Example 2.

[0051] Figure 5 Shows the particle size distribution map of the adsorbed tetanus vaccine prepared in Example 1. Detailed implementation mode

[0052] To make the purpose, technical solution and advantages of the present disclosure clearer, the following further details the present disclosure in conjunction with embodiments. The specific embodiments described herein are only used to explain the present disclosure and do not constitute any limitation to the present disclosure. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0053] In some embodiments, the present invention relates to a preparation process of a tetanus vaccine, including the following steps:

[0054] (1) Inoculate Clostridium tetani into a strain culture medium and culture to obtain a seed solution;

[0055] (2) Inoculate the seed solution into a fermentation medium and carry out fermentation culture;

[0056] (3) Add formaldehyde to the harvested fermentation broth to inactivate the bacteria; after inactivation, carry out solid-liquid separation and harvest the supernatant;

[0057] (4) Use a membrane package to concentrate and ultrafilter the filtrate containing tetanus toxin, add ammonium sulfate to the filtrate for salting out to obtain purified tetanus toxin protein, and use a membrane package to ultrafilter and desalt the dissolved tetanus toxin;

[0058] (5) Dilute the toxin and add formaldehyde for detoxification, and add L-lysine protectant during the detoxification process;

[0059] (6) Concentrate and ultrafilter the detoxified tetanus toxin detoxification solution to remove formaldehyde, and then carry out sterile filtration to obtain the purified tetanus toxoid stock solution;

[0060] (7) Further purify the refined tetanus toxoid stock solution using chromatography technology to obtain a chromatographed refined toxoid stock solution with further improved purity.

[0061] (8) Dilute the chromatographed tetanus toxoid, add aluminum adjuvant and sodium chloride solution, and mix well to prepare adsorbed tetanus vaccine.

[0062] Among them, in step (1), Clostridium tetani is purchased from the National Institutes for Food and Drug Control, China, and the strain number is: CMCC 64008. The culture conditions of the strain are: 35 ± 1.0 °C, 44 - 52 h, culture for 2 - 3 generations, preferably 48 h, 3 generations.

[0063] Among them, in step (2), the fermentation medium is tetanus double peptone toxin production medium.

[0064] Among them, in step (2), the fermentation culture conditions are: 35 ± 1.5 °C, not exceeding 144 h, pH value at 7.0 ± 0.6, intermittent stirring every 2 h for the first 48 h, and intermittent stirring every 4 h after 48 h, preferably the culture time is 120 h.

[0065] Among them, in step (3), the formaldehyde inactivation conditions are: final concentration of formaldehyde: 0.30 ± 0.05% (w / w), stir and inactivate for 1 h.

[0066] Among them, in step (3), the bacterial liquid separation is carried out by means of large-capacity centrifugation and / or disc centrifugation and / or depth filtration.

[0067] Among them, in step (4), the pore size of the ultrafiltration membrane package is: 50KD, the fermentation broth concentration and ultrafiltration buffer is 5 mM PBS pH7.0 buffer, and the ultrafiltration desalting buffer is 5 mM PBS pH7.0 buffer.

[0068] In step (4), the salting-out conditions are: salting-out is carried out in two-stage precipitation. The salt is ammonium sulfate. First, precipitate and remove miscellaneous proteins at low salt concentration, and then add ammonium sulfate to precipitate and separate the target tetanus toxin; after the salting-out operation is completed, harvest the target precipitate, redissolve it with 5 mM PBS pH7.0 buffer, and then remove the residual ammonium sulfate by ultrafiltration and buffer exchange.

[0069] Among them, in step (4), the salting-out conditions are: the final concentration of the first-stage ammonium sulfate precipitation is 10% - 18% (w / w), the feed liquid is controlled below 15 °C, stirred for 30 min, and the centrifugation conditions are 6900 rpm, 10 ± 2 °C, 40 ± 1 min, preferably the ammonium sulfate concentration is 12 - 18% (w / w).

[0070] Among them, in step (4), the salting-out conditions are as follows: the final concentration of ammonium sulfate precipitation in the second stage is 20%-28% (w / w), the feed liquid is controlled below 15°C, stirred for 30 min, and the centrifugation conditions are 6900 rpm, 10±2°C, 40±1 min. The preferred ammonium sulfate concentration is 26±2% (w / w).

[0071] Among them, in step (5), the toxin detoxification conditions are as follows: 37±1.0°C, 21-40 days, with intermittent stirring; the detoxification system: the toxin concentration is 500±100 Lf / ml, the buffer solution is 0.005 mol / L PB buffer solution with pH 7.0-7.2, the final concentration of formaldehyde is 0.060-0.075% (w / w), add L-lysine solution until the final concentration of L-lysine is 0.025 mol / L, adjust the pH value of the feed liquid to 7.40±0.10 with glacial acetic acid. The preferred toxin detoxification time is 30 days, and the preferred toxin concentration is 500 Lf / ml. The preferred detoxifying agent concentration is 0.060%-0.075%.

[0072] Among them, in step (6), the pore size of the concentration ultrafiltration membrane package is: 50 KD, the ultrafiltration buffer solution is 5 mM PBS pH7.0 solution, and the pore size of the sterile filtration filter element is: 0.2 μm.

[0073] Among them, in step (7), the chromatography uses gel filtration chromatography. After the chromatography column is equilibrated with 5 mM PBS pH7.0 solution, the sample is loaded. The loading volume does not exceed 5% of the column volume. Elution is carried out with 5 mM PBS pH7.0 solution, and the monomeric tetanus toxoid components are collected in segments according to the elution sequence; the chromatography medium can be selected from one or several of Superdex 200 and / or S-300 and / or S-200. The chromatography medium selected based on the same principle also belongs to the protection scope of the present invention. The tetanus toxoid stock solution after chromatography typically has a purity inspection not less than 90%.

[0074] Among them, in step (8), the adsorbed tetanus vaccine formulation is as follows: each 1 ml of adsorbed vaccine contains 7-10 Lf of toxoid, the aluminum hydroxide content is 1.0-1.5 mg, the sodium chloride content is 7.5-9.5 mg, and the pH value is 6.0-7.0. The preferred toxin concentration is 8.5 Lf / ml, the aluminum hydroxide content is 1.35 mg / ml, the sodium chloride content is 8.5 mg / ml, and the pH value is 6.30-6.50.

[0075] The aluminum adjuvant used in the preparation process is specifically small particle aluminum adjuvant with a particle size in the nanometer level, and the preferred adjuvant particle size is 100-400 nm.

[0076] In some preferred embodiments, the preparation method includes the following steps:

[0077] (1) Inoculate the Clostridium tetani strain into the strain culture medium. Culture conditions: 35 ± 1.0 °C, 48 h, culture for 3 generations, static culture.

[0078] (2) Inoculate the strain culture solution into the fermentation medium. During the culture process, control the temperature at 35.0 ± 1.5 °C, and control the pH value of the fermentation process at 7.0 ± 0.6.

[0079] (3) After the fermentation culture is completed, add formaldehyde solution until the final concentration of formaldehyde is 0.30% (w / w). Stir and let stand, and separate the fermentation broth by disc centrifuge to obtain the bacterial liquid.

[0080] (4) Concentrate the fermentation broth. Add PBS buffer to the concentrated fermentation broth for equal-volume dialysis. Slowly add ammonium sulfate to the concentrated and ultrafiltered fermentation broth, stir, centrifuge the broth, collect the supernatant. Slowly add ammonium sulfate to the broth, stir, centrifuge the broth, collect the precipitate, dissolve it with PB buffer, and perform equal-volume dialysis.

[0081] (5) Dilute it with PB buffer according to the flocculation unit of the broth. Add formaldehyde solution, mix well, and incubate. Add L-lysine solution to the broth, adjust the pH value of the broth with glacial acetic acid, and start detoxification.

[0082] (6) Concentrate the original toxoid solution. Add pBS solution to the broth for equal-volume dialysis. Filter and store the refined tetanus toxoid original solution through a sterilizing filtration device.

[0083] (7) Load the refined original toxoid solution onto a gel filtration chromatography column with a suitable separation range, and further purify the refined tetanus toxoid original solution using the gel filtration chromatography technical scheme to obtain a chromatographed refined toxoid original solution with further improved purity.

[0084] (8) Weigh the aluminum hydroxide adjuvant, turn on the magnetic stirrer to stir the aluminum adjuvant solution, slowly add the chromatographed refined tetanus toxoid original solution to it, and then add sodium chloride solution.

[0085] In a further preferred embodiment, the preparation method includes the following steps:

[0086] (1) Inoculate the Clostridium tetani strain into the strain culture medium. Culture conditions: 35 ± 1.0 °C, 48 h, culture for 3 generations, static culture. Microscopic examination of the strain should be carried out for each generation. If contaminated with miscellaneous bacteria, it should be discarded.

[0087] (2) Inoculate the bacterial strain culture solution into the fermentation medium. The fermentation culture process is divided into two stages: the early stage (before 48 hours) and the middle and late stages (after 48 hours). During the culture process, control the temperature at 35.0 ± 1.5 °C. In the early stage of fermentation, stir for 1 minute every 2 hours at a stirring speed of 60 rpm. In the middle and late stages, stir for 2 minutes every 4 hours at a stirring speed of 60 rpm. Control the pH value during the fermentation process at 7.0 ± 0.6. Culture for 120 h. Regularly sample during the fermentation process for microscopic examination of the bacterial strain and detection of floc units. If contaminated with miscellaneous bacteria, it should be discarded;

[0088] (3) After the fermentation culture is completed, add formaldehyde solution until the final concentration of formaldehyde is 0.30% (w / w), stir at 60 rpm for 60 min, and separate the bacterial liquid from the fermentation broth by disc centrifuge;

[0089] (4) Concentrate and ultrafilter the fermentation broth using a 50KD membrane package, concentrate the fermentation broth about 20 times, add 5 mM PBS pH7.0 buffer solution for equal-volume dialysis of the feed liquid. Stop ultrafiltration when the cumulative permeation flow rate reaches 10 times the volume of the concentrated liquid. Slowly add ammonium sulfate to the feed liquid after concentration and ultrafiltration until the final concentration is 12% (w / w), stir for 30 min, control the feed liquid below 15 °C, stir for 30 min, centrifuge the feed liquid. Centrifugation conditions: 4000 rpm, 10 ± 2 °C, 40 min. Collect the supernatant. Slowly add ammonium sulfate to the feed liquid until the final concentration is 22% (w / w), stir for 30 min, control the feed liquid below 15 °C, stir for 30 min, centrifuge the feed liquid. Centrifugation conditions: 4000 rpm, 10 ± 2 °C, 40 min. Collect the precipitate and dissolve it with 5 mM PBS pH7.0 buffer solution, and perform equal-volume dialysis using a 50KD membrane package. Stop ultrafiltration when the cumulative permeation flow rate reaches 10 times the volume of the concentrated liquid;

[0090] (5) According to the floc units of the feed liquid, dilute it to 500 Lf / ml with 5 mM PBS pH7.0 buffer solution, add formaldehyde solution until the final concentration of formaldehyde is 0.06% (w / w), mix well, and incubate for 30 min. Add 1M L-lysine solution to the feed liquid to make its final concentration 0.025 mol / L, adjust the pH value of the feed liquid to 7.40 ± 0.10 with glacial acetic acid, and start detoxification. The detoxification temperature is 37 °C ± 1.0 °C, and the detoxification time is 30 days. Stir intermittently during the detoxification process;

[0091] (6) Concentrate and ultrafilter the original toxin solution using a 50KD membrane package and perform equal-volume dialysis. Stop ultrafiltration when the cumulative permeation flow rate reaches 10 times the volume of the concentrated liquid. Filter the refined tetanus toxoid original solution through a sterilized 0.2 μm bacteria-removing filter device and store it. The storage period at 2 - 8 °C does not exceed 76 months;

[0092] (7) After the chromatography column was equilibrated with 5 mM PBS pH 7.0 buffer for 3 column volumes, sample loading was started. The sample loading volume did not exceed 5% of the column volume. It was eluted with 5 mM PBS pH 7.0 buffer for 2 column volumes: collection started when UV280 was greater than 5 mAu, and fractions were collected when the inflection point of the elution peak appeared. A total of four elution peaks, PEAK I, PEAK II, PEAK III, and PEAK IV, were collected, corresponding to four states of polymers, oligomers, monomers, and degradation components respectively;

[0093] (8) Weigh aluminum hydroxide adjuvant, turn on the magnetic stirrer to stir the nano-aluminum adjuvant solution, slowly add the refined tetanus toxoid stock solution (the monomeric toxoid stock solution corresponding to PEAK III) to it, and then add 8.5% sodium chloride solution. Make up to the following with water for injection: each 1 ml of adsorbed vaccine contains 8.5 Lf of toxoid, 1.35 mg of aluminum hydroxide, and 8.5 mg of sodium chloride. Adjust the solution pH to 6.50. Stir for a total of 30 min during the whole process, and the tetanus vaccine adsorption was successful.

[0094] Examples and drawings are provided below to help understand the present disclosure. It should be understood that these examples and drawings are only for illustrating the present disclosure, but do not constitute any limitation. The actual protection scope of the present disclosure is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present disclosure.

[0095] Example 1

[0096] 1. Preparation of refined tetanus toxoid stock solution

[0097] (1) The Clostridium tetani strain (purchased from the National Institutes for Food and Drug Control, strain number: CMCC64008) was inoculated into the strain culture medium. The culture conditions were: 35 ± 1.0 °C, 48 h, cultured for 3 generations, static culture, and the strain was microscopically examined for each generation. If contaminated with miscellaneous bacteria, it was discarded to obtain the strain culture solution.

[0098] (2) The strain culture solution was inoculated into the fermentation medium (tetanus double peptone toxin-producing medium). The fermentation culture process was divided into two stages: the early stage (before 48 hours) and the middle and late stages (after 48 hours). The temperature was controlled at 35.0 ± 1.5 °C during the culture process. In the early stage of fermentation, it was stirred for 1 minute every 2 hours, and the stirring speed was 60 rpm. In the middle and late stages, it was stirred for 2 minutes every 4 hours, and the stirring speed was 60 rpm. The pH value during the fermentation process was controlled at 7.0 ± 0.6, and cultured for 120 h. Samples were taken regularly during the fermentation process for strain microscopic examination and flocculation unit detection. If contaminated with miscellaneous bacteria, it should be discarded.

[0099] (3) After the fermentation culture was completed, formaldehyde solution was added to a final formaldehyde concentration of 0.30% (w / w), stirred at 60 rpm for 60 min. The fermentation broth was separated by disc centrifugation.

[0100] (4) The filtrate after disc centrifugation is concentrated and ultrafiltered using a 50KD membrane package (PALL). The filtrate is concentrated about 20 times, and 5mmol / L pH7.0 PBS buffer is added to perform an equal-volume liquid exchange on the feed liquid. Ultrafiltration is stopped when the cumulative permeation flux reaches 10 times the volume of the concentrated liquid. Ammonium sulfate is slowly added to the feed liquid after concentration and ultrafiltration to a final concentration of 12.5% (w / w) for the first-stage salting-out. Stir for 30 min, control the feed liquid below 15°C, and stir for 30 min. Centrifuge the feed liquid under the following conditions: 6900 rpm, 10±2°C, 40 min. Collect the supernatant and slowly add ammonium sulfate to the feed liquid to a final concentration of 22%

[0101] (w / w) for the second-stage salting-out. Stir for 30 min, control the feed liquid below 15°C, and stir for 30 min. Centrifuge the feed liquid under the following conditions: 6900 rpm, 10±2°C, 40 min. Collect the precipitate and dissolve it with 5mmol / L pH7.0 PBS buffer. Perform equal-volume dialysis using a 50KD membrane package. Ultrafiltration is stopped when the cumulative permeation flux reaches 10 times the volume of the concentrated liquid.

[0102] (5) According to the flocculation unit of the feed liquid, add 5mM PBS pH7.0 buffer to dilute it to 500Lf / ml, add formaldehyde solution until the final concentration of formaldehyde is 0.06% (w / w), mix well, and incubate for 30 min. Add 1M L-lysine solution to the feed liquid to a final concentration of 0.025mol / L, adjust the pH value of the feed liquid to 7.40±0.10 with glacial acetic acid, and start detoxification. The detoxification temperature is 37°C±1.0°C, and the detoxification time is 30 days. Stir intermittently during the detoxification process; obtain the toxoid stock solution.

[0103] (6) The toxoid stock solution is concentrated and ultrafiltered using a 50KD membrane package to remove formaldehyde. The filtrate is concentrated about 20 times, and 5mmol / L pH7.0 PBS buffer is added to perform an equal-volume dialysis on the feed liquid. Ultrafiltration is stopped when the cumulative permeation flux reaches 10 times the volume of the concentrated liquid. Filter the refined tetanus toxoid stock solution through a sterilized 0.2μm bacteria-removing filter device and store it. The storage period at 2-8°C shall not exceed 76 months;

[0104] (7) The chromatography column medium is packed with Sephacryl S-300 packing material. After the packed chromatography column is equilibrated with 5 mM PBS pH 7.0 buffer for 3 column volumes, sample loading is started. The sample loading volume does not exceed 5% of the column volume. Elution is carried out with 5 mM PBS pH 7.0 buffer for 2 column volumes: collection starts when UV280 is greater than 5 mAu, and fractions are collected separately when the inflection point of the elution peak appears. A total of four elution peaks, PEAK i, PEAK ii, PEAK iii, and PEAK iv, are collected, corresponding to four states of polymer, oligomer, monomer, and degradation components respectively. The chromatography collection pattern is as shown in Figure 1 shown, and the purity inspection results are as shown in Figure 2 , Figure 3 shown;

[0105] 2. Preparation of adsorbed tetanus vaccine

[0106] Weigh aluminum hydroxide adjuvant with a particle size distribution of less than 1 μm. Turn on the magnetic stirrer to stir the nano-aluminum adjuvant solution, slowly add the purified tetanus toxoid stock solution (the monomer toxoid stock solution corresponding to PEAK iii) to it, and then add 8.5% (w / w) sodium chloride solution. Make up to: each 1 mL of adsorbed vaccine contains 8.5 Lf of toxoid, 1.35 mg of aluminum hydroxide, and 8.5 mg of sodium chloride. Adjust the solution pH to 6.50. Stir for a total of 30 min during the whole process, and the adsorption of tetanus vaccine is successful.

[0107] Example 2

[0108] Compared with Example 1 in step (7), the chromatography medium used is Supedex 200, and other conditions are the same as those in Example 1 and will not be elaborated here. The chromatography collection pattern is as shown in Figure 4 shown.

[0109] Example 3

[0110] Compared with Example 1, the aluminum adjuvant used for adsorption is 10-μm grade micro-aluminum adjuvant, and other conditions are the same as those in Example 1 and will not be elaborated here.

[0111] Example 4

[0112] Compared with Example 1, the purified toxoid without chromatography (i.e., without performing step (7)) is used to prepare the vaccine, and other conditions are the same as those in Example 1 and will not be elaborated here.

[0113] Example 5

[0114] Compared with Example 1, the purified toxoid without chromatography (i.e., without performing step (7)) is used to prepare the vaccine, and the aluminum adjuvant used for adsorption is 10-μm grade micro-aluminum adjuvant, and other conditions are the same as those in Example 1 and will not be elaborated here.

[0115] Example 6 - Purity Inspection of Chromatographic Collection Solution

[0116] The refined tetanus toxoid components of different polymerization degrees obtained by chromatographic separation were respectively subjected to flocculation purity, HPLC purity and SDS - PAGE purity inspections to analyze the activity / purity characteristics of each separated component:

[0117] 1. Purity Inspection Method A:

[0118] According to General Rule 3506 of the Third Part of the Chinese Pharmacopoeia, the flocculation concentration of each component was determined; the protein nitrogen content of each collected component was detected by the Kjeldahl method. The flocculation purity was determined according to the detection results:

[0119] Flocculation purity = flocculation value (Lf / ml) / protein nitrogen content (mg / ml) = purity value (Lf / mg PN).

[0120] 2. Purification Inspection Method B:

[0121] The purity analysis of each separated component obtained by harvesting was carried out by SEC - HPLC (TSK - G3000SWxl). The experimental steps are as follows:

[0122] Chromatographic column preparation: The chromatographic column was fully equilibrated with the mobile phase 5 mM PBS (pH 7.0). After the baseline was stable, the equilibrium reached the standard.

[0123] Preparation of test sample: Take 2 mL of each separated component sample obtained by harvesting, filter it with a 0.45 μm filter membrane, and set aside.

[0124] Operating parameter setting: Set the data acquisition time to 50 min, the sample loading volume to 100 μL, the UV detection wavelength parameter to 280 nm, the column oven temperature to 25 °C, and the flow rate to 0.6 mL / min to detect the test sample.

[0125] Data processing: The area percentage was statistically analyzed by the area normalization method to calculate the purity.

[0126] 3. Purity Inspection Method C:

[0127] Inspection was carried out according to the fifth method of General Rule 0541 of the Third Part of the Chinese Pharmacopoeia. SDS polyacrylamide gel electrophoresis was used for determination, and the sample loading volume was about 10 μg. Generally, after adjusting the protein concentration, protein loading buffer (6×) was added, and the protein amount was 0.1 - 30 μg / 5 - 30 μL.

[0128] Take an appropriate amount of the adjusted protein sample, add it to one - fifth volume of protein loading buffer (6×), and mix well.

[0129] Boil for 5 - 10 minutes, and boil the Marker for 3 - 5 minutes before use.

[0130] Loading: Fix the electrophoresis gel in the electrophoresis tank, add the electrophoresis buffer, and take an appropriate amount of sample for loading.

[0131] Electrophoresis: Cover the electrophoresis tank and perform electrophoresis at 60 - 100 V for the stacking gel and 100 - 150 V for the separating gel.

[0132] Staining: Remove the gel and make marks. Wash the gel with purified water for 30 minutes, changing the water every 10 minutes, on a shaker. Discard the water, add an appropriate amount of blue staining solution, and shake on the shaker for 2 hours - overnight for staining.

[0133] Decolorization: Discard the staining solution, add an appropriate amount of purified water, and shake for decolorization until the background color is lighter or colorless.

[0134] Result analysis: Scan the electrophoresis gel with a scanner and use software to analyze the percentage of the target band in the whole band to calculate the purity.

[0135] The purity analysis of each collected peak in chromatography is shown in Table 1 below.

[0136] Table 1. Purity analysis of each collected peak in chromatography

[0137]

[0138]

[0139] As can be seen from Table 1 above, there are significant differences in the purity of the components treated by the chromatography process. The sequentially harvested components are polymers, oligomers, monomers, and degradation components. Among the three - dimensional purity inspections, the component with the best purity is the TT - type toxin stock solution chromatography collected fraction - peak iii. Based on this, the TT - type toxin stock solution chromatography collected fraction - peak iii is defined as the refined tetanus toxoid stock solution after chromatography.

[0140] Example 7 - Purity inspection of consecutive production batches

[0141] Perform flocculent purity, HPLC purity, and SDS - PAGE purity inspections on the refined tetanus toxoid stock solutions after chromatography of consecutive batches, and analyze the activity / purity characteristics of each separated component:

[0142] 1. Purity inspection method A:

[0143] According to General Rule 3506 of the Third Edition of the Chinese Pharmacopoeia, determine the flocculent concentration of the refined tetanus toxoid stock solutions after chromatography of each batch; use the Kjeldahl method to detect the protein nitrogen content of each collected component. Determine the flocculent purity according to the test results:

[0144] Flocculent purity = Flocculent value (Lf / ml) / Protein nitrogen content (mg / ml) = Purity value (Lf / mg PN).

[0145] The purity standard should be > 1500 Lf / mg PN.

[0146] 2. Purification inspection method B:

[0147] For the purified tetanus toxoid stock solution after chromatography of the harvested batch, SEC-HPLC (TSK-G3000SWxl) is used for purity analysis. The experimental steps are as follows:

[0148] Column preparation: The column is fully equilibrated with the mobile phase 5 mM PBS (PH 7.0). After the baseline is stable, the equilibrium reaches the standard.

[0149] Preparation of test sample: Take 2 ml of the purified tetanus toxoid stock solution sample after chromatography of the harvested batch, filter it with a 0.45 μm filter membrane, and set aside.

[0150] Setting of operating parameters: Set the data acquisition time to 50 min, the sample injection volume to 100 μl, the UV detection wavelength parameter to 280 nm, the column oven temperature to 25 °C, and the flow rate to 0.6 ml / min to detect the test sample.

[0151] Data processing: Area normalization method is used for area percentage statistics to calculate the purity. The purity standard should be > 90%. 3. Purity inspection method C:

[0152] Examine according to the fifth method of General Rule 0541 in Part III of the Chinese Pharmacopoeia, and determine by SDS polyacrylamide gel electrophoresis. The sample injection volume is about 10 μg. Generally, after adjusting the protein concentration, add protein loading buffer (6×), and the protein amount is 0.1 - 30 μg / 5 - 30 μl.

[0153] Take an appropriate amount of the adjusted protein sample, add it to one-fifth volume of protein loading buffer (6×), and mix well.

[0154] Boil in boiling water for 5 - 10 minutes, and boil the Marker in boiling water for 3 - 5 minutes before use.

[0155] Sample injection: Fix the electrophoresis gel in the electrophoresis tank, add electrophoresis buffer, and take an appropriate amount of the sample for injection.

[0156] Electrophoresis: Cover the electrophoresis tank and perform electrophoresis. The stacking gel is at 60 - 100 V, and the separating gel is at 100 - 150 V.

[0157] Staining: Remove the gel and make marks. Wash the gel with purified water for 30 minutes, change the water every 10 minutes, and perform on a shaker. Discard the water, add an appropriate amount of blue staining solution, and shake on a shaker for staining for 2 hours - overnight.

[0158] Decoloration: Discard the staining solution, add an appropriate amount of purified water, and shake to decolorize until the background color is light or colorless.

[0159] Result analysis: Scan the electrophoresis gel with a scanner, and use software to analyze the percentage of the target band in the whole band to calculate the purity. The purity standard should be > 90%.

[0160] The purity analysis of the refined tetanus toxoid stock solution after continuous batch chromatography is shown in Table 2 below.

[0161] Table 2. Purity analysis of the refined tetanus toxoid stock solution after continuous batch chromatography

[0162]

[0163] It can be seen from Table 2 above that the purity of the refined tetanus toxoid stock solution obtained after treatment with the chromatography process has been significantly improved, the preparation process is stable, continuous and stable production can be achieved, and it has the feasibility of popularization for production applications.

[0164] Example 8 - Particle size uniformity verification of the finished product

[0165] For the adsorbed tetanus vaccine prepared by the present invention, the particle size of the vaccine finished product is detected using a Malvern 3000 nano particle size analyzer. The detection steps are as follows:

[0166] Turn on the power switch, and the instrument performs self - inspection. The instrument status indicator light flashes alternately red and green. Preheat for 20 - 30 minutes. Open the software DTS (Nano) on the computer, and the instrument indicator light turns green.

[0167] Check the sample cell, which should be intact and without damage. Hold the sample cell with the mouth downwards and blow it with compressed air for 3 seconds to remove any possible residual dust.

[0168] Prepare the adsorbed tetanus vaccine to be tested and mix it well. Use a disposable syringe to draw the test sample and slowly inject it into the sample cell. The liquid level height is about 10 mm - 15 mm. Note that there should be no bubbles on the wall of the sample cell. If there are bubbles, gently tap with your finger to make the bubbles escape, or use a disposable syringe to suck out the sample and inject it again.

[0169] Place the front of the sample cell (with an inverted triangle shape) forward into the detection slot, cover the lid, click the file option in the software, create a new file and save it. Click measurement type, and select nano particle size (size). Set and measure according to the Standard Operating Procedure of Nano ZS Nano Particle Size Analyzer, and measure each sample 3 times.

[0170] After the detection is completed, the software automatically generates a report, and save and print the relevant data.

[0171] In the printed result report, when the item "Result Quality" is "Good", it indicates that the test result is valid. Record the average particle size value (Z-Average) of each measurement, and calculate the average of the three measurement values as the final result.

[0172] The finished product prepared with the small particle size adjuvant according to the present invention has a particle size distribution as Figure 5 shown. The results show that the finished product prepared with the adjuvant within 1 micron according to the present invention has the advantages of a more uniform particle size distribution and a smaller absolute value of the particle size distribution. It is expected to avoid adverse reactions caused by large particle substances, and the product quality is better.

[0173] Results of the animal experiment of adsorbed tetanus vaccine in Example 9

[0174] The results of the animal experiment of 5 batches of adsorbed tetanus vaccines prepared in Examples 1-5 of the present invention are shown in Table 3 below. The immunogenicity was detected by the method of General Principles 3504 in the Chinese Pharmacopoeia 2020 Edition, and the qualified quality standard is not less than 80 IU / ml.

[0175] Table 3. Tetanus potency of adsorbed tetanus vaccine

[0176]

[0177] It can be seen from Table 3 above that the potency of the adsorbed tetanus vaccine prepared with the chromatographically purified tetanus toxoid stock solution prepared by the chromatography process and the aluminum adjuvant within 1 micron with a more uniform particle size distribution is not inferior to that of the vaccine prepared with the traditional large particle aluminum adjuvant and / or purified toxoid, and is at a relatively high level (greater than 80 IU / mL indicates that the effectiveness of the vaccine can meet the quality standard).

[0178] For the muscle stimulation animal experiment of 5 batches of adsorbed tetanus vaccines prepared in Examples 1-5 of the present invention, the stimulation experimental protocol is shown in Table 4 below, and the results are shown in Table 5.

[0179] Table 4. Muscle stimulation experimental protocol of adsorbed tetanus vaccine

[0180]

[0181] Table 5 Muscle stimulation experimental results of adsorbed tetanus vaccine

[0182] Sample batch number Observation results after 3 days Observation results after 10 days Negative control No irritation reaction No irritation reaction Example 5 Mild irritation reaction No irritation reaction Example 4 No irritation reaction No irritation reaction Example 3 Mild irritation reaction No irritation reaction Example 2 No irritation reaction No irritation reaction Example 1 No irritation reaction No irritation reaction

[0183] As can be seen from Table 5 above, the results of muscle stimulation of adsorbed tetanus vaccine prepared with the refined tetanus toxoid stock solution prepared by chromatography process and aluminum adjuvant with a more uniform particle size distribution within 1 micron show that the product prepared with an adjuvant with a more uniform particle size distribution has less irritation and better safety.

[0184] The technical solutions of the present disclosure are not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solutions of the present disclosure falls within the protection scope of the present disclosure.

Claims

1. A tetanus toxoid, wherein the flocculent purity of the tetanus toxoid is greater than 2000 Lf / mg PN, preferably greater than 2200 Lf / mg PN, more preferably greater than 2300 Lf / mg PN.

2. The tetanus toxoid according to claim 1, characterized in that, The HPLC purity of the tetanus toxoid is greater than 70%, preferably greater than 85%, more preferably greater than 90%, and further preferably greater than 95%; and / or The SDS-PAGE purity of the tetanus toxoid is greater than 80%, preferably greater than 85%, more preferably greater than 90%, and further preferably greater than 93%.

3. A method for preparing the tetanus toxoid according to claim 1 or 2, comprising subjecting the stock solution of the tetanus toxoid after detoxification of the toxin to chromatography.

4. The preparation method according to claim 3, characterized in that, The chromatography is gel filtration chromatography. Preferably, the chromatography medium for the chromatography is selected from one or more of Superdex 200 and / or S-300 and / or S-200. Preferably, the equilibration solution and / or elution solution for the chromatography is selected from PBS solutions, and preferably the concentration of the PBS solution is 4 - 6 mM, and / or the pH is 7.0 - 7.

2.

5. The preparation method according to claim 3, characterized in that, The preparation method further comprises the following steps before the chromatography: (1) Inactivating the bacteria in the fermentation broth of Clostridium tetani with formaldehyde, followed by solid-liquid separation to obtain a filtrate containing tetanus toxin. (2) After concentrating and ultrafiltering the filtrate containing tetanus toxin, salting out with ammonium sulfate, and then ultrafiltering for desalting to obtain a diluted feed solution of the toxin. (3) Detoxifying the diluted feed solution of the toxin with formaldehyde to obtain a detoxified solution of tetanus toxin. (4) Concentrating and ultrafiltering the detoxified solution of tetanus toxin to remove formaldehyde, and then performing sterile filtration to obtain the stock solution of the tetanus toxoid.

6. According to the preparation method of claim 5, wherein In step (1), the conditions for bacteria inactivation include: the final concentration of formaldehyde is 0.2 - 0.4% (w / w), preferably 0.25 - 0.35% (w / w), and / or the time for bacteria inactivation is 0.5 - 2 h, preferably 45 - 75 min; and / or In step (1), the solid-liquid separation is performed by centrifugation, and preferably, it is performed by at least one of large-capacity centrifugation, disc centrifugation or depth filtration; and / or In step (2), the salting out is performed by two-stage precipitation; preferably, the conditions for the first-stage precipitation include: the final concentration of ammonium sulfate is 10% - 18% (w / w), preferably 12 - 18% (w / w); and / or the temperature < 15°C, the time is 20 - 60 min; and / or the centrifugation conditions are 6500 - 7000 rpm, 8 - 12°C, 30 - 50 min; preferably, the conditions for the second-stage precipitation include: the final concentration of ammonium sulfate is 20% - 28% (w / w), preferably 24 - 28% (w / w); and / or the temperature < 15°C, the time is 20 - 60 min; and / or the centrifugation conditions are 6500 - 7000 rpm, 8 - 12°C, 30 - 50 min; and / or In step (2), the ultrafiltration membrane package used for the concentration ultrafiltration and / or the ultrafiltration desalting has a pore size of 30 - 70KD, and / or In step (2), the buffer solution for the concentration ultrafiltration and / or the ultrafiltration desalting is selected from PBS solution, preferably the concentration of the PBS solution is 4 - 6 mM, and / or the pH is 7.0 - 7.2; and / or In step (3), the toxin detoxification is carried out in the presence of L-lysine. Preferably, the conditions for the toxin detoxification include: the toxin concentration is 300 - 800 Lf / ml, preferably 400 - 600 Lf / ml; and / or the final concentration of formaldehyde is 0.050 - 0.080% (w / w), preferably 0.060% - 0.075%; and / or the final concentration of L-lysine is 0.02 - 0.05 mol / L, preferably 0.02 - 0.03 mol / L; and / or the buffer solution is selected from PBS solution, preferably the concentration of the PBS solution is 4 - 6 mM, and / or the pH is 7.0 - 7.2; and / or the pH value of the toxin detoxification is 7.0 - 7.5, preferably 7.3 - 7.5; and / or the temperature of the toxin detoxification is 35 - 40 °C, preferably 36 - 38 °C; and / or the time is 21 - 40 days, preferably 28 - 32 days; and / or In step (4), the ultrafiltration membrane package used for the concentration ultrafiltration has a pore size of 30 - 70KD; and / or the buffer solution is selected from PBS solution, preferably the concentration of the PBS solution is 4 - 6 mM, and / or the pH is 7.0 - 7.2; and / or In step (4), the pore size of the filter element for the sterile filtration is 0.2 μm.

7. A vaccine composition, comprising the tetanus toxoid according to claim 1 or 2 or the tetanus toxoid obtained by the preparation method according to any one of claims 3 - 6, and an adjuvant.

8. The vaccine composition according to claim 7, characterized in that, The particle size of the adjuvant is less than 1 μm, preferably 100 - 400 nm; and / or The adjuvant is an aluminum adjuvant, preferably an aluminum hydroxide adjuvant.

9. The vaccine composition according to claim 7 or 8, characterized in that, The tetanus vaccine composition further comprises a sodium chloride solution, Preferably, in each 1 mL of the vaccine composition, the content of the tetanus toxoid is 7 - 10 Lf, preferably 8 - 9 Lf; Preferably, in each 1 mL of the vaccine composition, the content of the adjuvant is 1.0 - 1.5 mg, preferably 1.3 - 1.4 mg; Preferably, in each 1 mL of the vaccine composition, the content of the sodium chloride is 7.5 - 9.5 mg, preferably 8 - 9 mg; Preferably, the pH value of the vaccine composition is 6.0 - 7.0, preferably 6.30 - 6.

50.

10. Use of the tetanus toxoid according to claim 1 or 2 or the tetanus toxoid obtained by the preparation method according to any one of claims 3 - 6 or the vaccine composition according to any one of claims 7 - 9 in the preparation of a vaccine for preventing a disease caused by tetanus toxin.

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