A method for preparing capsular polysaccharide of Haemophilus influenzae type b

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a method for preparing capsular polysaccharides of Haemophilus influenzae type b. The present invention provides a method for preparing capsular polysaccharides of Haemophilus influenzae type b that is simple to operate, comprising sterilizing a fermentation broth to obtain a sterilizing liquid, clarifying and ultrafiltering the sterilizing liquid, removing proteins, nucleic acids, and residual hexadecyltrimethylammonium bromide, ultrafiltration concentration, and freeze-drying. Using the preparation method of the present invention, the various indicators of the prepared capsular polysaccharide meet the requirements of the pharmacopoeia, the purity is high, and the entire preparation process does not use organic solvents such as phenol, acetone, and ethanol. It has the advantages of being simple and efficient, economical and environmentally friendly, having low equipment requirements, and being suitable for large-scale industrial production, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing capsular polysaccharide of Haemophilus influenzae type b. Background Art

[0002] Haemophilus Influenzae Type b (Hib) is a Gram-negative bacterium that can be divided into non-capsulated strains and encapsulated strains. The disease caused by non-capsulated bacteria in children is usually mild, and severe infections are generally caused by encapsulated strains. Encapsulated strains can be divided into 6 serotypes: a, b, c, d, e, and f. Among these 6 serotypes, Hib has the strongest pathogenicity and is one of the main pathogens of purulent meningitis, sepsis, pneumonia and otitis media in children in my country. More than 90% of Hib invasive diseases occur in children under 5 years old.

[0003] Since the advent of the Hib vaccine in the early 1990s, vaccination has been the only public health measure to prevent the vast majority of severe cases of Hib. Antibiotics are necessary for treatment but have little effect on prevention, and the increasing resistance of bacteria to effective antibiotics further underscores the importance of prevention. Vaccination remains an effective way to reduce antibiotic resistance in some bacterial pathogens, including Hib.

[0004] Hib capsular polysaccharide is a polymer formed by repeated PRP, which is the main component of Hib virulence. Hib polysaccharide vaccine can form non-T cell-dependent protection, and children under 2 years old cannot form immune protection. The conjugate vaccine formed by the combination of PRP and carrier protein can stimulate the body to produce T cell-dependent protection and form long-term immune memory in infants.

[0005] The World Health Organization (WHO) recommends that Hib be included in the Expanded Program on Immunization (EPI) as a member of the quadrivalent vaccine (tetanus-diphtheria-pertussis-Hib). However, due to the complex production process, the cost of Hib conjugate vaccine is very high, mainly including: bacterial culture polysaccharide production, protein production (generally diphtheria or tetanus toxin), ribose and protein purification, and further separation of ribose-protein conjugates from free protein and ribose. Improving the efficiency of any step will reduce the cost of vaccine production.

[0006] Traditional Hib polysaccharide purification mainly includes: collecting the supernatant after removing the bacteria by centrifugation of the sterilization solution, collecting the complex polysaccharide by precipitation with hexadecyltrimethylammonium bromide (CTAB), precipitating by ethanol graded precipitation and washing the precipitate with ethanol, acetone, etc., drying to obtain the crude polysaccharide, and finally extracting with phenol, precipitating the polysaccharide with ethanol, washing with ethanol and acetone, and drying to obtain the refined polysaccharide. The patent states that the purification of ribose begins with inactivating the cells with phenol, formaldehyde or thimerosal, and then separating the culture fluid by centrifugation. Next, the clarified supernatant is concentrated by tangential flow ultrafiltration using a 50kD ultrafiltration membrane package, and then the polysaccharide is precipitated with CTAB. The supernatant of the complex of ribose and CTAB dissociated with calcium chloride is precipitated with ethanol several times, and the protein is removed by phenol extraction and further ethanol precipitation. Finally, lipopolysaccharide is separated from ribose by ultracentrifugation. Mickie Takagi established a purification method for Hib polysaccharide: the ethanol precipitation process is simplified to two steps. Phenol treatment is replaced by ultrafiltration and enzymatic hydrolysis. Lipopolysaccharide is removed by adding detergents and chelating agents combined with ultrafiltration. However, the purification process always involves the use of organic solvents such as phenol and ethanol, as well as chromatography columns that are difficult to scale up for production.

[0007] Based on the problems existing in the above-mentioned Hib polysaccharide preparation method, it is urgent to find a Hib polysaccharide preparation method that is simple, efficient, economical, environmentally friendly, has low equipment requirements, is low-cost, and is suitable for large-scale production. Summary of the invention

[0008] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing capsular polysaccharide of Haemophilus influenzae type b.

[0009] A capsular polysaccharide of Haemophilus influenzae type b is prepared according to the following steps:

[0010] (1) fermenting and culturing Haemophilus influenzae type b seed solution to obtain a sterilization solution, and ultrafiltration to obtain a first ultrafiltrate;

[0011] (2) adding sodium chloride and sodium deoxycholate solution to the first ultrafiltrate, mixing and adjusting the pH, centrifuging and ultrafiltration to obtain a second ultrafiltrate;

[0012] (3) adding phosphate buffer and hexadecyltrimethylammonium bromide to the second ultrafiltrate, centrifuging after the reaction, and collecting the precipitate;

[0013] (4) adding sodium chloride solution to dissociate the precipitate, and collecting the supernatant by centrifugation;

[0014] (5) adding sodium iodide to the supernatant, centrifuging, collecting the supernatant, filtering, and obtaining a filtrate;

[0015] (6) The filtrate is concentrated by ultrafiltration to obtain the third ultrafiltrate, which is freeze-dried.

[0016] Preferably, in step (2), the concentration of sodium chloride is 0.8 to 2.0 mol / L.

[0017] Preferably, the concentration of sodium chloride is 0.8 mol / L.

[0018] Preferably, in step (4), the concentration of the sodium chloride solution is 0.25 to 0.3 mol / L.

[0019] Preferably, in step (4), the concentration of the sodium chloride solution is 0.28 mol / L.

[0020] Preferably, in step (1), the bactericidal solution is obtained by adding a bactericide; the bactericide is selected from at least one of 0.12% sodium deoxycholate and 1% formaldehyde.

[0021] Preferably, in step (1), the cut-off pore size of the membrane package used for ultrafiltration is 30 to 50 kD;

[0022] And / or, in step (2), the final concentration of the sodium deoxycholate solution is 0.1-0.5% by volume; the pH is 3.6-4.0;

[0023] And / or, in step (2), the centrifugal speed is 10000-18000g, the centrifugal temperature is 2-10°C, and the centrifugal time is 30-60min.

[0024] Preferably, in step (3), the pH of the phosphate buffer is 6.6 to 7.0, and the final concentration is 10 to 20 mmol / L; the final concentration of the hexadecyltrimethylammonium bromide is 0.1 to 0.5% by volume;

[0025] And / or, in step (3), the centrifugal speed is 10000-18000g, the centrifugal temperature is 18-30°C, and the centrifugal time is 30-60min;

[0026] And / or, in step (4), the centrifugal speed is 10000-18000g, the centrifugal temperature is 18-30°C, and the centrifugal time is 30-60min;

[0027] And / or, in step (5), the final concentration of sodium iodide is 0.1 to 0.5% by volume;

[0028] And / or, in step (5), the centrifugal speed is 10000-18000g, the centrifugal temperature is 18-30°C, and the centrifugal time is 30-60min.

[0029] The present invention also provides a method for preparing the above-mentioned Haemophilus influenzae type b capsular polysaccharide, comprising the following steps:

[0030] (1) fermenting and culturing Haemophilus influenzae type b seed solution to obtain a sterilization solution, and ultrafiltration to obtain a first ultrafiltrate;

[0031] (2) adding sodium chloride and sodium deoxycholate solution to the first ultrafiltrate, mixing and adjusting the pH, centrifuging and ultrafiltration to obtain a second ultrafiltrate;

[0032] (3) adding phosphate buffer and hexadecyltrimethylammonium bromide to the second ultrafiltrate, centrifuging after the reaction, and collecting the precipitate;

[0033] (4) adding sodium chloride solution to dissociate the precipitate, and collecting the supernatant by centrifugation;

[0034] (5) adding sodium iodide to the supernatant, centrifuging, collecting the supernatant, filtering, and obtaining a filtrate;

[0035] (6) The filtrate is concentrated by ultrafiltration to obtain the third ultrafiltrate, which is freeze-dried.

[0036] The present invention also provides use of the above-mentioned Haemophilus influenzae type b capsular polysaccharide in preparing Haemophilus influenzae type b vaccine.

[0037] The invention optimizes the preparation method of Hib polysaccharide, increases the recovery rate of Haemophilus influenzae type b capsular polysaccharide by adding sodium chloride during the preparation process, uses sodium deoxycholate to form flocculent precipitation in a low pH environment to remove impurities such as protein, and uses sodium chloride solution for dissociation to separate impurities and target polysaccharide. The Hib polysaccharide obtained by the method has high yield, low impurity content and stable quality.

[0038] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0039] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a diagram showing the effects of protein removal, nucleic acid removal and polysaccharide recovery in Example 4.

[0041] Figure 2The identification results of prepared Haemophilus influenzae type b capsular polysaccharide (bidirectional diffusion method). 1 is Hib antiserum; 2 is water for injection; 3 is positive control; 4 is 25ug / ml Hib capsular polysaccharide; 5 is 50ug / ml Hib capsular polysaccharide; 6 is 100ug / ml Hib capsular polysaccharide.

[0042] Figure 3 This is the full spectrum of the H NMR spectrum of the capsular polysaccharide of Haemophilus influenzae type b prepared in Example 4 (3 batches). A is the literature "Xu Meifeng. Quantitative analysis of the capsular polysaccharide of Haemophilus influenzae type b 1 Establishment and verification of H-NMR method." A is the full spectrum of H-NMR spectrum of Haemophilus influenzae type b capsular polysaccharide prepared with batch number HibDG20220402; C is the full spectrum of H-NMR spectrum of Haemophilus influenzae type b capsular polysaccharide prepared with batch number HibDG20220703; D is the full spectrum of H-NMR spectrum of Haemophilus influenzae type b capsular polysaccharide prepared with batch number HibDG20220804.

[0043] Figure 4 The H NMR fingerprints of the capsular polysaccharide of Haemophilus influenzae type b prepared in Example 4 (3 batches). A is the H NMR fingerprint of the capsular polysaccharide of Haemophilus influenzae type b prepared with batch number HibDG20220402; B is the H NMR fingerprint of the capsular polysaccharide of Haemophilus influenzae type b prepared with batch number HibDG20220703; C is the H NMR fingerprint of the capsular polysaccharide of Haemophilus influenzae type b prepared with batch number HibDG20220804.

[0044] Figure 5 This is a graph showing the effect of sodium chloride ion strength on protein removal and polysaccharide recovery.

[0045] Figure 6 The polysaccharide recovery rate and nucleic acid removal rate as well as the nucleic acid-polysaccharide ratio of the dissociated precipitate with different sodium chloride concentrations are shown. DETAILED DESCRIPTION

[0046] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0047] Example 1 Preparation method of Haemophilus influenzae type b capsular polysaccharide 1

[0048] 1. Fermentation culture to obtain sterilization solution

[0049] The seed liquid of Haemophilus influenzae type b was inoculated into a fermenter, and after culturing to a stable period, 0.12% sodium deoxycholate was added for sterilization to obtain a sterilized liquid.

[0050] 2. Clarification and ultrafiltration of sterilizing liquid

[0051] The sterilization solution was centrifuged at 17000g and 25°C for 30 minutes, the precipitate was discarded, and the supernatant was collected; the collected supernatant was ultrafiltered with an equal volume of injection water using an ultrafiltration membrane package with a cutoff pore size of 30kD to obtain the first ultrafiltrate.

[0052] 3. Protein removal

[0053] Cool the ultrafiltrate to below 15°C, add solid sodium chloride to a final concentration of 0.8 mol / L, stir thoroughly to dissolve, add 10% sodium deoxycholate solution to a volume fraction of 0.5%, mix well, adjust the pH to 4.0 with glacial acetic acid, and let stand at 2-8°C. Centrifuge at 17000g, 4°C for 60 minutes, discard the precipitate, and collect the supernatant. Adjust the pH of the supernatant to neutral. Use an ultrafiltration membrane with a cutoff pore size of 30kD to ultrafilter with an equal volume of injection water to obtain a second ultrafiltrate.

[0054] 4. Removal of nucleic acid

[0055] Add 0.2 mol / L phosphate buffer at pH 7.00 to the second ultrafiltrate to a final concentration of 10 mmol / L, then add 10% cetyltrimethylammonium bromide to a final volume fraction of 0.5%. After fully mixing and reacting at room temperature for 90 minutes, centrifuge at 17000g, 25°C for 30 minutes to collect the precipitate.

[0056] 5. Dissociation of precipitate

[0057] The collected precipitate was dissociated with 0.25 mol / L sodium chloride solution. After complete dissociation, the precipitate was centrifuged at 17000 g and 25° C. for 30 minutes, and the supernatant was collected.

[0058] 6. Remove hexadecyl trimethyl ammonium bromide

[0059] Solid sodium iodide was added to the supernatant to a final volume fraction of 0.5%, and the mixture was fully reacted at room temperature and centrifuged at 17000 g and 25° C. for 30 minutes to collect the supernatant. The supernatant was clarified and filtered through 0.45 μm to obtain a filtrate.

[0060] 7. Ultrafiltration concentration and freeze drying

[0061] The obtained filtrate was ultrafiltered in equal volume through an ultrafiltration membrane package with a cutoff pore size of 30 kD, and finally concentrated to obtain the third ultrafiltrate, which was freeze-dried to obtain freeze-dried Haemophilus influenzae type b capsular polysaccharide.

[0062] Example 2 Preparation method of Haemophilus influenzae type b capsular polysaccharide

[0063] 1. Fermentation culture to obtain sterilization solution

[0064] The seed liquid of Haemophilus influenzae type b was inoculated into a fermenter, and after culturing to a stable period, 0.12% sodium deoxycholate was added for sterilization to obtain a sterilized liquid.

[0065] 2. Clarification and ultrafiltration of sterilizing liquid

[0066] The sterilization solution was centrifuged at 17000g and 25°C for 30 minutes, the precipitate was discarded, and the supernatant was collected; the collected supernatant was ultrafiltered with an equal volume of injection water using an ultrafiltration membrane package with a cutoff pore size of 30kD to obtain the first ultrafiltrate.

[0067] 3. Protein removal

[0068] Cool the ultrafiltrate to below 15°C, add solid sodium chloride to a final concentration of 0.8 mol / L, stir thoroughly to dissolve, add 10% sodium deoxycholate solution to a volume fraction of 0.25%, mix well, adjust the pH to 3.7 with glacial acetic acid, and let stand at 2-8°C. Centrifuge at 17000g, 4°C for 60 minutes, discard the precipitate, and collect the supernatant. Adjust the pH of the supernatant to neutral. Use an ultrafiltration membrane with a cutoff pore size of 30kD to ultrafilter with an equal volume of injection water to obtain a second ultrafiltrate.

[0069] 4. Removal of nucleic acid

[0070] Add 0.2 mol / L phosphate buffer with pH=6.6 to the second ultrafiltrate to a final concentration of 10 mmol / L, then add 10% cetyltrimethylammonium bromide to a final volume fraction of 0.5%. After fully mixing and reacting at room temperature for 90 minutes, centrifuge at 17000g, 25°C for 30 minutes to collect the precipitate.

[0071] 5. Dissociation of precipitate

[0072] The collected precipitate was dissociated with 0.25 mol / L sodium chloride solution. After complete dissociation, the precipitate was centrifuged at 17000 g and 25° C. for 30 minutes, and the supernatant was collected.

[0073] 6. Remove hexadecyl trimethyl ammonium bromide

[0074] Solid sodium iodide was added to the supernatant to a final volume fraction of 0.5%, and the mixture was fully reacted at room temperature and centrifuged at 17000 g and 25° C. for 30 minutes to collect the supernatant. The supernatant was clarified and filtered through 0.45 μm to obtain a filtrate.

[0075] 7. Ultrafiltration concentration and freeze drying

[0076] The obtained filtrate was ultrafiltered with an ultrafiltration membrane package with a cutoff pore size of 30 kD and concentrated to obtain the third ultrafiltrate, which was freeze-dried to obtain freeze-dried Haemophilus influenzae type b capsular polysaccharide, freeze-dried and stored.

[0077] Example 3 Preparation method of Haemophilus influenzae type b capsular polysaccharide

[0078] 1. Fermentation culture to obtain sterilization solution

[0079] The seed liquid of Haemophilus influenzae type b was inoculated into a fermenter, and after culturing to a stable period, 0.12% sodium deoxycholate was added for sterilization to obtain a sterilized liquid.

[0080] 2. Clarification and ultrafiltration of sterilizing liquid

[0081] The sterilization solution was centrifuged at 17000g and 25°C for 30 minutes, the precipitate was discarded, and the supernatant was collected; the collected supernatant was ultrafiltered with an equal volume of injection water using an ultrafiltration membrane package with a cutoff pore size of 30kD to obtain the first ultrafiltrate.

[0082] 3. Protein removal

[0083] Cool the ultrafiltrate to below 15°C, add solid sodium chloride to a final concentration of 0.8 mol / L, stir thoroughly to dissolve, add 10% sodium deoxycholate solution to a volume fraction of 0.5%, mix well, adjust the pH to 4.0 with glacial acetic acid, and let stand at 2-8°C. Centrifuge at 17000g, 4°C for 60 minutes, discard the precipitate, and collect the supernatant. Adjust the pH of the supernatant to neutral. Use an ultrafiltration membrane with a cutoff pore size of 30kD to ultrafilter with an equal volume of injection water to obtain a second ultrafiltrate.

[0084] 4. Removal of nucleic acid

[0085] Add 0.2 mol / L phosphate buffer with pH=6.7 to the second ultrafiltrate to a final concentration of 10 mmol / L, then add 10% cetyltrimethylammonium bromide to a final volume fraction of 0.5%. After fully mixing and reacting at room temperature for 90 minutes, centrifuge at 17000g, 25°C for 30 minutes to collect the precipitate.

[0086] 5. Dissociation of precipitate

[0087] The collected precipitate was dissociated with 0.3 mol / L sodium chloride solution. After complete dissociation, it was centrifuged at 17000 g and 25° C. for 30 minutes to collect the supernatant.

[0088] 6. Remove hexadecyl trimethyl ammonium bromide

[0089] Solid sodium iodide was added to the supernatant to a final volume fraction of 0.5%, and the mixture was fully reacted at room temperature and centrifuged at 17000 g and 25° C. for 30 minutes to collect the supernatant. The supernatant was clarified and filtered through 0.45 μm to obtain a filtrate.

[0090] 7. Ultrafiltration concentration and freeze drying

[0091] The obtained filtrate was ultrafiltered with an ultrafiltration membrane package with a cutoff pore size of 30 kD and concentrated to obtain the third ultrafiltrate, which was freeze-dried to obtain freeze-dried Haemophilus influenzae type b capsular polysaccharide, freeze-dried and stored.

[0092] Example 4 Preparation method of Haemophilus influenzae type b capsular polysaccharide

[0093] 1. Fermentation culture to obtain sterilization solution

[0094] The seed liquid of Haemophilus influenzae type b was inoculated into a fermenter, and after being cultured to a stable period, 1% formaldehyde was added for sterilization to obtain a sterilization liquid.

[0095] 2. Clarification and ultrafiltration of sterilizing liquid

[0096] The sterilization solution was centrifuged at 17000g and 25°C for 30 minutes, the precipitate was discarded, and the supernatant was collected; the collected supernatant was ultrafiltered with an equal volume of injection water using an ultrafiltration membrane package with a cutoff pore size of 50kD to obtain the first ultrafiltrate.

[0097] 3. Protein removal

[0098] Cool the ultrafiltrate to below 15°C, add solid sodium chloride to a final concentration of 0.8 mol / L, stir thoroughly to dissolve, add 10% sodium deoxycholate solution to a volume fraction of 0.5%, mix well, adjust the pH to 3.6 with glacial acetic acid, and let stand at 2-8°C. Centrifuge at 17000g, 4°C for 60 minutes, discard the precipitate, and collect the supernatant. Adjust the pH of the supernatant to neutral. Use an ultrafiltration membrane with a cutoff pore size of 30kD to ultrafilter with an equal volume of injection water to obtain a second ultrafiltrate.

[0099] 4. Removal of nucleic acid

[0100] Add 0.2 mol / L phosphate buffer with pH=6.62 to the second ultrafiltrate to a final concentration of 10 mmol / L, then add 10% cetyltrimethylammonium bromide to a final volume fraction of 0.5%. After fully mixing and reacting at room temperature for 90 minutes, centrifuge at 17000g, 25°C for 30 minutes to collect the precipitate.

[0101] 5. Dissociation of precipitate

[0102] The collected precipitate was dissociated with 0.28 mol / L sodium chloride solution. After complete dissociation, the precipitate was centrifuged at 17000 g and 25° C. for 30 minutes, and the supernatant was collected.

[0103] 6. Remove hexadecyl trimethyl ammonium bromide

[0104] Solid sodium iodide was added to the supernatant to a final volume fraction of 0.5%, and the mixture was fully reacted at room temperature and centrifuged at 17000 g and 25° C. for 30 minutes to collect the supernatant. The supernatant was clarified and filtered through 0.45 μm to obtain a filtrate.

[0105] 7. Ultrafiltration concentration and freeze drying

[0106] The obtained filtrate was ultrafiltered with an ultrafiltration membrane package with a cutoff pore size of 30 kD and concentrated to obtain the third ultrafiltrate, which was freeze-dried to obtain freeze-dried Haemophilus influenzae type b capsular polysaccharide, freeze-dried and stored.

[0107] The technical solution of the present invention is further illustrated by experiments below.

[0108] Experimental Example 1: Quality test results of Haemophilus influenzae type b capsular polysaccharide prepared in Examples 1 to 4 The comparison of conditions in Examples 1 to 4 is summarized in Table 1

[0109] Table 1

[0110]

[0111] According to the relevant requirements and methods of the current edition of the Pharmacopoeia of the People's Republic of China, the quality of the capsular polysaccharides of Haemophilus influenzae type b prepared continuously in Examples 1-3 and Example 4 (batch numbers are HibDG20220402, HibDG20220703, and HibDG20220804, respectively) was tested, and the results are shown in Tables 2 and 3. The identification and hydrogen nuclear magnetic resonance spectra of the capsular polysaccharide prepared in Example 4 are shown in Tables 2 and 3. Figure 2-4 As shown in Table 2-3, it is shown that the capsular polysaccharide prepared by Example 4 has a high purity and no other organic process impurities remain. Figure 1 It can be seen that the capsular polysaccharide of Haemophilus influenzae type b prepared by the present invention meets the requirements of the pharmacopoeia in all test items, has good protein removal and nucleic acid removal effects, high purity, and high polysaccharide recovery rate.

[0112] Table 2 Quality test results of Haemophilus influenzae type b capsular polysaccharide prepared in Examples 1 to 3

[0113]

[0114] Table 3 Example 4 Three consecutive batches of Haemophilus influenzae type b capsular polysaccharide quality test results

[0115]

[0116] Experimental Example 2: Comparison of the bactericidal effects of two bactericidal agents

[0117] As shown in Table 4 and Table 5, the contents of protein, nucleic acid, phosphorus, ribose and endotoxin in the purified polysaccharide in the sterilization solution treated with the two sterilization reagents all meet the quality standards, indicating that either 0.12% sodium deoxycholate or 1% formaldehyde can be used as the sterilizing agent.

[0118] Table 4 Comparison of the bactericidal effects of two bactericidal agents

[0119]

[0120] Table 5 Chemical test results of purified polysaccharides from sterilization liquid after treatment with two sterilization reagents

[0121]

[0122] Experimental Example 3: Effects of different NaCl concentrations on protein removal and polysaccharide recovery

[0123] According to Table 6 and Figure 5 It can be seen that with the increase of ionic strength, the polysaccharide recovery rate also increases. Too high NaCl concentration will cause particle precipitation, affecting the protein removal effect. When the NaCl concentration is ≥0.8mol / L, the polysaccharide recovery is high, and the proteoglycan is relatively low. Therefore, 0.8mol / LNaCl is selected based on the principle of energy saving. As can be seen from Table 7, without adding sodium chloride, the protein precipitation effect is poor, the centrifugation effect is poor, and the protein cannot be removed; adding low concentrations of sodium chloride can remove proteins to a certain extent, but the polysaccharide recovery rate is low, and the freeze-dried polysaccharide protein obtained is unqualified; therefore, adding high concentrations of sodium chloride (0.8mol / L) can ensure the recovery of polysaccharides while removing proteins.

[0124] Table 6 Effect of different NaCl concentrations on the recovery of Hib polysaccharide

[0125]

[0126]

[0127] Table 7

[0128]

[0129] Experimental Example 4: Polysaccharide recovery rate and nucleic acid removal rate of dissociated precipitates at different sodium chloride concentrations and nucleic acid-polysaccharide ratio

[0130] Different concentrations of NaCl were used for dissociation precipitation. At a certain concentration, as the NaCl concentration increased, the recovery rate of Hib polysaccharide increased, while the removal rate of nucleic acid decreased. According to Table 1, a NaCl concentration of 0.25 mol / L-0.3 mol / L can obtain Hib freeze-dried polysaccharide with qualified nucleic acid; as shown in Table 8, Figure 6It can be seen that when the sodium chloride concentration is low, the dissociation effect is poor and the polysaccharide recovery rate is low. The higher the sodium chloride concentration, the more thorough the dissociation, but the nucleic acid removal effect is not good. After freeze-drying, the polysaccharide nucleic acid is as high as 128.4 mg / g. Therefore, the optimal NaCl concentration range for dissociation is 0.28-0.3 mol / L, and the further preferred optimal value is 0.28 mol / L.

[0131] Table 8 Effect of different sodium chloride concentrations on polysaccharide recovery

[0132]

[0133] Experimental Example 5: Effect of swapping protein removal and nucleic acid removal steps on Hib polysaccharide recovery

[0134] The process of the present invention (process 2): first ultrafiltrate - protein removal - nucleic acid removal. Process 1: first ultrafiltrate - nucleic acid removal - protein removal, the other process flow is consistent with Example 4 and the parameters are within the required range of the present invention. According to the results of Tables 9 and 10, it can be seen that the protein content of the freeze-dried polysaccharide obtained by the steps of process 1 does not meet the standard; the protein content of the freeze-dried polysaccharide obtained by the steps of process 2 meets the standard and the content is relatively low. Therefore, in the preparation process of the capsular polysaccharide of Haemophilus influenzae type b, the process sequence of the protein removal and nucleic acid removal steps using the process parameters is extremely important and affects the recovery of Hib polysaccharide.

[0135] Table 9 Effect of the steps of process 1 on the recovery of Hib polysaccharide

[0136]

[0137] Note: The first ultrafiltration, the second ultrafiltration and the third ultrafiltration are named relative to the steps of the present invention; Not applicable: indicates that the step was not detected or could not be detected, the same below.

[0138] Table 10 Effect of the steps of process 2 on the recovery of Hib polysaccharide

[0139]

[0140] In summary, the present invention optimizes the concentration of bactericide, NaCl and the process sequence of protein removal and nucleic acid removal steps to obtain a method for preparing capsular polysaccharide of Haemophilus influenzae type b. The capsular polysaccharide prepared by this method meets the requirements of the pharmacopoeia in various indicators, has high yield, low impurity content, stable quality, and does not use organic solvents such as phenol, acetone, ethanol, etc. in the entire preparation process. The method is economical and efficient, environmentally friendly, simple to operate, has low equipment requirements, and is suitable for large-scale industrial production.

Claims

1. A capsular polysaccharide of Haemophilus influenzae type b, characterized in that: It is prepared according to the following steps: (1) fermenting and culturing Haemophilus influenzae type b seed solution to obtain a sterilization solution, and ultrafiltration to obtain a first ultrafiltrate; (2) adding sodium chloride and sodium deoxycholate solution to the first ultrafiltrate, mixing and adjusting the pH, centrifuging and ultrafiltration to obtain a second ultrafiltrate; (3) adding phosphate buffer and hexadecyltrimethylammonium bromide to the second ultrafiltrate, centrifuging after the reaction, and collecting the precipitate; (4) adding sodium chloride solution to dissociate the precipitate, and collecting the supernatant by centrifugation; (5) adding sodium iodide to the supernatant, centrifuging, collecting the supernatant, filtering, and obtaining a filtrate; (6) The filtrate is concentrated by ultrafiltration to obtain the third ultrafiltrate, which is freeze-dried.

2. The capsular polysaccharide of Haemophilus influenzae type b according to claim 1, characterized in that In step (2), the concentration of sodium chloride is 0.8-2.0 mol / L.

3. The capsular polysaccharide of Haemophilus influenzae type b according to claim 2, characterized in that The concentration of the sodium chloride is 0.8 mol / L.

4. The capsular polysaccharide of Haemophilus influenzae type b according to claim 1, characterized in that In step (4), the concentration of the sodium chloride solution is 0.25-0.3 mol / L.

5. The capsular polysaccharide of Haemophilus influenzae type b according to claim 4, characterized in that In step (4), the concentration of the sodium chloride solution is 0.28 mol / L.

6. The capsular polysaccharide of Haemophilus influenzae type b according to claim 1, characterized in that In step (1), the bactericidal solution is obtained by adding a bactericide; the bactericide is selected from at least one of 0.12% sodium deoxycholate and 1% formaldehyde.

7. The capsular polysaccharide of Haemophilus influenzae type b according to claim 1, characterized in that In step (1), the cut-off pore size of the membrane package used for ultrafiltration is 30 to 50 kD; And / or, in step (2), the final concentration of the sodium deoxycholate solution is 0.1-0.5% by volume; the pH is 3.6-4.0; And / or, in step (2), the centrifugal speed is 10000-18000g, the centrifugal temperature is 2-10°C, and the centrifugal time is 30-60min.

8. The capsular polysaccharide of Haemophilus influenzae type b according to claim 1, characterized in that In step (3), the pH of the phosphate buffer is 6.6 to 7.0, and the final concentration is 10 to 20 mmol / L; the final concentration of the hexadecyltrimethylammonium bromide is 0.1 to 0.5% by volume; And / or, in step (3), the centrifugal speed is 10000-18000g, the centrifugal temperature is 18-30°C, and the centrifugal time is 30-60min; And / or, in step (4), the centrifugal speed is 10000-18000g, the centrifugal temperature is 18-30°C, and the centrifugal time is 30-60min; And / or, in step (5), the final concentration of sodium iodide is 0.1 to 0.5% by volume; And / or, in step (5), the centrifugal speed is 10000-18000g, the centrifugal temperature is 18-30°C, and the centrifugal time is 30-60min.

9. The method for preparing the capsular polysaccharide of Haemophilus influenzae type b according to any one of claims 1 to 8, characterized in that: The steps include: (1) fermenting and culturing Haemophilus influenzae type b seed solution to obtain a sterilization solution, and ultrafiltration to obtain a first ultrafiltrate; (2) adding sodium chloride and sodium deoxycholate solution to the first ultrafiltrate, mixing and adjusting the pH, centrifuging and ultrafiltration to obtain a second ultrafiltrate; (3) adding phosphate buffer and hexadecyltrimethylammonium bromide to the second ultrafiltrate, centrifuging after the reaction, and collecting the precipitate; (4) adding sodium chloride solution to dissociate the precipitate, and collecting the supernatant by centrifugation; (5) adding sodium iodide to the supernatant, centrifuging, collecting the supernatant, filtering, and obtaining a filtrate; (6) The filtrate is concentrated by ultrafiltration to obtain the third ultrafiltrate, which is freeze-dried.

10. Use of the Haemophilus influenzae type b capsular polysaccharide according to any one of claims 1 to 8 in the preparation of a Haemophilus influenzae type b vaccine.

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