CN112741901B - Vaccine containing streptococcus pneumoniae capsular polysaccharide type 5 and preparation method thereof - Google Patents
Vaccine containing streptococcus pneumoniae capsular polysaccharide type 5 and preparation method thereof Download PDFInfo
- Publication number
- CN112741901B CN112741901B CN201911053113.7A CN201911053113A CN112741901B CN 112741901 B CN112741901 B CN 112741901B CN 201911053113 A CN201911053113 A CN 201911053113A CN 112741901 B CN112741901 B CN 112741901B
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- streptococcus pneumoniae
- polysaccharide
- capsular polysaccharide
- pneumoniae capsular
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- 230000000269 nucleophilic effect Effects 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 108010040473 pneumococcal surface protein A Proteins 0.000 description 1
- 102000007739 porin activity proteins Human genes 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
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- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
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- A61K39/02—Bacterial antigens
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
- A61K39/092—Streptococcus
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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Abstract
The invention provides a vaccine containing streptococcus pneumoniae capsular polysaccharide 5 and a preparation method thereof, belonging to the technical field of biological product preparation. The preparation method provided by the invention comprises the steps of degrading capsular polysaccharides of each serotype at least containing streptococcus pneumoniae type 5 respectively; activating; freeze-drying or precipitation; re-dissolving or dissolving activated streptococcus pneumoniae capsular polysaccharide and combining with carrier protein; purifying the combined vaccine stock solution. The antigen specific epitope in the vaccine stock solution prepared by the method is not lost, the integrity of the antigen is maintained, the immunogenicity in the preparation process of the polysaccharide-protein conjugate is ensured not to be reduced to the maximum extent, and the generation of protein self-polymers is avoided. The streptococcus pneumoniae capsular polysaccharide-protein conjugate vaccine stock solution prepared by the method can be used for preparing a monovalent and multivalent conjugate vaccine of streptococcus pneumoniae capsular polysaccharide, and can also be used for preparing a conjugate vaccine containing the streptococcus pneumoniae capsular polysaccharide.
Description
Technical Field
The invention belongs to the technical field of biological product preparation, and particularly relates to a vaccine containing streptococcus pneumoniae capsular polysaccharide 5 and a preparation method thereof.
Background
Streptococcus pneumoniae (Streptococcus pneumoniae) can cause invasive diseases such as meningitis and pneumonia, and non-invasive diseases such as otitis media. Currently, the main defenses against streptococcus pneumoniae are polysaccharide vaccines and conjugate vaccines, the protective effect of the former in adults has been demonstrated, and the protective power in infants and young children is weak for the main susceptible population of streptococcus pneumoniae. Since the first time in 2000, conjugate vaccines were introduced into immunization programs or approved for marketing in many countries around the world, and have recently been recommended for adults, as they can cause effective protection and immune memory in infants. The multivalent pneumococcal conjugate vaccines currently available in the market are mainly 7-valent pneumococcal conjugate vaccine (trade name Pel, prevnar) produced by Hui (Hui's) and 13-valent pneumococcal conjugate vaccine (trade name Pel 13, prevnar 13) produced by upgrade products and 11-valent pneumococcal conjugate vaccine (trade name Synforix) produced by GSK.
The immunogenicity of polysaccharide-protein conjugates depends on the molecular weight of the polysaccharide, the activation and conjugation process, the type of carrier protein, the purification method of the conjugate, the quality control criteria of the conjugate, and the influence of multiple factors such as adjuvants, which are interrelated, wherein each serotype pneumococcal polysaccharide has completely different polysaccharide repeating units, and the substituent groups on the polysaccharide repeating units also have great differences, which makes the process research and the quality criteria research of the polysaccharide conjugate vaccine of the multiple pneumococcal polysaccharide face great technical challenges.
The activation and binding processes are also most critical to the immunogenicity of the conjugate vaccine, and many methods for binding polysaccharides and proteins are reported in literature, such as reductive amination, amide condensation, thioether condensation, disulfide reaction, thiocarbamoylation, O-alkylisourea reaction, diazo binding reaction, etc., with reductive amination and amide condensation being the most common.
Reductive amination is the reaction between aldehyde groups on a polysaccharide and amino groups of a carrier protein to form the corresponding schiff base, which can be selectively reduced to a very stable saturated carbon-nitrogen bond in the presence of sodium cyanoborohydride (NaCNBH 3). In addition, the reductive amination is carried out in aqueous solution under milder conditions. After binding, unreacted aldehyde groups can be subsequently blocked by reduction via sodium borohydride (NaBH 4). The conjugate can then be purified (e.g., by ultrafiltration/diafiltration) to produce the final bulk polysaccharide-protein conjugate in succinate buffered saline, using this method for pyrotechnical 13 and V114 of merck, usa.
The best patent (CN 103599529B) mentions that the conjugate can be prepared by reductive amination using NaIO 4 for 16-18 hours at room temperature, followed by ultrafiltration to give an activated polysaccharide, lyophilization of the activated polysaccharide, reconstitution with buffer, and reaction at ph7.2 for 96 hours at 30 ℃.
The merck company patent (CN 102858365A) mentions that the type 5 polysaccharide is first subjected to molecular weight degradation using a pressure of 0-1000bar, the degradation products are then activated for 3-20 hours at 2-6 ℃, the activated polysaccharide is obtained by ultrafiltration concentration, and then the activated polysaccharide is reacted with carrier protein according to a ratio of 0.2-2:1, and reacting at 2-8 ℃ for 48-120 hours under the condition of pH6.3-8.4, and preparing the conjugate by a reductive amination method.
Both cases are water phase combination reaction, and the reaction time exceeds 96 hours, and as the reductive amination reaction is mild, the combination reaction time is generally longer, and the overlong reaction time is a test on the stability of the activated polysaccharide, the polysaccharide groups are lost in the combination process, and the cost and risk of the prolonged reaction time are increased.
The amide condensation is generally carried out by activating capsular saccharide with cyanogen bromide (CNBr) or cyanating agent (1-cyano-4-dimethylamino-pyridine tetrafluoroboric acid, CDAP), adding adipoyl hydrazine (ADH) or other difunctional nucleophilic spacer into the activated capsular saccharide to form capsular saccharide-ADH derivative, and covalently binding with carboxyl group on protein carrier via carbodiimide (EDAC) mediated condensation. The production process of the Hib-TT combined vaccine of the marketed vaccine adopts the circuit. The activation reaction is carried out for 10-30 minutes, the binding reaction is carried out for 2-4 hours, the reaction is rapid, but the carrier protein has amino groups besides carboxyl groups, and the self-polymerization or cross-linking products of the proteins are easy to occur in the amide condensation reaction.
The two methods of reductive amination and amide condensation have advantages and disadvantages respectively: the reductive amination reaction is mild, the protein self-polymerization phenomenon can not be generated, the oxidation of NaIO 4 can possibly lead to polysaccharide skeleton cracking and epitope group loss, antigen damage can be caused in the combining process, the amide condensation reaction is rapid, antigen damage can not be caused in the activation stage, but the polysaccharide-protein conjugate can be generated in the combining stage, the conjugate with poor effect of polysaccharide-protein-polysaccharide can be generated, and meanwhile, the increase of the amount of carrier protein also brings risks to immunogenicity in multivalent vaccines. The specific selection of which binding method is also determined by the nature of the polysaccharide, protein.
The pneumococcal type 5 polysaccharide has a specific structure, as shown in FIG. 1, and contains three ortho-dihydroxyl groups in the molecule, one in the main chain (beta-D-ManpNAc), two in the side chains (alpha-L-PnepNAc, beta-D-GlcpA), and a six-membered cyclic carbonyl group (beta-D-Sugp). According to the polysaccharide structure, three ortho-dihydroxyl groups of the 5-type capsular polysaccharide can be used as oxidation sites of periodate, wherein alpha-L-PnepNAc of a side chain is cis-ortho-dihydroxyl, the other two sites are trans-ortho-dihydroxyl, the activity of the three sites is different when NaIO 4 is oxidized, the cis-ortho-dihydroxyl of the side chain has the maximum reaction activity, the obtained active product has the loss of active groups in the subsequent process steps, the structural integrity of an antigen is damaged, and the existence of a hexacyclic ring carbonyl also interferes with the detection of aldehyde group content generated by the oxidation of NaIO 4.
To overcome the interference of six-membered ring carbonyl groups on aldehyde group detection and avoid generation of new substances X, the Pasteur patent (US 20040170638A 1) mentions that NaBH 4 can be used for reducing ring carbonyl groups contained in molecules into alcohols under alkaline conditions, then the ortho-dihydroxyl groups in the sugar structure are oxidized by NaIO 4 to produce aldehyde groups, and then the aldehyde groups are condensed with amino groups of carrier proteins under the catalysis of NaCNBH 3 to generate a Schiff base structure. This approach of first reducing is a disruption to the polysaccharide antigen, leading to loss of specific epitopes, possibly leading to loss of immunogenicity and protective effects, and after first reducing, the polysaccharide loss is nearly half.
In view of the foregoing, it is desirable to develop a method for preparing streptococcus pneumoniae capsular polysaccharide vaccines that ensures antigen integrity and immunogenicity of the polysaccharide carrier protein conjugate and avoids the production of protein self-polymers, directed against the specific structure of the serotype 5 polysaccharide.
Disclosure of Invention
The invention aims to provide a vaccine containing streptococcus pneumoniae capsular polysaccharide 5 and a preparation method thereof. In the vaccine prepared by the method, streptococcus pneumoniae capsular polysaccharide and carrier protein form good combination, the integrity of polysaccharide antigen is well maintained in the preparation process flow, and no protein self-polymer is generated in the combination.
In order to achieve the aim of the invention, the inventor has found that by searching and optimizing each step and parameter in the process flow through a large number of experiments, the method of adopting low-temperature activation and organic phase to participate in the combination process can reduce the damage degree of polysaccharide groups as much as possible in the activation stage of polysaccharide and reduce the loss degree of groups in the combination process, and the obtained polysaccharide-protein conjugate has good antigen integrity and no protein self-poly (polysaccharide-protein-polysaccharide) product. The method is applicable to the preparation of the special structure of the streptococcus pneumoniae capsular polysaccharide and protein combination, and is also applicable to the combination of the streptococcus pneumoniae capsular polysaccharide and carrier protein of other serotypes except the type 5. The specific technical scheme is as follows:
The invention provides a preparation method of streptococcus pneumoniae capsular polysaccharide-protein combined vaccine stock solution, which comprises the steps of (1) degrading streptococcus pneumoniae capsular polysaccharide respectively; (2) activation; (3) lyophilization or precipitation; (4) Re-dissolving the lyophilized or precipitated activated streptococcus pneumoniae capsular polysaccharide, combining with a carrier protein and blocking; (5) purifying the conjugate vaccine stock solution.
The streptococcus pneumoniae capsular polysaccharide according to the invention is prepared according to the prior art or is commercially available, e.g. by culturing streptococcus pneumoniae, inactivating, extracting the inactivated streptococcus pneumoniae capsular polysaccharide and purifying it.
Degradation is to reduce viscosity and avoid other factors affecting the filtration effect. The streptococcus pneumoniae capsular polysaccharides are degraded to polysaccharides having a molecular weight of 100-850kDa, preferably 150-650kDa, by degradation methods well known in the art, e.g. using mild acid or physical methods. The acid includes, but is not limited to, acetic acid, citric acid, or carbonic acid; such physical methods include, but are not limited to, high pressure homogeneous or ultrasonic disruption. In the examples of the present invention, high pressure homogeneous mode is preferably used to degrade polysaccharide.
In the method, the activation in the step (2) is carried out by adding buffer solution and water for injection into the degraded streptococcus pneumoniae capsular polysaccharide stock solution obtained in the step (1), and then adding oxidant for activation; the oxidizing agent is capable of oxidizing the ortho-hydroxyl group to an aldehyde.
The oxidant is sodium periodate, potassium periodate or periodic acid.
The buffer solution is phosphate buffer solution, HEPES, MOPS or acetic acid-sodium acetate buffer solution; the concentration of the buffer solution is 10-100mM, and the pH value is 3-7.
After the buffer solution and the water for injection are added, the final concentration of the streptococcus pneumoniae capsular polysaccharide is 1-3g/L.
Preferably, the activation of step (2) is by adding 0.1 to 0.5 molar equivalents of oxidizing agent to the Streptococcus pneumoniae capsular polysaccharide solution after addition of buffer and WFI.
The conditions for the activation by adding the oxidant are: the temperature is 2-8 ℃, the pH value is 3.0-7.2, and the time is 4-24 hours.
Preferably, the conditions for activation by addition of the oxidizing agent are: the temperature is 2-8 ℃ and 4-16 hours.
More preferably, the conditions for activation by addition of the oxidizing agent are: the temperature is 2-8 ℃, the pH value is 4.0-6.5, and the time is 4-8 hours.
The activation method of the step (2) can effectively reduce the damage and loss of the groups of all the serotype streptococcus pneumoniae capsular polysaccharides including Pn5 and furthest maintain the integrity of capsular polysaccharide antigens. The capsular polysaccharide activated by the method of the invention has an activation degree of 1-25, preferably 5-15, and a molecular weight of 100-850kDa, preferably 150-650 kDa.
And (3) after the activation in the step (2), sterilizing, filtering and washing to obtain the purified activated streptococcus pneumoniae capsular polysaccharide.
The activated polysaccharide is added with a lyoprotectant such as sucrose or other substances which have filling/supporting function and are inert in chemical reaction as the lyoprotectant, and is frozen into a shell structure (shell) after being pre-frozen. And calculating the required amount of carrier protein according to the feeding ratio, pre-freezing into a shell structure, and freeze-drying. The freeze-drying in the step (3) of the method comprises the steps of mixing activated streptococcus pneumoniae capsular polysaccharide with excipient according to a mass ratio of 1:10-50, and freeze-drying the excipient, wherein the excipient is non-reducing neutral chemical substances such as sucrose, trehalose, mannitol, sorbitol and the like, and the activated polysaccharide and carrier protein required by combination can be independently freeze-dried respectively, or the liquid activated polysaccharide and the carrier protein can be mixed and freeze-dried. The activated polysaccharide and/or carrier protein after lyophilization is stored at-20+ -5deg.C or below.
Or preparing polysaccharide precipitate by ethanol precipitation without freeze-drying, and storing at-20+ -5deg.C or below. When taken out for use, it is dissolved in an organic solvent and then bound to a carrier protein.
And (4) re-dissolving the freeze-dried activated streptococcus pneumoniae capsular polysaccharide with an organic solvent, and re-dissolving or dissolving the carrier protein with the same organic solvent. Or reconstituting a lyophilized mixture of the polysaccharide and carrier protein with an organic solvent.
According to the invention, repeated experiments are carried out on a plurality of organic solvents, and the fact that DMF or DMSO is selected as the organic solvent is found that the polysaccharide and protein have the best binding effect, so that the group loss of all serotypes of streptococcus pneumoniae capsular polysaccharide including pneumopolysaccharide 5 and carrier protein in the binding process can be effectively reduced, the antigen integrity is maintained, and the binding yield is improved.
The re-dissolved streptococcus pneumoniae capsular polysaccharide is mixed with re-dissolved or dissolved carrier protein according to the mass ratio of 1:2-2:1, and after complete dissolution, 1.0-1.5 molar equivalent sodium cyanoborohydride is added for initial binding, and the mixture is incubated for 12-48 hours at 23+/-2 ℃.
In the combination reaction system, the final concentration of the streptococcus pneumoniae capsular polysaccharide is 0.5-4g/L.
Further, the binding reaction is terminated by adding 1.0 to 1.5 molar equivalents of sodium cyanoborohydride to block unreacted aldehyde groups, and this blocking reaction is continued at 23.+ -. 2 ℃ for 18.+ -. 6 hours.
The conjugate after the combination in the step (4) has a molecular weight of 2000-20000kDa, and the mass ratio of polysaccharide to carrier protein is 0.5-2.5.
The carrier proteins are CRM197, tetanus toxoid TT, pertussis toxoid, cholera toxoid, escherichia coli LT, escherichia coli ST, and purified protein derivatives from pseudomonas aeruginosa exotoxin a, outer membrane complex C, porin, transferrin binding protein, pneumolysin, pneumococcal surface protein a, pneumococcal adhesin protein, C5a peptidase from group a or group B streptococcus, haemophilus influenzae protein D, ovalbumin, keyhole limpet hemocyanin, bovine serum albumin, or tuberculin.
CRM197 or tetanus toxoid TT are preferred.
In the preparation method provided by the invention, the purification in the step (5) is to dilute the solution combined and sealed in the step (4) and remove an organic phase, then filter, purify by column chromatography, elute, collect the eluent, ultrafiltrate, concentrate, sterilize and filter.
According to the properties of the conjugate, free polysaccharide and free protein, the conjugate can be subjected to ultrafiltration dialysis or precipitation technology or chromatography technology such as molecular sieve, hydrophobic chromatography, ion exchange and the like or a combination of several of the above technologies to remove unbound protein and polysaccharide, and after purification, the conjugate is subjected to sterilization filtration by a 0.2 μm filter, thus obtaining the pneumococcal capsular polysaccharide-protein monovalent conjugate.
One specific purification method presented in the examples of the present invention is:
diluting the solution after the step (4) is combined and blocked by using cooled 5mM succinate with pH of 6.0 and 0.9% saline with the concentration of 1:6-15, then using 100-300kDa MWCO cellulose membrane tangential flow ultrafiltration to purify and remove organic phase solvents, filtering the diluted conjugate solution by a 5 mu m filter, purifying by using a Sepharose 4FF chromatographic medium, eluting by using a physiological saline solution, monitoring the wavelength to be 206nm and 280nm, collecting eluent, and performing ultrafiltration concentration and sterilization filtration to obtain the combined vaccine stock solution.
In the above preparation method of the present invention, the streptococcus pneumoniae capsular polysaccharide is selected from capsular polysaccharides produced by streptococcus pneumoniae of one or more of the following serotypes: type 1, type 2, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 8, type 9N, type 9V, type 10A, type 11A, type 12F, type 14, type 15B, type 15C, type 17F, type 18C, type 19A, type 19F, type 20, type 22F, type 23A, type 23F, type 24F, type 33F, type 45, and type 46.
Preferably, the streptococcus pneumoniae capsular polysaccharide is a capsular polysaccharide produced by streptococcus pneumoniae serotype 5.
More preferably, the streptococcus pneumoniae capsular polysaccharide is selected from type 1, type 2, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 8, type 9N, type 9V, type 10A, type 11A, type 12F, type 14, type 15B, type 17F, type 18C, type 19A, type 19F, type 20, type 22F, type 23F, and type 33F, to produce a type 24 streptococcus pneumoniae capsular polysaccharide.
The streptococcus pneumoniae capsular polysaccharide-protein conjugate vaccine stock solution prepared by the preparation method belongs to the protection scope of the invention.
The invention also provides a streptococcus pneumoniae capsular polysaccharide-protein conjugate, which is prepared by the following steps: degrading streptococcus pneumoniae capsular polysaccharides respectively (1); (2) activation; (3) lyophilization or precipitation; (4) The lyophilized or precipitated activated streptococcus pneumoniae capsular polysaccharide is reconstituted, combined with a carrier protein and blocked.
In the invention, the prepared activated streptococcus pneumoniae capsular polysaccharide can be used for preparing polysaccharide precipitate by technical means such as ethanol precipitation, and the polysaccharide precipitate is stored at-20+/-5 ℃ and below for standby after being washed by absolute ethanol.
The preparation steps (1) - (4) of the streptococcus pneumoniae capsular polysaccharide-protein conjugate follow the steps (1) - (4) and specific and preferred technical schemes of the preparation method of the streptococcus pneumoniae capsular polysaccharide-protein conjugate vaccine stock solution.
Preferably, the present invention provides a capsular polysaccharide-protein conjugate comprising Streptococcus pneumoniae type 5.
More preferably, the present invention provides a streptococcus pneumoniae capsular polysaccharide-protein conjugate of type 5.
The invention provides the application of the streptococcus pneumoniae capsular polysaccharide-protein conjugate vaccine stock solution or the streptococcus pneumoniae capsular polysaccharide-protein conjugate prepared by the preparation method in preparing medicines for preventing or treating diseases caused by streptococcus pneumoniae.
The invention also provides application of the streptococcus pneumoniae capsular polysaccharide-protein conjugate vaccine stock solution or the streptococcus pneumoniae capsular polysaccharide-protein conjugate prepared by the preparation method in preparation of biological products, wherein the biological products are monovalent vaccines, multivalent vaccines or combined vaccines.
The invention provides a vaccine containing the streptococcus pneumoniae capsular polysaccharide-protein conjugate vaccine stock solution or the streptococcus pneumoniae capsular polysaccharide-protein conjugate.
Preferably, the vaccine is a monovalent vaccine having a streptococcus pneumoniae capsular polysaccharide selected from capsular polysaccharides produced by streptococcus pneumoniae of any one of the following serotypes: type 1, type 2, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 8, type 9N, type 9V, type 10A, type 11A, type 12F, type 14, type 15B, type 15C, type 17F, type 18C, type 19A, type 19F, type 20, type 22F, type 23A, type 23F, type 24F, type 33F, type 45, and type 46. More preferably, the streptococcus pneumoniae capsular polysaccharide of the monovalent vaccine is selected from capsular polysaccharides produced by streptococcus pneumoniae serotype 5.
Preferably, the vaccine is a multivalent vaccine, the streptococcus pneumoniae capsular polysaccharide of which is selected from capsular polysaccharides produced by streptococcus pneumoniae of any two or more of the following serotypes: type 1, type 2, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 8, type 9N, type 9V, type 10A, type 11A, type 12F, type 14, type 15B, type 15C, type 17F, type 18C, type 19A, type 19F, type 20, type 22F, type 23A, type 23F, type 24F, type 33F, type 45, and type 46. More preferably, the streptococcus pneumoniae capsular polysaccharide of the multivalent vaccine is at least selected from capsular polysaccharides produced by streptococcus pneumoniae serotype 5.
It can be appreciated in the art that based on the prior art, the multivalent vaccine provided by the invention is:
24-valent vaccine: type 1, type 2, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 8, type 9N, type 9V, type 10A, type 11A, type 12F, type 14, type 15B, type 17F, type 18C, type 19A, type 19F, type 20, type 22F, type 23F, and type 33F; or (b)
Preferably, the vaccine may also be a combination vaccine comprising at least capsular polysaccharide from streptococcus pneumoniae serotype 5.
In the various vaccines provided by the invention, the content of streptococcus pneumoniae capsular polysaccharide of each serotype is 2-8 mug/mL. Preferably 4. Mu.g/mL.
If the vaccine provided by the invention contains 6B serotype streptococcus pneumoniae capsular polysaccharide, the content of the serotype streptococcus pneumoniae capsular polysaccharide is 4-16 mug/mL, and the content of the other serotype streptococcus pneumoniae capsular polysaccharides is 2-8 mug/mL. Preferably, the amount of Streptococcus pneumoniae capsular polysaccharide 6B is 8 μg/mL.
In the vaccine provided by the invention, the content of carrier protein is 30-90 mug/mL.
The vaccine of the invention further comprises an excipient and/or adjuvant; the excipient is sodium chloride, succinic acid or phosphate buffer; the adjuvant is an aluminum-based adjuvant, preferably aluminum hydroxide, aluminum phosphate, aluminum sulfate; the final concentration of the aluminium-based adjuvant in the vaccine is 0.2-1.0mg/mL, preferably 0.25-0.5mg/mL.
The vaccine of the present invention may be injected by intramuscular, subcutaneous, intradermal routes or administered via mucosal routes.
The vaccine of the present invention is suitable for vaccinating infants, children and/or adults of ages 2,4, 6 and 12-15 months of age.
The invention has the beneficial effects that: aiming at the problems that in the prior art, antigen immune epitopes are lost in the process of preparing streptococcus pneumoniae capsular polysaccharide-protein conjugate, protein self-polymers are easy to form, so that immunogenicity is reduced, periodate is adopted to activate polysaccharide to form aldehyde groups, the pH, temperature and time during activation are strictly controlled, the group damage and loss in the process of activating streptococcus pneumoniae type 5 capsular polysaccharide are effectively reduced, and the antigen integrity is maintained to a greater extent.
The activated polysaccharide is freeze-dried and precipitated to obtain solid activated polysaccharide, and after re-dissolution, the solid activated polysaccharide and carrier protein (CRM 197) are subjected to reductive amination reaction in an organic solvent DMSO or DMF to form a polysaccharide-protein conjugate. Compared with the prior art that reductive amination reaction is carried out in an aqueous phase, the reductive amination reaction is carried out by adopting an organic phase DMSO or DMF, so that the antigen integrity can be ensured, the binding efficiency of polysaccharide and carrier protein can be improved, and the stability of the obtained conjugate is good. The reductive amination reaction product is free of protein-protein ineffective polymers such as polysaccharide-protein-polysaccharide compared to the amide condensation reaction. Research shows that the polysaccharide-protein conjugate prepared by the method has a molecular weight of 2000-20000kDa, the polysaccharide/protein ratio is between 0.5 and 2.5, and the concentration of the antibody in an animal body is obviously higher than that of polysaccharide-protein conjugates prepared by other processes.
Drawings
FIG. 1 is a block diagram of pneumococcal capsular polysaccharide 5. Wherein A is a sugar structural formula, and B is a sugar structural formula.
FIG. 2 shows HNMR patterns of pneumococcal capsular polysaccharide 5 and pneumococcal activated capsular polysaccharide type 5, wherein A is the HNMR pattern of pneumococcal capsular polysaccharide and B is the HNMR pattern of the activated capsular polysaccharide.
FIG. 3 shows an immunodouble-spread gel of the conjugate, wherein 1 is a normal saline control, 2 is a polysaccharide stock solution, 3 is a pretreated polysaccharide, 4 is an activated polysaccharide, and 5-6 are binding stock solutions (polysaccharide-protein conjugates).
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The examples we follow use standard techniques, well known and conventional to those skilled in the art unless otherwise specified. The examples are illustrative, but do not limit the invention.
EXAMPLE 1 preparation of Streptococcus pneumoniae serotype 5 capsular polysaccharide-protein conjugates
Serotype 5 capsular polysaccharides can be obtained directly from bacteria using isolation methods known to those skilled in the art. Serotype 5 pneumococcal (Streptococcus pneumoniae) strain was derived from the national food and drug verification institute. And carrying out passage and library establishment on the strains. Inoculating the strain into liquid culture medium, culturing at 35+ -2deg.C, adding sodium deoxycholate to lyse bacteria and release capsular polysaccharide at late stage of logarithmic growth, centrifuging, and collecting supernatant. The supernatant is concentrated by ultrafiltration 3-10 times through an ultrafiltration membrane bag with a molecular weight cutoff of 100 or 300 kDa. Ultrafiltering with phosphate buffer solution of pH7.0, adding absolute alcohol to reach alcohol concentration of 25-45%, stirring, and centrifuging to remove nucleic acid. Adding ethanol into the supernatant to reach the final concentration of 55% -80%, stirring, centrifuging, removing the supernatant, and collecting the precipitate.
Adding water to dissolve the precipitate, adding phenol with the volume 1-3 times of the solution volume for multiple extraction, and removing protein. Ethanol is added into the supernatant to lead the final concentration to reach 55% -80%, the precipitate is collected after centrifugation, the precipitate is redissolved by adopting injection water, and the pneumococcal capsular polysaccharide of 5 type is obtained after sterilization and filtration by a sterile filter of 0.2 mu m. Polysaccharide identification adopts HNMR method (figure 2) and immune double diffusion method (figure 3). The HNMR pattern is consistent with the report of the literature, and the immune double diffusion shows positive, which indicates that the pneumococcal capsular polysaccharide type 5 is obtained by purification.
1. Degradation of streptococcus pneumoniae serotype 5 capsular polysaccharides
Prior to activation, the polysaccharide is degraded in molecular weight of the Pn5 capsular polysaccharide stock using acid hydrolysis or high pressure homogenizers to facilitate subsequent activation and binding reactions.
Acid hydrolysis: diluting the polysaccharide liquid with injectable water to 1-5mg/ml, adding hydrochloric acid or glacial acetic acid or other non-oxidizing acid, heating to 85-94 deg.C, incubating for 1-6 hr, sampling during hydrolysis, rapidly cooling with ice water bath, regulating pH to neutrality with alkali, and monitoring molecular weight degradation with SEC-MALLS-RI.
High pressure homogeneous phase: diluting the polysaccharide solution to 1-5mg/ml with injectable water or physiological saline, allowing to act under 1000-1500psi pressure for 10-20 cycles with high pressure homogenizer, sampling at the end of each cycle, and monitoring molecular weight degradation with SEC-MALLS-RI.
SEC-MALLS-RI is used to determine the molecular weight of polysaccharides and polysaccharide-protein conjugates. The polysaccharide was separated by hydrodynamic volume using SEC. Molecular weight was determined using Refractive Index (RI) and multi-angle laser light scattering (MALLS) detectors. When light interacts with a substance, it scatters and the amount of scattered light is related to the concentration of the substance, the square of dn/dc (specific index increment) and the molar mass. The molecular weight measurement is calculated based on the light signal scattered from the MALLS detector and the reading of the concentration signal from the RI detector.
The results are shown in the following table 1, from table 1, the molecular weight of streptococcus pneumoniae serotype 5 capsular polysaccharide (hereinafter referred to as type 5 capsular polysaccharide) is more than 2000kDa, the molecular weight of the polysaccharide is reduced to 685.1kDa-136.10kDa after acid hydrolysis for 1-6 hours, the dispersity Mw/Mn is reduced from 1.302 to 1.250 and 1.107, and the polysaccharide molecules are more uniform; as seen from Table 2, the high pressure was homogeneous for 10-20 cycles, the molecular weight was reduced to 810.80kDa and 254.80kDa, the dispersity Mw/Mn was reduced from 1.591 to 1.274 and 1.166, and the polysaccharide molecules were more uniform. The 5-type capsular polysaccharide before and after degradation meets the European pharmacopoeia requirements, and the immune double diffusion results are positive, so that the integrity of the antigen is maintained (figure 3).
Table 15 comparison of quality before and after degradation of capsular polysaccharide
Table 25 comparison of quality before and after degradation of capsular polysaccharide
WFI is water for injection.
2. Activation of streptococcus pneumoniae serotype 5 capsular polysaccharides
The molecular weight of the degraded type 5 capsular polysaccharide solution was added with 10-100mM acetic acid-sodium acetate buffer (pH 4.8.+ -. 0.5) and WFI to obtain a final polysaccharide concentration of 1-3g/L, the reaction temperature was adjusted to 2-8deg.C, and the oxidation reaction was started by adding about 0.1-0.5 molar equivalents of sodium periodate. The oxidation reaction was carried out at 2-8deg.C for about 4-24 hours, after activation, 10-20 times the volume of the activated polysaccharide was washed with WFI using a 10kDa MWCO membrane, and the activated polysaccharide was obtained by sterilization and filtration, the results of which are shown in Table 3.
TABLE 3 activation results
* Degree of activation = polysaccharide content (nmol/ml)/reducing sugar content (nmol/ml)
The detection method of the activated polysaccharide comprises the following steps: (i) sugar concentration as determined by colorimetry; (ii) aldehyde concentration by colorimetric determination; (iii) Degree of activation (degree of oxidation) (iv) molecular weight by SEC-MALLS.
The degree of oxidation (do=moles of saccharide repeat units/moles of aldehyde groups) of the activated polysaccharide was determined as follows: the number of moles of saccharide repeat units is determined by a variety of colorimetric methods, for example by using the aldonic acid method. The polysaccharide is hydrolyzed with sulfuric acid solution containing sodium tetraborate under the action of high temperature, and the hydrolysis product is further reacted with m-hydroxy biphenyl to generate pink derivative, which is absorbed by ultraviolet, and the absorbance of the pink derivative is read out by spectrophotometry at 524 nm. Within the range of the assay, the absorbance is proportional to the amount of uronic acid present.
MBTH colorimetry was also used to determine the moles of aldehyde. MBTH assays involve the formation of azine compounds by reacting an aldehyde group (from a given sample) with 3-methyl-2-benzothiazolone hydrazone (MBTH assay reagent). Excess 3-methyl-2-benzothiazolone hydrazone is oxidized to form the active cation. The active cations and azine react to form a blue chromophore. The chromophore formed was then spectrally read at 650 nm.
Capsular polysaccharide structural integrity assay: the content of polysaccharide solution, activated polysaccharide, and PnepNAc sugar was analyzed by ion chromatography using literature method (Talaga P,Vialle S,Moreau M.Development of a high-performance anion-exchange chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates[J].Vaccine,2002,20(19):2474-2484.),, and the structural integrity was calculated using Fuc as an internal reference, and the results are shown in table 4.
Relative antigenicity detection: the antigenicity of the known polysaccharide and the activated polysaccharide is detected by an immune turbidity method, and the antigenicity of the activated polysaccharide is compared with the antigenicity of the initial polysaccharide to obtain relative antigenicity, so that the retention of the epitope of the activated polysaccharide is evaluated.
Activated polysaccharide-1 and activated polysaccharide-2 were prepared by the method in example 1. Activated polysaccharide-3 was prepared using the conditions of patent CN103599529B, and the resulting activated polysaccharide was characterized and the results are shown in table 4.
TABLE 4 structural integrity comparison of different activation processes
As shown in the above table, the low temperature activation gave better structural integrity than the room temperature activation to give the activated polysaccharide. 3. Lyophilization or precipitation of activated streptococcus pneumoniae serotype 5 capsular polysaccharide
The activated polysaccharide was mixed with sucrose to a ratio of 25 grams of sucrose per gram of activated polysaccharide. The bottles of the mixed mixture were then freeze-dried. Calculated amounts of CRM197 protein were separately shell frozen (shell-frozen) and lyophilized. The lyophilized samples were stored at-20.+ -. 5 ℃.
Adding 20-50% ethanol at 2-8deg.C for precipitating for 12-24 hr, centrifuging to obtain precipitate, washing the precipitate with anhydrous ethanol for 3-4 times, collecting precipitate, volatilizing, weighing to obtain solid activated polysaccharide, and preserving at-20+ -5deg.C or below.
4. Binding to CRM197 after reconstitution of Streptococcus pneumoniae serotype 5 capsular polysaccharide
(1) Redissolving activated polysaccharide and CRM197 protein
The lyophilized activated polysaccharide was reconstituted in Dimethylsulfoxide (DMSO) at a saccharide concentration of 1-8mg/ml, and once the polysaccharide was completely dissolved, lyophilized CRM197 was dissolved in DMSO at a protein concentration of 1-8 mg/ml.
The solid activated polysaccharide obtained by ethanol precipitation is redissolved in dimethyl sulfoxide (DMSO) according to sugar concentration of 1-8mg/ml, and once the polysaccharide is completely dissolved, lyophilized CRM197 is dissolved in DMSO according to protein concentration of 1-8 mg/ml.
(2) Bonding and sealing
The reconstituted activated polysaccharide was combined with reconstituted CRM197 in a reaction vessel (feed ratio 0.8:1) and after complete dissolution, sodium cyanoborohydride was added to initiate binding. The final polysaccharide concentration in the reaction solution is about 0.5-4g/L. Binding was initiated by adding 1.0-1.5 molar equivalents of sodium cyanoborohydride to the reaction mixture and incubating at 23±2 ℃ for 24-48 hours. The conjugation reaction was terminated by adding 1.0-1.5 molar equivalents of sodium cyanoborohydride to block unreacted aldehyde groups. The blocking reaction was continued at 23.+ -. 2 ℃ for 18.+ -. 6 hours.
Meanwhile, the water phase combination product is used as a control, and the reaction conditions are as follows: the lyophilized activated polysaccharide was reconstituted with PB buffer and lyophilized CRM197 was also reconstituted with PB buffer, and the reconstituted activated polysaccharide was combined with reconstituted CRM197 in a reaction vessel (feed ratio 0.8:1) and after complete dissolution, sodium cyanoborohydride was added to initiate binding. The final polysaccharide concentration in the reaction solution was about 10-12g/L. Binding was initiated by adding 1.0-1.5 molar equivalents of sodium cyanoborohydride to the reaction mixture and incubating at 30±2 ℃ for 72-96 hours. The conjugation reaction was terminated by adding 1.0-1.5 molar equivalents of sodium cyanoborohydride to block unreacted aldehyde groups. The blocking reaction was continued at 23.+ -. 2 ℃ for 18.+ -. 6 hours.
The DMSO-bound product and the water-bound reaction product were analyzed for PnepNAc sugar content by ion chromatography, respectively, and the structural integrity was calculated using Fuc as an internal reference, and the results are shown in table 5. In Table 5, conjugate-1 is DMSO-bound and conjugate-2 is conventional water-bound.
Table 5 comparison of structural integrity of different bonding processes
As shown in the above table, the DMSO-phase binding product has higher antigen integrity, lower polysaccharide/protein ratio, higher protein yield, and higher bound saccharide yield than the water-phase binding product. The better polysaccharide structural integrity ensures the better polysaccharide identification of the generated antibody against streptococcus pneumoniae, and can obtain better protection effect in clinical use.
5. Purification of conjugates
The blocked conjugate solution was then cooled with 5mM succinate-0.9% saline (pH 6.0) 1:10-1:15 diluting, then using 100-300kDa MWCO cellulose membrane tangential flow filtration to purify and remove DMSO, filtering the diluted conjugate solution through a 5 mu m filter, purifying by using a Sepharose 4FF chromatographic medium, eluting with physiological saline solution, monitoring the wavelength to be 206nm and 280nm, collecting eluent near the external water, and performing ultrafiltration concentration, sterilization and filtration to obtain the conjugate stock solution. The results of three consecutive purification runs are shown in Table 6.
TABLE 6 Streptococcus pneumoniae serotype 5 capsular polysaccharide-CRM 197 conjugate results
The stock solution of the monovalent conjugate requires detection of the polysaccharide/protein ratio, the free polysaccharide content, the free protein content:
the ratio of the polysaccharide concentration to the protein concentration in the polysaccharide/protein ratio conjugate stock solution is the polysaccharide/protein ratio.
B determination of free polysaccharide content free sugar content: 1ml of the combined sample is added with 1% DOC solution, mixed evenly and kept stand in ice water bath for 30min. Mu.l of 100% trichloroacetic acid was added, and after mixing, the mixture was centrifuged at 12000rpm at 4℃for 15 minutes. The content of free sugar in the supernatant was measured by carbazole sulfate method or ELISA method.
C free protein content the free protein content was analyzed by HPLC and UV detected (214 nm). The mobile phase was 0.2M NaCl, flow rate was 1ml/min. The elution conditions should allow separation of the free protein from the conjugate.
The free protein in the conjugate was determined by a calibration curve (from 0-50. Mu.g/ml carrier protein). Free protein content% = free protein (μg/ml)/total protein content x 100% determined using Lowry method (μg/ml).
EXAMPLE 2 evaluation of Streptococcus pneumoniae serotype 5 capsular polysaccharide-protein conjugates
Capsular polysaccharide structural integrity assay: the content of PnepNAc saccharide in the activated polysaccharide, the conjugated polysaccharide, was analyzed by ion chromatography using literature method (Talaga P,Vialle S,Moreau M.Development of a high-performance anion-exchange chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates[J].Vaccine,2002,20(19):2474-2484.),, and the structural integrity was calculated using Fuc as an internal reference, and the results are shown in table 7.
Evaluation of immunogenicity of capsular polysaccharide-protein conjugates: 18-22g BALB/c mice were immunized, and the polysaccharide content of each immunizing substance was 2. Mu.g/ml, 10 mice per group. 0. Two needles were used for intraperitoneal immunization at 14 days, 0.5ml each time, blood was collected at 28 days, antibody titer of the serotypes 5 pneumococcal capsular polysaccharide was detected by ELISA method, and bactericidal titer was detected by opsonophagocytosis assay (OPA) method.
The ELISA method is based on UAB method, adopting indirect enzyme-linked immunosorbent assay, diluting 5-type pneumococcal polysaccharide with PBS until polysaccharide concentration is about 1-10 mug/ml, respectively coating 100 mug/hole on an ELISA plate, coating at 2-8 ℃ overnight, adding blocking liquid after PBST washing the plate, blocking at 37 ℃ for 2 hours, serial diluting serum to be detected with 10% calf serum/PBS, adding 100 mug/hole on the blocked ELISA plate, reacting at 37 ℃ for 1 hour, adding HRP-marked anti-mouse IgG secondary antibody, reacting at 37 ℃ for 1 hour, washing the plate, developing, stopping reading at 450 nm. Serum positive transfer standard: negative control OD 2.1 times, serum antibody titer results were judged: after serial 10-fold dilution of serum, the highest serum positive transfer dilution was judged as the serum antibody titer of 10 X of the antibody.
The OPA method is based on the UAB method and is modified as follows: test serum was serially diluted 2.5-fold and added to the microtiter assay plates. Live serotype 5 target bacteria were added to the wells and the plates were shaken at 37 ℃ for 30 minutes. Differentiated HL-60 cells (phagocytes) and baby rabbit serum (3-4 weeks old, 6.25% final concentration) were added to the wells and the plates were shaken for 45 min at 37 ℃. To terminate the reaction, 80 μl of 0.9% nacl was added to all wells, mixed, and 10 μl aliquots were transferred to wells of MultiScreen HTS HV filter plates containing 200 μl of water. The liquid was filtered through the plate under vacuum and 150 μl of HySoy media was added to each well and filtered through. The filter plates were then incubated overnight at 37℃with 5% CO2 and then fixed with Destain solution (Bio-Rad). The plates were then stained with coomassie blue and destained once. Colonies were imaged and counted on Cellular Technology Limited (CTL) ImmunoSpot. The killing curve was plotted using the original colony count and OPA titers were calculated.
TABLE 7 structural integrity and effect comparison of different technologies
Conjugate 1 was prepared by the method disclosed in example 1. Conjugate 2 was prepared using the conditions of patent CN103599529B and the resulting conjugate was characterized and the results are shown in table 7. The polysaccharide/protein ratio was different for the 2 conjugates, with conjugate 1 having a lower ratio than conjugate 2; both polysaccharide and protein yields were higher for conjugate 1 than conjugate 2; in terms of structural integrity, conjugate 1 was higher than conjugate 2, and in terms of immunogenicity, both conjugates were able to produce antibodies with similar titers in mice, with conjugate 1 being higher than conjugate 2.
EXAMPLE 3 preparation of multivalent pneumococcal capsular polysaccharide conjugate vaccine
The 5-type monovalent conjugate prepared above was mixed with 1,3,4,6A,6B,7F,9V,14, 18C,19A,19F,23F-type monovalent conjugates, each conjugate was mixed at a polysaccharide final concentration of 4. Mu.g/ml and 6B-type conjugate at a polysaccharide final concentration of 8. Mu.g/ml, and the mixture was prepared by mixing with physiological saline as a solvent to prepare a 13-valent pneumococcal polysaccharide conjugate vaccine preparation for injection. The prepared 13-valent pneumococcal capsular polysaccharide conjugate vaccine is prepared into a freeze-dried preparation by adopting a low-temperature drying mode, and is re-dissolved by normal saline before use.
EXAMPLE 4 aluminium adsorption of multivalent pneumococcal capsular polysaccharide-protein conjugate
Three ways are used to adsorb multivalent pneumococcal capsular polysaccharide-protein conjugate to prepare vaccines.
(1) The purified pneumococcal capsular polysaccharide-protein monovalent conjugates of serotypes 1, 3,4, 5, 6A, 6B, 9V, 14, 18C, 19A, 19F and 23F were mixed and then with 0.5 μg/ml of aluminium phosphate adjuvant to give a polysaccharide content of: serotypes 6B 8. Mu.g/ml, serotypes 1, 3,4, 5, 6A, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F were each 4. Mu.g/ml, and a 13-valent pneumococcal capsular polysaccharide conjugate vaccine (13 vPnC) was prepared with a final aluminum adjuvant concentration of 0.25mg/ml.
(2) Pneumococcal capsular polysaccharide-protein monovalent conjugates of serotypes 1,3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A, 19F and 23F were mixed with 0.5mg/ml aluminium phosphate adjuvant to give polysaccharide content: the 6B serotypes were 8. Mu.g/ml and the remaining serotypes were 4. Mu.g/ml (the remaining serotypes were mixed in the comparative example) with an aluminum adjuvant concentration of 0.25mg/ml and the adsorbed solutions were then mixed in equal volumes to make the final product.
(3) To 0.5mg/ml of aluminium phosphate adjuvant, pneumococcal capsular polysaccharide-protein monovalent conjugates of serotypes 1,3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A, 19F and 23F were added sequentially one by one to mix with the adjuvant, the final polysaccharide content being: the 6B serotypes were 8. Mu.g/ml and the remaining serotypes were 4. Mu.g/ml (the remaining serotypes were mixed in the comparative example) with an aluminum adjuvant concentration of 0.25mg/ml and the adsorbed solutions were then mixed in equal volumes to make the final product.
The adsorption completeness of 13vPnC prepared by the adsorption mode and the recovery rate of the content of each polysaccharide are detected. The adsorption effect and the content of each type of polysaccharide of 13vPnC are detected by an immunonephelometry.
The calculation method of the adsorption rate of each type of polysaccharide and the recovery rate of each type of polysaccharide is as follows:
polysaccharide adsorption rate= (1-supernatant polysaccharide content/theoretical polysaccharide content) ×100%;
The polysaccharide adsorption rate of the vaccine reflects the ratio of the content of free polysaccharide types in the vaccine, which are not captured by the aluminum adjuvant, to the total theoretical polysaccharide types in the vaccine.
Recovery of polysaccharide from vaccine types: after the vaccine is dissociated, the content of each type of polysaccharide is detected, and the detected value is compared with the theoretical value of the content of the serotype polysaccharide, so that the recovery rate is required to be 70-130% of the theoretical value. The detection results of the mixing and then adsorbing proportioning mode are shown in table 8, the detection results of the mixing and proportioning mode are shown in table 9, and the detection results of the sequence adsorption of each type of conjugate are shown in table 10.
TABLE 8 adsorption completeness of polysaccharide of 13vPnC types and results of polysaccharide content of each type
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TABLE 9 adsorption completeness of polysaccharide of 13vPnC types and results of polysaccharide content of each type
TABLE 10 adsorption completeness of polysaccharide of 13vPnC types and results of polysaccharide content of each type
As can be seen from tables 8 to 10, the adsorption rate of the 13vPnC composition (vaccine) prepared by the above method was excellent, exceeding 75% (detection limit); the content of each polysaccharide after the vaccine dissociation is between 70% and 130%. The content of the vaccine is accurate, and the adsorption efficiency is high. The three adsorption modes are not different.
Adjusting the final concentration of aluminum content of the aluminum gel adjuvant to 0.25mg/ml and 4.0mg/ml by adopting aluminum hydroxide and aluminum sulfate; similar results were obtained for the preparation of multivalent pneumococcal conjugate compositions having an aluminium content of 0.125mg/ml and 2.0 mg/ml.
EXAMPLE 5 multivalent pneumococcal capsular polysaccharide conjugate vaccine immunogenicity analysis
The vaccine group 1 and the vaccine group 2 are respectively obtained by using the aluminum adsorption vaccine of the 13-valent pneumococcal capsular polysaccharide conjugate vaccine prepared in the examples 3 and 4, the type 5 polysaccharide-protein conjugate is respectively prepared by adopting a low-temperature activated organic phase binding process and a room-temperature activated water phase binding process, and the pneumococcal capsular polysaccharide mixture of the 13 serotypes and a physiological saline negative control group are respectively immunized 18-22g of BALB/c mice, the polysaccharide content of each immune substance is 6B 8 mug/ml, and the other serotypes are 4 mug/ml. 10 immunized mice per group. 0. Two needles were used for intraperitoneal immunization at day 14, 0.5ml each time, blood was collected at day 28, and the antibody titer of serum against all of the above-mentioned serotype pneumococcal capsular polysaccharides was measured by ELISA method, and the results are shown in table 11.
TABLE 11
The results show that after 2-needle immunization of mice with the pneumococcal capsular polysaccharide mixture of 13 serotypes, the antibody IgG levels of the mice are not different from those of the negative control group; after mice are immunized by the aluminum adsorption vaccine of the pneumococcal capsular polysaccharide conjugate vaccine of 13 price, the anti-IgG of serum is remarkably improved compared with a negative control, the antibody titer of other serotypes except for type 5 in the vaccine group 1 and the vaccine group 2 is not greatly different, the titer of polysaccharide-protein conjugate prepared by combining the two types 5 conjugates is higher than that of polysaccharide-protein conjugate prepared by combining the room temperature activated water phase, and the titer of the polysaccharide-protein conjugate is between 0.9 and 1.2.
EXAMPLE 6 formulation of multivalent pneumococcal capsular polysaccharide conjugate vaccine
According to the similar procedure as in example 1,2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F and other monovalent conjugates were prepared, each conjugate was mixed according to a polysaccharide final concentration of 2-4. Mu.g/ml and 6B conjugate was mixed according to a polysaccharide final concentration of 4-8. Mu.g/ml, and the mixture was prepared by mixing with physiological saline as a solvent to prepare a 24-valent pneumococcal conjugate vaccine injection preparation; and adsorbing the prepared 13-valent pneumococcal capsular polysaccharide conjugate vaccine with an aluminum adjuvant to prepare the aluminum-containing 13-valent pneumococcal conjugate vaccine.
18-22G BALB/c mice were immunized with the above 24-valent pneumococcal conjugate vaccine and 24 serotypes of pneumococcal capsular polysaccharide mixture, physiological saline negative control group, and 13-valent pneumococcal conjugate vaccine of example 5, each immunization having a polysaccharide content of 6B 8 μg/ml, and the remaining serotypes of 4 μg/ml. 10 immunized mice per group. 0. Two needles were used for intraperitoneal immunization at day 14, 0.5ml each time, blood was collected at day 28, and the antibody titer of serum against all of the above-mentioned serotype pneumococcal capsular polysaccharides was measured by ELISA method, and the results are shown in table 12.
Table 12
The results show that after 2-needle immunization of mice with pneumococcal capsular polysaccharide mixtures of 24 serotypes, the antibody IgG levels of the mice were not different from those of the negative control group; after the 24-valent pneumococcal capsular polysaccharide conjugate vaccine aluminum adsorption vaccine is used for immunizing mice, the anti-IgG of serum is obviously improved compared with a negative control, the common serotype antibody titer with the 13-valent pneumococcal capsular polysaccharide conjugate vaccine has no statistical difference, and the positive conversion rate of 11 types of antibodies which are more than the 24-valent pneumococcal capsular polysaccharide conjugate vaccine is 100%, which indicates that the immunogenicity is good.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (27)
1. A method for preparing a combined streptococcus pneumoniae capsular polysaccharide-protein vaccine stock comprising type 5 streptococcus pneumoniae capsular polysaccharides, comprising separately administering to each serotype streptococcus pneumoniae capsular polysaccharide comprising at least type 5 streptococcus pneumoniae capsular polysaccharide: (1) degradation; (2) activation; (3) lyophilization or precipitation; (4) Re-dissolving the freeze-dried or precipitated activated streptococcus pneumoniae capsular polysaccharide, and then combining and sealing with carrier protein; (5) purifying the conjugate vaccine stock solution;
the degradation in the step (1) is to degrade streptococcus pneumoniae capsular polysaccharide into polysaccharide with molecular weight of 100-850 kDa;
the activation in the step (2) is to add buffer solution and water for injection into the degraded streptococcus pneumoniae capsular polysaccharide stock solution obtained in the step (1), and then add oxidant for activation; the oxidizing agent is capable of oxidizing ortho-dihydroxyl to aldehyde; the oxidant is sodium periodate, potassium periodate or periodic acid;
the condition for the activation by adding the oxidant is as follows: the temperature is 2-8 ℃, the pH value is 4.0-6.5, and the time is 4-8 hours;
step (4) re-dissolving the freeze-dried activated streptococcus pneumoniae capsular polysaccharide with an organic solvent, and re-dissolving or dissolving the carrier protein with the same organic solvent; the organic solvent is DMF or DMSO; the concentration of the organic solvent before redissolution is 50% -100%.
2. The method of claim 1, wherein the degradation of step (1) is degradation of streptococcus pneumoniae capsular polysaccharides to polysaccharides having a molecular weight of 150-650 kDa.
3. The method of claim 1, wherein the degradation in step (1) is degradation of streptococcus pneumoniae capsular polysaccharides with mild acid or physical means; the acid is acetic acid, citric acid or carbonic acid; the physical method is high-pressure homogeneous phase or ultrasonic crushing.
4. The method of claim 1, wherein the buffer is phosphate buffer, HEPES, MOPS, or acetate-sodium acetate buffer; the concentration of the buffer solution is 10-100 mM, and the pH value is 3-7.
5. The method of claim 1, wherein the final concentration of streptococcus pneumoniae capsular polysaccharide is 1-3 g/L after addition of buffer and water for injection.
6. The method of claim 1, wherein the activating in step (2) is performed by adding 0.1 to 0.5 molar equivalent of an oxidizing agent to the streptococcus pneumoniae capsular polysaccharide solution after adding the buffer and water for injection.
7. The method of claim 6, wherein the activated polysaccharide has an activation degree of 1 to 25.
8. The method of claim 7, wherein the activated polysaccharide has an activation degree of 5 to 15.
9. The process according to claim 1, wherein the purified activated streptococcus pneumoniae capsular polysaccharide is obtained by ultrafiltration, aseptic filtration after completion of the activation in step (2).
10. The method of claim 1, wherein the step (3) of lyophilizing is performed by mixing the activated streptococcus pneumoniae capsular polysaccharide with an excipient, wherein the excipient is sucrose, trehalose, mannitol or sorbitol, in a mass ratio of 1:10-50.
11. The method of claim 1, wherein the activated streptococcus pneumoniae capsular polysaccharide produced in step (2) is precipitated with ethanol to produce an activated polysaccharide precipitate, and the activated polysaccharide precipitate is solubilized with DMF or DMSO, passed directly to step (4), combined with a carrier protein and blocked.
12. The preparation method according to claim 1, wherein the reconstituted streptococcus pneumoniae capsular polysaccharide is mixed with the reconstituted or dissolved carrier protein according to a mass ratio of 1:2-2:1, and after complete dissolution, 1.0-1.5 molar equivalents of sodium cyanoborohydride are added to initiate binding, and incubated at 23+ -2 ℃ for 12-48 hours.
13. The method of claim 12, wherein the final concentration of streptococcus pneumoniae capsular polysaccharide in the binding reaction system is between 0.5 and 4 g/L.
14. The method of claim 12, wherein the conjugation reaction is terminated by further adding 1.0 to 1.5 molar equivalents of sodium cyanoborohydride to block unreacted aldehyde groups, the blocking reaction continuing at 23±2 ℃ for 18±6 hours.
15. The method of any one of claims 1 to 14, wherein the conjugate after the binding in step (4) has a molecular weight of 2000-20000kDa and a mass ratio of polysaccharide to carrier protein of 0.5-2.5.
16. The method of claim 1, wherein the carrier protein is CRM197.
17. The process of claim 1, wherein the purification in step (5) is performed by diluting the solution after the combination and blocking in step (4) to remove the organic phase, then filtering, purifying by column chromatography, eluting, collecting the eluate, concentrating by ultrafiltration, sterilizing, and filtering.
18. The method of claim 17, wherein the combined and blocked solution of step (4) is diluted with pre-chilled 5mM succinate-0.9% brine at ph6.0, 1:6-15, then purified to remove the organic phase using 100-300kDa MWCO cellulose membrane tangential flow filtration, the diluted combined solution is filtered through a 5 μm filter, purified using Sepharose 4FF chromatographic medium, eluting with physiological saline solution, monitoring wavelengths of 206 nm and 280nm, and the eluate is collected, concentrated by ultrafiltration and sterile filtered to obtain the combined vaccine stock solution.
19. The method of claim 1, wherein the streptococcus pneumoniae capsular polysaccharide further comprises capsular polysaccharides from streptococcus pneumoniae of one or more of the following serotypes: type 1, type 2, type 3, type 4, type 6A, type 6B, type 7F, type 8, type 9N, type 9V, type 10A, type 11A, type 12F, type 14, type 15B, type 15C, type 17F, type 18C, type 19A, type 19F, type 20, type 22F, type 23A, type 23F, type 24F, type 33F, type 45, and type 46.
20. A streptococcus pneumoniae capsular polysaccharide-protein conjugate vaccine stock solution prepared by the method of any one of claims 1-19.
21. Use of a streptococcus pneumoniae capsular polysaccharide-protein conjugate vaccine stock solution according to claim 20 in the manufacture of a medicament for the prophylaxis or treatment of a disease caused by streptococcus pneumoniae.
22. Use of a streptococcus pneumoniae capsular polysaccharide-protein conjugate vaccine stock solution prepared by the method of any one of claims 1-18 in the preparation of a biologic, said biologic being a monovalent vaccine.
23. Use of a streptococcus pneumoniae capsular polysaccharide-protein conjugate vaccine stock according to claim 20 in the preparation of a biologic which is a multivalent vaccine or combination vaccine, the streptococcus pneumoniae capsular polysaccharide of which further comprises capsular polysaccharides from streptococcus pneumoniae of any one or more of the following serotypes: type 1, type 2, type 3, type 4, type 6A, type 6B, type 7F, type 8, type 9N, type 9V, type 10A, type 11A, type 12F, type 14, type 15B, type 15C, type 17F, type 18C, type 19A, type 19F, type 20, type 22F, type 23A, type 23F, type 24F, type 33F, type 45, and type 46.
24. Vaccine comprising a streptococcus pneumoniae capsular polysaccharide-protein conjugate vaccine stock prepared by the method of any one of claims 1-18, characterised in that it is a monovalent vaccine.
25. A vaccine comprising a streptococcus pneumoniae capsular polysaccharide-protein conjugate vaccine stock according to claim 20, characterised in that it is a multivalent vaccine, the streptococcus pneumoniae capsular polysaccharide of which also comprises capsular polysaccharides from streptococcus pneumoniae of any one or more of the following serotypes: type 1, type 2, type 3, type 4, type 6A, type 6B, type 7F, type 8, type 9N, type 9V, type 10A, type 11A, type 12F, type 14, type 15B, type 15C, type 17F, type 18C, type 19A, type 19F, type 20, type 22F, type 23A, type 23F, type 24F, type 33F, type 45, and type 46.
26. A vaccine comprising a streptococcus pneumoniae capsular polysaccharide-protein conjugate vaccine stock according to claim 20, characterised in that it is a combination vaccine, the streptococcus pneumoniae capsular polysaccharide of which further comprises capsular polysaccharides from streptococcus pneumoniae of any one or more of the following serotypes: type 1, type 2, type 3, type 4, type 6A, type 6B, type 7F, type 8, type 9N, type 9V, type 10A, type 11A, type 12F, type 14, type 15B, type 15C, type 17F, type 18C, type 19A, type 19F, type 20, type 22F, type 23A, type 23F, type 24F, type 33F, type 45, and type 46.
27. The vaccine of any one of claims 24-26, wherein the carrier protein is present in an amount of 30-90 μg/mL; the content of the streptococcus pneumoniae capsular polysaccharide of serotype 5 is 2-8 mug/mL; when the vaccine contains 6B serotype streptococcus pneumoniae capsular polysaccharide, the content of the 6B serotype streptococcus pneumoniae capsular polysaccharide is 4-16 mug/mL; when the vaccine contains streptococcus pneumoniae capsular polysaccharides of each of the other serotypes, the content of streptococcus pneumoniae capsular polysaccharides of each of the other serotypes is 2-8 μg/mL.
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| CN113607939A (en) * | 2021-08-05 | 2021-11-05 | 艾美探索者生命科学研发有限公司 | Method for measuring adsorption rate of various combined stock solutions of pneumococcal polysaccharide combined vaccine |
| CN115721709A (en) * | 2021-08-27 | 2023-03-03 | 康希诺生物股份公司 | Preparation method of pneumococcal conjugate vaccine |
| CN114965784B (en) * | 2022-06-01 | 2023-10-27 | 艾美探索者生命科学研发有限公司 | Method for measuring polysaccharide activation degree |
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