Hepatitis b vaccine and method of production thereof

Abstract

The present disclosure relates to a method for inducing expression of hepatitis B surface antigen, a method for purifying hepatitis B surface antigen, a method for producing hepatitis B vaccine and hepatitis B vaccine. The method for inducing expression of hepatitis B surface antigen: the yeast expressing hepatitis B surface antigen is subjected to three-stage fermentation to OD 600 100-200, methanol is added, expression of hepatitis B surface antigen is induced, and fermentation product is harvested. The method for purifying hepatitis B surface antigen comprises: primary purification of the fermentation product by PEG sedimentation and silica gel adsorption to obtain crude purified liquid with less impurities, further purification by anion exchange and gel filtration chromatography to obtain antigen extract, and in-situ adsorption by potassium aluminum sulfate solution to obtain hepatitis B vaccine with high in-vitro potency. The method for inducing expression of hepatitis B surface antigen provided by the present disclosure can improve the yield of hepatitis B surface antigen; the method for purifying hepatitis B antigen improves the recovery rate of antigen, shortens the production time, and is conducive to process scale-up production.

Description

Technical Field

[0001] This disclosure belongs to the field of biopharmaceutical technology, specifically relating to a method for inducing hepatitis B surface antigen expression, a method for purifying hepatitis B surface antigen, a method for producing hepatitis B vaccine, and a hepatitis B vaccine. Background Technology

[0002] Hepatitis B virus (HBV) is a DNA virus with a genome of 3.2KB double-stranded DNA molecule. It can transmit the virus through blood or other bodily fluids. HBV can survive outside the body for at least 7 days. During this time, if HBV enters an unvaccinated individual, that individual is likely to become infected. The incubation period for HBV is 30 to 180 days.

[0003] HBV is a hepatotropic virus that primarily resides within liver cells and damages them, causing inflammation, necrosis, and fibrosis, ultimately leading to hepatitis B (commonly known as HBV infection). HBV can be detected within 30 to 60 days of infection and can persist, potentially developing into chronic hepatitis B, especially if contracted in infancy or childhood. Chronic hepatitis B may progress to cirrhosis and even liver cancer.

[0004] The World Health Organization (WHO) clearly states that hepatitis B vaccination is the primary method of preventing hepatitis B and recommends that infants be vaccinated against hepatitis B as soon as possible after birth (ideally within 24 hours). Recombinant yeast hepatitis B vaccine is a subunit vaccine for hepatitis B surface antigen (HBsAg). It is produced by constructing a plasmid containing the gene expressing the hepatitis B surface antigen using a transgenic method, transferring it into yeast to create recombinant yeast, and then culturing this recombinant yeast to express the hepatitis B surface antigen subunit.

[0005] In May 2022, the 75th World Health Assembly adopted the 2030 Agenda for Sustainable Development, which set the goal of eliminating hepatitis globally. Therefore, producing a high-yield, low-cost recombinant hepatitis B vaccine is an important way to achieve this goal. Summary of the Invention

[0006] To address the aforementioned technical problems, this disclosure provides a method for inducing hepatitis B surface antigen expression, a method for purifying hepatitis B surface antigen, a method for producing a hepatitis B vaccine, and a hepatitis B vaccine.

[0007] In a first aspect, this disclosure provides a method for inducing the expression of hepatitis B surface antigen, the method comprising:

[0008] Yeast expressing hepatitis B surface antigen was inoculated into the first-stage culture medium at a ratio of 2.0–6.0% for primary fermentation to obtain the first-stage culture medium;

[0009] Take the first-stage culture medium and inoculate it into the second-stage culture medium at a ratio of 2.0-6.0% for secondary fermentation to obtain the second-stage culture medium;

[0010] Take the second-stage culture medium and inoculate it into the third-stage culture medium at a ratio of 1.5–3.0% for tertiary fermentation. When the bacterial density in the fermentation broth reaches OD... 600 The value is 100-200. Methanol is added initially to induce the expression of hepatitis B surface antigen, and the fermentation product is harvested.

[0011] In some implementations, the temperature of the primary fermentation is 33–35°C.

[0012] In some embodiments, the temperature of the secondary fermentation is 33-35°C, preferably 33°C.

[0013] In some embodiments, the temperature of the tertiary fermentation is 30–35°C, preferably 33–35°C, and more preferably 33°C.

[0014] In some implementations, the OD 600 It ranges from 130 to 170.

[0015] In some implementation schemes, methanol (inducer) is added in a continuous constant-flow manner.

[0016] In some implementation schemes, during the induction culture process, the amount of methanol added varies with the OD of the fermentation broth. 600 The value and the amount of fermentation broth increased proportionally.

[0017] In some implementation schemes, methanol is added at a rate of 1.0-2.0 mL / 100 OD during the induction culture process. 600 / L fermentation broth / hour (i.e., if the fermentation broth OD) 600 The concentration is 100, and 1.0-2.0 ml of methanol is continuously added to the fermentation broth per hour; if the OD... 600 The concentration is 170, and 1.7-3.4 ml of methanol is continuously added per liter of fermentation broth per hour, meaning that the amount of methanol added increases proportionally with the increase of the OD value and the volume of fermentation broth.

[0018] In some implementations, the pH of the third-level culture medium is controlled to be 4.0–6.0 during induction.

[0019] In some implementations, the yeast is selected from Hansenula polymorpha, Pichia pastoris, and brewer's yeast.

[0020] In some implementations, the amino acid sequence of the hepatitis B surface antigen is shown in SEQ ID No:1:

[0021] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGS TTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI.

[0022] In some implementations, the HBsAg gene is ligated into the pMD19-T vector (Takara Bio Inc.) to obtain the plasmid pHBsAg, and the plasmid pHBsAg is introduced into Hansenula polymorpha to obtain an engineered strain of Hansenula polymorpha hepatitis B.

[0023] Secondly, this disclosure provides a method for purifying hepatitis B surface antigen, the method comprising:

[0024] 1) The fermentation product of the first aspect is disrupted by cell disruption, and the first supernatant is collected after centrifugation;

[0025] 2) Add polyethylene glycol to the first supernatant, let it stand to precipitate, centrifuge, and collect the second supernatant;

[0026] 3) Add silica gel to the second supernatant to adsorb hepatitis B surface antigen in the second supernatant, and collect the silica gel precipitate adsorbed with hepatitis B surface antigen by centrifugation;

[0027] 4) Add desorption buffer to the silica gel precipitate adsorbed with hepatitis B surface antigen to desorb the hepatitis B surface antigen from the silica gel and obtain a third supernatant containing hepatitis B surface antigen.

[0028] 5) The third supernatant was subjected to first ultrafiltration, ion exchange chromatography, second ultrafiltration and gel filtration chromatography in sequence to obtain the hepatitis B surface antigen stock solution.

[0029] In some implementations, in step 2), the pH of the first supernatant is first adjusted to 5.0–8.0, and then polyethylene glycol is added; and / or

[0030] The polyethylene glycol added in step 2) is selected from polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, or polyethylene glycol 10000, preferably polyethylene glycol 6000 or polyethylene glycol 8000; and / or

[0031] In step 2), the final concentration of polyethylene glycol is 3% to 6%, preferably 4% to 5%.

[0032] In some embodiments, in step 3), the silica gel is added in the form of fumed silica, with a final concentration of 0.5% to 2.0%.

[0033] In some embodiments, in step 3), the adsorption conditions are 2–8°C for 12–20 h.

[0034] In some embodiments, in step 4), the temperature for releasing the silica gel adsorption is 50-62°C, preferably 52-62°C.

[0035] In some embodiments, the desorption buffer contains 0.8-3.4 g / L of sodium deoxycholate, preferably 2-3 g / L.

[0036] In some embodiments, in step 5), the first ultrafiltration uses an ultrafiltration membrane with a cutoff pore size of 50-1000 kDa, more preferably an ultrafiltration membrane of 300-500 kDa.

[0037] In some implementations, in step 5), the ion exchange chromatography is anion exchange chromatography.

[0038] In some embodiments, the chromatography medium used in the ion exchange chromatography is selected from DEAE Sepharose Fast Flow and MoSphere. TM IEX-50Q, MoSphere TM IEX-50DEAE, GP DEAE-60, or DEAE sephardexA-50, preferably MoSphere TM IEX-50DEAE or DEAE Sepharose Fast Flow.

[0039] In some embodiments, the equilibration solution used for the ion exchange chromatography contains 0.05–0.20 mol / L sodium chloride, preferably a borate buffer, phosphate buffer, or Tris-HCl buffer with a sodium chloride content of 0.1–0.15 mol / L, with phosphate buffer being the most preferred.

[0040] In some embodiments, the concentration of the buffer salt in the equilibration solution is 10-100 mM, more preferably 10-60 mM.

[0041] In some embodiments, the eluent used in the ion exchange chromatography contains 0.1 to 0.55 mol / L sodium chloride, preferably a borate buffer, phosphate buffer, or Tris-HCl buffer with a sodium chloride content of 0.25 to 0.55 mol / L, with phosphate buffer being the most preferred.

[0042] In some embodiments, the concentration of the buffer salt in the eluent is 10-100 mM, more preferably 10-60 mM.

[0043] In some embodiments, the eluent used in the ion exchange chromatography has a pH of 6.0-8.0.

[0044] In some embodiments, in step 5), the second ultrafiltration uses an ultrafiltration membrane with a cutoff pore size of 50-1000 kDa, more preferably an ultrafiltration membrane of 300-500 kDa.

[0045] In some embodiments, in step 5), the chromatography medium used for gel filtration chromatography is selected from Sephacryl S-300HR, Sephacryl S-400HR, Sephacryl S-500HR, Sepharose 4FastFlow, Sepharose 6FastFlow, or Sepharose 2B, preferably Sephacryl S-500HR or Sepharose 4FastFlow.

[0046] In some embodiments, the eluent used for the gel filtration chromatography is selected from 10–100 mM, preferably 10–60 mM, of borate buffer, phosphate buffer, or Tris-HCl buffer.

[0047] In some embodiments, the eluent used in the gel filtration chromatography has a pH of 6.0-8.0.

[0048] In some implementations, the antigen is extracted by homogenizing Hansenula polymorpha cells under low temperature and high pressure or by cryogenic ball milling. The antigen extract is then purified using a stepwise sedimentation method. First, impurities are precipitated in a neutral environment with 0.3–0.6 mol / L salt solution and 3%–6% PEG. After centrifugation, the supernatant is collected. Then, 0.5%–2.0% fumed silica is added to the supernatant to adsorb the antigen. After collecting the silica precipitate, the antigen is purified using a solution containing 0.8–3.4 g / L sodium deoxycholate. Antigen desorption is performed on the precipitate, and the supernatant solution after centrifugation is collected. The crudely purified antigen raw material solution is concentrated by ultrafiltration using an ultrafiltration membrane with a cutoff pore size of 50-1000 kDa. The ultrafiltration solution is then purified by ion exchange chromatography using borate buffer, phosphate buffer, or Tris-HCl buffer at a pH of 6.0-8.0 to obtain chromatography solution 1. Chromatography solution 1 is then subjected to another ultrafiltration and further purified by gel filtration chromatography using 20-100 mM phosphate buffer (pH 7.0) to obtain a purified solution. The purified solution is then subjected to in-situ adsorption using potassium aluminum sulfate solution at pH 3.0-4.0 to obtain the aluminum adsorption product, which is then diluted with vaccine diluent to prepare a semi-finished product. The method for producing hepatitis B vaccine disclosed in this paper can significantly reduce product contamination rate, improve antigen recovery rate, accelerate industrialization process, reduce manual labor intensity, save equipment investment and maintenance costs, shorten production time, and realize fully closed pipeline production.

[0049] Thirdly, this disclosure provides a method for producing a hepatitis B vaccine, the method comprising:

[0050] The hepatitis B surface antigen stock solution is prepared by the method described in the second aspect, and the hepatitis B surface antigen stock solution is treated with formaldehyde to obtain hepatitis B surface antigen formaldehyde-treated solution.

[0051] Under stirring conditions, a potassium aluminum sulfate solution with a pH of 3.0–4.2 and an aluminum ion concentration of 0.12–0.21 mol / L is added to the hepatitis B surface antigen formaldehyde treatment solution. Then, the pH is adjusted to 6.0–8.0, preferably 6.5–7.5, using a 0.5–2.0 mol / L sodium hydroxide solution to obtain the hepatitis B aluminum adsorption product, which is then diluted with vaccine diluent to prepare a hepatitis B vaccine semi-finished product.

[0052] In some implementation schemes, the concentration of hepatitis B surface antigen in the formaldehyde-treated solution is 260-340 μg / mL.

[0053] In some embodiments, the stirring speed is 100–700 rpm, preferably 400–600 rpm.

[0054] In some embodiments, the pH of the potassium aluminum sulfate solution is 3.5–4.0, the concentration of aluminum ions is 0.14–0.18 mol / L, and the concentration of the sodium hydroxide solution is 1.0–1.5 mol / L.

[0055] In some embodiments, the pH of the potassium aluminum sulfate solution is 3.0 to 4.2, preferably 3.5 to 4.0.

[0056] Fourthly, this disclosure provides a hepatitis B vaccine prepared by the method of the third aspect.

[0057] In some implementations, the hepatitis B surface antigen content in the hepatitis B vaccine is 20-24 μg / mL.

[0058] In some embodiments, the aluminum ion content in the hepatitis B vaccine is 0.35-0.60 mg / mL.

[0059] In some implementations, the pH of the hepatitis B vaccine semi-finished product is 6.5 to 7.5.

[0060] The method for producing hepatitis B vaccine disclosed herein can increase the yield of hepatitis B surface antigen through three-stage fermentation. Furthermore, the production process is easy to scale up and industrialize, shortening production time and facilitating large-scale production of hepatitis B vaccine. Moreover, this method for producing hepatitis B vaccine significantly reduces the production costs of both antigen and vaccine.

[0061] This disclosure utilizes a three-stage fermentation process to obtain Hansenula polymorpha cells expressing HBsAg, rapidly and efficiently obtaining HBsAg-containing Hansenula polymorpha. The yeast cells are collected via hollow fiber, and a large number of impurities in the antigen extract are removed in the crude purification step using stepwise sedimentation (PEG sedimentation) and specific adsorption (silica gel adsorption process). The refining step utilizes two-step ultrafiltration to concentrate the solution, removing impurities and reducing purification time. The antigen extract is further purified using two-step chromatography (anion exchange and gel filtration chromatography), ultimately yielding a high-potency hepatitis B vaccine in vitro. The operation is convenient, quick, easily reproducible, and easily scaled up for production, reducing production time and costs. Attached Figure Description

[0062] Figure 1 The growth curve of Hansenula yeast cultured in the second stage of fermentation in Example 1 is shown.

[0063] Figure 2 The chromatogram of ion exchange chromatography of hepatitis B surface antigen in Example 7 is shown.

[0064] Figure 3 The chromatogram of hepatitis B surface antigen obtained by gel filtration chromatography in Example 8 is shown. Detailed Implementation

[0065] The present disclosure will be further illustrated below by way of examples. It should be understood that the examples of the present disclosure are only for illustration and not for limitation. Simple improvements to the present disclosure under the concept of the present disclosure are within the scope of protection claimed by the present disclosure.

[0066] definition

[0067] Unless otherwise defined, all terms used herein (including technical and scientific terms) shall have the meanings commonly understood by those skilled in the art. It should be noted that the terms used herein shall be interpreted in a manner consistent with the context of this specification and shall not be interpreted in an idealized or overly rigid manner.

[0068] The term "about" as used herein is as understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is unfamiliar with the use of this term in the context in which it is used, "about" will mean a particular value plus or minus 10%.

[0069] As used herein, the term "vaccine" is intended to include both preventative and therapeutic vaccines. A preventative vaccine is given to stimulate an immune response to an antigen, so that if an individual is subsequently exposed to the antigen, the pre-existing immunity will protect the individual from the corresponding disease associated with said antigen. A therapeutic vaccine is given to an individual who already has an antigen-related disease, wherein said vaccine can elicit an immune response to the antigen or enhance the individual's existing immunity to said antigen to treat and / or improve disease symptoms.

[0070] The crude purification process route for hepatitis B antigen purification (i.e., vaccine stock solution preparation) is as follows: three-stage fermentation of bacterial strain → cell disruption → centrifugation clarification → polyethylene glycol precipitation and silica gel adsorption → silica gel desorption → first ultrafiltration → ion exchange chromatography → second ultrafiltration → gel filtration chromatography → sterile filtration.

[0071] The main reagents used in the examples include YNB (manufacturer: BD), methanol (manufacturer: Xilong Chemical), soybean peptone (manufacturer: SOLABIA), polyethylene glycol (manufacturer: Aladdin or SIGMA), silica gel (manufacturer: Degussa or Aladdin), sodium deoxycholate (manufacturer: Solarbio, SIGMA), borate buffer (manufacturer: Sinopharm or SIGMA), phosphate buffer (manufacturer: Sinopharm or SIGMA), and potassium aluminum sulfate (manufacturer: Sinopharm or Merck).

[0072] The main equipment used in the examples includes a bacterial fermenter (manufacturer: Shanghai Baoxing Biotechnology, model: Biotech-3000), a pH meter (manufacturer: Shanghai Precision Scientific Instruments, model: PHS-3C), a high-pressure homogenizer (manufacturer: ATS, model: AH-PILOT), a large-capacity high-speed centrifuge (manufacturer: Backman, model: J-26XP), an ELISA reader (manufacturer: Bio-Rad, model: Model 680), and a protein purification system (manufacturer: GE, model: PURIFIER).

[0073] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0074] Example

[0075] Example 1. Fermentation culture of Hansenula polymorpha.

[0076] This invention employs a three-stage fermentation process for Hansenula polymorpha cultivation, using working seed culture of Hansenula polymorpha, and controlling different OD values ​​before induction during the three-stage fermentation stage. 600 .

[0077] The first-stage fermentation culture was conducted in 500ml shake flasks with a culture volume of 20ml and an inoculum size of 2.0%. The culture was carried out at 33.0℃ and 250rpm until the OD reached [value missing]. 600 The primary fermentation was carried out in three parallel batches, named high, medium and low, respectively, and then secondary fermentation was carried out.

[0078] The second-stage fermentation was carried out in 2L shake flasks with a culture volume of 800ml and an inoculum size of 2.0%. The culture was conducted at 35.0℃ under 200rpm conditions until OD reached [the desired fermentation temperature]. 600 The values ​​are 13, 12.5, and 11.5 respectively. The secondary fermentation bar chart is shown below. Figure 1 As shown, after secondary fermentation, the high, medium, and low levels were each divided into three groups for tertiary fermentation, and named 1-high, 2-high, 3-high, 1-medium, 2-medium, 3-medium, 1-low, 2-low, and 3-low, respectively.

[0079] In the third stage of fermentation, a 60L fermenter was used for the initial induction culture, with a culture volume of 15L, an inoculum size of 1.5%, and a pH of 5. The culture was carried out at 35℃ until the fermentation broth reached its OD value. 600 The concentrations were 208, 220, 240, 142, 160, 148, 90, 80, and 88, respectively. Methanol was added continuously at a constant flow rate to induce induction, maintaining a stable methanol addition rate of 2.0 mL / 100 OD throughout the induction process. 600The fermentation broth was kept at a constant rate of / L fermentation broth / hour. During induction, the pH of the fermentation broth was adjusted to 5.5, and the induction was carried out at 33℃ for 72 hours. After induction, 9 samples were obtained as shown in Table 1, and the OD values ​​after induction were recorded. 600 Values ​​were determined. After each sample was lysed, the hepatitis B surface antigen content and protein content were measured, and the specific activity was calculated. The test results are shown in Table 1.

[0080] Table 1 Comparison of fermentation induction results at different cell densities

[0081]

[0082] In Example 1, the fermentation process of *Hansenula polymorpha* induced by different cell densities was continuously tested nine times, and the experimental groups with different density levels were compared. The results showed that when the cell density was between 130-170 for induction (samples 1-medium, 2-medium, and 3-medium), the average antigen expression level was 380 μg / ml, which was much higher than that of low-density induction (samples 1-low, 2-low, and 3-low). When the induction density was higher than 200 (samples 1-high, 2-high, and 3-high), although the total protein content increased, the antigen content did not increase effectively. Compared with the decrease in expression when the cell density was between 130-170, this indicates that induction at a cell density higher than 200 cannot increase the hepatitis B surface antigen content, and the content of impurities in the expression product increases, increasing the difficulty of separation and purification, especially increasing the burden on subsequent purification processes. Therefore, high-density induction of OD... 600 The optimal value range is 130-170. Meanwhile, the test results for samples 1-, 2-, and 3- show that the initial induction batches are consistent under these conditions, indicating that the Hansenula yeast fermentation process is stable and controllable. Compared with general processes, it ensures a higher cell density, thereby increasing the yield of hepatitis B antigen and allowing for large-scale production of hepatitis B vaccine (Hansenula yeast).

[0083] Samples (1-medium, 2-medium, 3-medium) obtained when the bacterial cell density was between 130 and 170 at the initial induction stage were subjected to subsequent separation and purification processes.

[0084] Example 2. Cell disruption and centrifugation clarification process

[0085] Low-temperature high-pressure homogenization was used to lyse the cells. The yeast cells were suspended in 50mM phosphate buffer (pH=7) until there were no clumps in the bacterial culture. Then, the cells were lysed by a high-pressure homogenizer. The temperature during the lysing process was maintained at 10℃ and the lysing pressure was 1350 bar. The homogenization was repeated 3 times. After high-speed centrifugation, the supernatant was collected to obtain the antigen extract. The results of this process were repeated 3 times and are shown in Table 2.

[0086] Table 2 Results of Hansenula polymorpha cell disruption and centrifugation clarification

[0087]

[0088] In Example 2, the cell debris removal process was subjected to three consecutive pilot-scale cell disruption and centrifugation tests. The results showed that the average cell disruption rate was 89.6% and the average cell debris removal rate was 96.8%, which was consistent between batches. This indicates that the cell disruption and centrifugation process is stable and controllable and can be scaled up for large-scale production of hepatitis B vaccine (Hansenula polymorpha).

[0089] Example 3. Precipitation of polyethylene glycol (PEG)

[0090] To adjust the pH of the antigen extract to 8.0, 0.2 mol / L NaOH solution was added dropwise. Then, NaCl solution was added to adjust the final salt ion concentration to 0.3 mol / L. Polyethylene glycol 6000 was added to a final concentration of 4%. After stirring until the polyethylene glycol 6000 was evenly mixed, the mixture was allowed to stand at 2°C for 12 hours to precipitate. The precipitate was removed by high-speed centrifugation, and the supernatant was collected. The antigen recovery rate and protein removal rate in the supernatant were compared. This process was repeated three times, and the results are shown in Table 3.

[0091] Table 3. PEG precipitation results

[0092]

[0093] In Example 3, polyethylene glycol 6000 was chosen as the optimal PEG precipitation process. When conditions such as pH and ion concentration are fixed, the larger the protein molecular weight, the lower the concentration of PEG used for precipitation, resulting in lower requirements for process equipment and reduced costs. Furthermore, PEG6000 is non-toxic and non-irritating, widely used in various pharmaceutical formulations, and exhibits good selectivity for protein precipitation. Three batches of experiments were conducted, showing an average antigen recovery rate of 62.0% and an average total protein removal rate of 45.3%, consistent across batches. This indicates that the PEG precipitation process is stable and controllable, and can be scaled up for large-scale production of hepatitis B vaccine (Hansenula polymorpha).

[0094] Example 4. Silica gel adsorption

[0095] Under strong stirring conditions, fumed silica AEROSIL255 was slowly added to the supernatant after centrifugation of PEG precipitation until the final concentration was 1%. After stirring evenly, adsorption was carried out at 4°C for 15 hours. After the adsorption process was completed, the silica precipitate was collected by high-speed centrifugation. After desorption, the antigen recovery rate and protein removal rate in the sample were detected and calculated. The results of this process were repeated three times and are shown in Table 4.

[0096] Table 4. Silica gel adsorption results

[0097]

[0098] In Example 4, the silica gel adsorption process was tested in three batches. The results showed that the average antigen recovery rate was 57.1% and the average total protein removal rate was 67.9%, which were consistent between batches. This indicates that the silica gel adsorption process is stable and controllable and can be scaled up for large-scale production of hepatitis B vaccine (Hansenula polymorpha).

[0099] Example 5. Desorption process

[0100] The silica gel precipitate was added to the desorption buffer solution with a pH of 8.0 and a sodium deoxycholate concentration of 2 g / L. The solution was stirred vigorously at 50°C for 1 hour. After desorption, the solution was centrifuged at high speed and the supernatant was collected. The supernatant was the antigen desorption solution. The antigen recovery rate and protein removal rate were measured by taking samples of the desorption solution. The results of the process were repeated three times and are shown in Table 5.

[0101] Table 5 Desorption results

[0102]

[0103] In Example 5, the silica gel desorption process was tested in three batches. The results showed that the average antigen recovery rate was 77.5% and the average total protein removal rate was 75.9%, which were consistent between batches. This indicates that the silica gel adsorption process is stable and controllable and can be scaled up for large-scale production of hepatitis B vaccine (Hansenula polymorpha).

[0104] Example 6. Ultrafiltration Process

[0105] The desorbed solution was concentrated by ultrafiltration using a tangential flow filtration process. An ultrafiltration membrane with a cutoff pore size of 500 kDa was used for ultrafiltration concentration. The solution was washed with 6 times its volume of 50 mM phosphate buffer at pH 7.0. The results of this process were repeated three times and are shown in Table 6.

[0106] Table 6: Antigen recovery rate and protein removal rate during ultrafiltration process

[0107]

[0108] In Example 6, three consecutive batches of experiments were conducted on the ultrafiltration process. The results showed that the average antigen recovery rate was 90.8% and the average total protein removal rate was 57.5%, which were consistent between batches. This indicates that the ultrafiltration process is stable and controllable and can be scaled up for large-scale production of hepatitis B vaccine (Hansenula polymorpha).

[0109] Example 7. Ion exchange chromatography process

[0110] The antigen solution after the first ultrafiltration was loaded onto a DEAE Sepharose Fast Flow ion exchange chromatography column equilibrated with 0.15 mol / L phosphate buffer (pH = 7, 50 mM). During loading, the salt concentration in the sample should be controlled to be no higher than 0.15 mol / L, and the loading volume should be within 4 column volumes. After loading, the column was equilibrated with 0.10 mol / L phosphate buffer (pH = 7, 50 mM) at a volume no less than 5 column volumes until the effluent from the column reached UV. 280 When the monitored value is below 20 mAU, after equilibration, the target antigen is eluted using a 0.25 mol / L phosphate buffer (pH = 7, 50 mM) containing sodium chloride. The antigen is harvested based on the UV280 detection value; the harvested product is the antigen chromatography solution. The chromatogram is recorded, as follows: Figure 2 As shown in the figure, the ion exchange chromatography pattern in step 1 is displayed. The above steps were repeated three times, and the recovery rate of the target antigen and the removal rate of the protein after purification by ion exchange chromatography were compared. The results are shown in Table 7.

[0111] Table 7: Antigen recovery rate and protein removal rate during ion exchange chromatography

[0112]

[0113] In Example 7, DEAE Sepharose Fast Flow ion exchange chromatography was used for the first step of purification of the feed solution. Antigen harvesting was based on UV280 detection values. The results showed that the main protein elution peak appeared at the elution point using a 0.25 mol / L salt concentration elution buffer. The protein contained in the salt concentration elution peak was mainly hepatitis B surface antigen, while the antigen content was lower when eluted with other salt concentration elution buffers. Therefore, it can be determined that the salt concentration for antigen elution in the ion exchange chromatography purification of hepatitis B surface antigen is 0.25 mol / L. At the same time, a pilot-scale ion exchange chromatography production was carried out using this process. The results showed that the average antigen recovery rate was 33.4%, and the average total protein removal rate was 86.3%, which was consistent between batches. This indicates that the ion exchange chromatography process is stable and controllable and can be scaled up for large-scale production of hepatitis B vaccine (Hansenula polymorpha).

[0114] Example 8. Study on gel filtration chromatography process

[0115] Gel filtration chromatography was used to effectively purify antigen particles and remove aggregated particles. The chromatographic solution obtained after ion exchange chromatography was then subjected to ultrafiltration. The chromatographic solution was concentrated using an ultrafiltration membrane with a cutoff pore size of 500 kDa, followed by washing with 6 times its volume of 50 mM phosphate buffer at pH 7.0. The ultrafiltrate was then purified by gel filtration chromatography using Sephacryl S-500HR chromatography media. The sample loading volume was 1% of the column volume, and elution was performed at a linear rate of 0.5 cm / min using 50 mM phosphate buffer at pH 6.0. 280 For detection, continuously collect equal volumes of elution peaks, combine them for HPLC analysis, and the chromatographic solution with a purity greater than 99.0% is considered a pure solution. Record the chromatographic peak chromatogram, as shown below. Figure 3 As shown in the figure, the gel filtration chromatography pattern in step 1- is displayed. The above steps were repeated three times, and the antigen recovery rate and protein removal rate of the chromatography solvent were compared. The results are shown in Table 8.

[0116] HPLC detection was performed using a Hitachi L5000 series high-performance liquid chromatograph connected to a TOSOH TSK gelG5000PW liquid chromatograph column (7.5mm ID x 30cm, catalog number: 05764). 20mM PB (pH 7.0) buffer was used, the sample loading flow rate was 0.60 mL / min, and the detection wavelength was 280 nm. The instrument software was used to analyze the chromatogram of the test sample, integrate the characteristic peaks of hepatitis B surface antigen, and calculate the purity of the target protein.

[0117] Table 8: Antigen recovery rate and protein removal rate during gel filtration chromatography

[0118]

[0119] In Example 8, the feed solution was further purified by Sephacryl S-500HR gel filtration chromatography. The elution peaks were collected in equal volumes and analyzed by HPLC. The purity of the chromatographic solution was greater than 99.0%. The results showed that a large amount of target protein was recovered during the gel filtration chromatography process. At the same time, a pilot-scale ion exchange chromatography production was carried out using this process. The results showed that the average antigen recovery rate was 76.5% and the average total protein removal rate was 67.7%, which was consistent between batches. This indicates that the gel filtration chromatography process is stable and controllable and can be scaled up for large-scale production of hepatitis B vaccine (Hansenula polymorpha).

[0120] Example 9. Aluminum Adsorption

[0121] Formaldehyde treatment of antigen: Add formaldehyde solution to the original antigen solution with a protein content of 300 μg / ml until the final formaldehyde concentration is 100 μg / ml, and incubate at 33℃ for 60 h to obtain formaldehyde-treated hepatitis B surface antigen solution.

[0122] The above experiment was conducted by adding a 0.14 mol / L (aluminum ion concentration) potassium aluminum sulfate solution with a pH of 3.5 to the antigen formaldehyde treatment solution. After stirring (600 rpm) until homogeneous, a 1 mol / L sodium hydroxide solution was quickly added dropwise to adjust the pH of the sample solution to 7.0 ± 0.2, allowing the antigen and aluminum agent to adsorb in situ, forming an aluminum adsorption product. The relative potency, protein content, and antigen content of the supernatant were then measured in vitro, and the results are shown in Table 9. Finally, the final product was diluted to ensure that the hepatitis B surface antigen content was 20 μg / mL and the aluminum ion content was 0.60 mg / mL, which constituted the vaccine semi-finished product. The relative potency was then tested, and the semi-finished product was aseptically filled and packaged to obtain the finished hepatitis B vaccine.

[0123] Table 9: Antigen adsorption rate, protein adsorption rate, and relative efficacy of potassium aluminum sulfate in situ adsorption process

[0124]

[0125] In Example 9, the aluminum adsorption process was tested in three consecutive batches. The results showed that the average antigen adsorption rate was 97.6% and the average relative efficacy of the semi-finished product in vitro was 2.56, which was consistent between batches. This indicates that the aluminum adsorption process is stable and controllable and meets the standard of relative efficacy in vitro >1.0 in the current Chinese Pharmacopoeia, Part III. This shows that the determined process can be stably scaled up for large-scale production of hepatitis B vaccine.

[0126] The preferred embodiments of this disclosure have been described in detail above. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0127] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0128] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. A method for inducing the expression of hepatitis B surface antigen, characterized in that, The method includes: Hansenula polymorpha expressing hepatitis B surface antigen was inoculated into the first-stage culture medium at a ratio of 2.0-6.0% for primary fermentation to obtain the first-stage culture broth, wherein the temperature of the primary fermentation was 33-35℃. Take the first-stage culture medium and inoculate it into the second-stage culture medium at a ratio of 2.0~6.0% for secondary fermentation to obtain the second-stage culture medium. The temperature of the secondary fermentation is 33~35℃. Take the second-stage culture medium and inoculate it into the third-stage culture medium at a ratio of 1.5-3.0% for tertiary fermentation. When the bacterial density in the fermentation broth reaches OD... 600 When the OD value was 160, methanol was initially added to induce hepatitis B surface antigen expression, and the fermentation product was harvested. The methanol was added continuously at a constant flow rate, and the amount of methanol added during the induction culture increased with the OD value of the fermentation broth. 600 The value and the volume of fermentation broth increased proportionally, with methanol added at a rate of 2.0 mL / 100 OD. 600 / L fermentation broth / hour; the temperature of the tertiary fermentation is 33~35℃; the induction culture time is 72h; during the induction period, the pH of the tertiary culture medium is controlled at 4.0~6.

0.

2. The method according to claim 1, characterized in that, The temperature for the secondary fermentation is 33°C.

3. The method according to claim 2, characterized in that, The temperature for the three-stage fermentation is 33°C.

4. A method for purifying hepatitis B surface antigen, characterized in that, The method includes: 1) A fermentation product is prepared by any one of claims 1 to 3, the fermentation product is subjected to cell disruption, and the first supernatant is collected after centrifugation; 2) Add polyethylene glycol to the first supernatant, let it stand to precipitate, centrifuge, and collect the second supernatant; 3) Add silica gel to the second supernatant to adsorb hepatitis B surface antigen in the second supernatant, and collect the silica gel precipitate adsorbed with hepatitis B surface antigen by centrifugation; 4) Add desorption buffer to the silica gel precipitate adsorbed with hepatitis B surface antigen to desorb the hepatitis B surface antigen from the silica gel and obtain a third supernatant containing hepatitis B surface antigen. 5) The third supernatant was subjected to first ultrafiltration, ion exchange chromatography, second ultrafiltration and gel filtration chromatography in sequence to obtain the hepatitis B surface antigen stock solution; In step 2), the pH of the first supernatant is first adjusted to 5.0-8.0, and then polyethylene glycol 6000 with a final concentration of 3%-6% is added. In step 3), the silica gel is added in the form of fumed silica, and the final concentration of fumed silica is 0.5%~2.0%. In step 3), the adsorption conditions are 2~8℃ for 12~20h. In step 4), the temperature for releasing the silica gel adsorption is 50~62℃; The desorption buffer solution contains 0.8-3.4 g / L of sodium deoxycholate; In step 5), the ion exchange chromatography is anion exchange chromatography.

5. The method according to claim 4, characterized in that, In step 2), the final concentration of polyethylene glycol 6000 is 4% to 5%.

6. The method according to claim 4, characterized in that, The desorption buffer solution contains 2-3 g / L sodium deoxycholate.

7. The method according to claim 4, characterized in that, In step 5), the first ultrafiltration uses an ultrafiltration membrane with a cutoff pore size of 300-500 kDa; The sodium chloride content in the equilibration solution used in the ion exchange chromatography is 0.05~0.20 mol / L; The sodium chloride content in the eluent used in the ion exchange chromatography is 0.1~0.55 mol / L; The pH of the eluent used in the ion exchange chromatography is 6.0-8.0; In step 5), the second ultrafiltration uses an ultrafiltration membrane with a cutoff pore size of 300-500 kDa; In step 5), the chromatography medium used for gel filtration chromatography is Sephacryl S-500 HR; The elution buffer used in the gel filtration chromatography is selected from 10-100 mM borate buffer, phosphate buffer, or Tris-HCl buffer. The pH of the eluent used in the gel filtration chromatography is 6.0-8.

0.

8. The method according to claim 7, characterized in that, The chromatography medium used in the ion exchange chromatography was DEAE Sepharose Fast Flow. The equilibration solution used in the ion exchange chromatography is a borate buffer, phosphate buffer, or Tris-HCl buffer with a sodium chloride content of 0.1~0.15 mol / L. The concentration of the buffer salt in the equilibration solution used in the ion exchange chromatography is 10-100 mM; The elution buffer used in the ion exchange chromatography is a borate buffer, phosphate buffer, or Tris-HCl buffer with a sodium chloride content of 0.25-0.55 mol / L. The concentration of the buffer salt in the eluent used in the ion exchange chromatography is 10-100 mM; The elution buffer used in the gel filtration chromatography is 10-60 mM borate buffer, phosphate buffer, or Tris-HCl buffer.

9. The method according to claim 8, characterized in that, The equilibration solution used in the ion exchange chromatography was a phosphate buffer solution with a sodium chloride content of 0.1~0.15 mol / L; The concentration of the buffer salt in the equilibration solution used in the ion exchange chromatography is 10-60 mM; The eluent used in the ion exchange chromatography was a phosphate buffer solution with a sodium chloride content of 0.25-0.55 mol / L. The concentration of the buffer salt in the eluent used in the ion exchange chromatography is 10-60 mM; The elution buffer used in the gel filtration chromatography was 10-60 mM phosphate buffer.

10. A method for producing a hepatitis B vaccine, characterized in that, The method includes: Hepatitis B surface antigen stock solution is prepared by the method according to any one of claims 4 to 9, and the hepatitis B surface antigen stock solution is treated with formaldehyde to obtain hepatitis B surface antigen formaldehyde treated solution; Under stirring conditions, a potassium aluminum sulfate solution with a pH of 3.0–4.2 and an aluminum ion concentration of 0.12–0.21 mol / L was added to the hepatitis B surface antigen formaldehyde treatment solution. Then, the pH was adjusted to 6.0–8.0 using a 0.5–2.0 mol / L sodium hydroxide solution to obtain the hepatitis B aluminum adsorption product, which was then diluted with a vaccine diluent to prepare a hepatitis B vaccine semi-finished product.

11. The method according to claim 10, characterized in that, Adjust the pH to 6.5-7.5 using a 0.5-2.0 mol / L sodium hydroxide solution; The concentration of hepatitis B surface antigen in the formaldehyde-treated solution is 260-340 μg / mL; The stirring speed is 100~700 rpm; The pH of the potassium aluminum sulfate solution is 3.5~4.0, the concentration of aluminum ions is 0.14~0.18 mol / L, and the concentration of the sodium hydroxide solution is 1.0~1.5 mol / L.

12. The method according to claim 11, characterized in that, The stirring speed is 400~600 rpm.

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