TWI586363B - Multivalent pneumococcal polysaccharide-protein conjugate composition - Google Patents

Description

Multivalent pneumococcal polysaccharide-protein conjugate composition Field of invention

The present invention relates to a multivalent immunological composition comprising: 15 distinct polysaccharide-protein conjugates which are derived from S. pneumoniae serotype 1, 2, 3, 4, 5, 6A , 6B, 7F, 9N, 9V , 14,18C, 19A, 19F and 23F of the capsular polysaccharide to a carrier protein, for example CRM _197, prepared by binding the conjugate. The present invention relates generally to the field of medicine, and in particular to microbiology, immunology, vaccines, and pneumococcal disease prevention by infants, children, and adults by immunization.

Background of the invention

The main cause of pneumococcal pneumonia. According to the National Bureau of Statistics's 2010 death cause trend, pneumonia is one of the top ten causes of death, and 14.9 deaths per 100,000 people, an increase of 82.9% since 2000. The World Health Organization (WHO) also estimates that in 2012, 476,000 HIV-negative children younger than 5 years of age will die from pneumococcal infection, which accounts for 5% of all-cause mortality in children younger than 5 years of age.

In 1977, Dr. Robert Austrian developed a 14-valent pneumococcal polysaccharide vaccine to prevent pneumococcal disease, which was then evolved into a 23-valent polysaccharide vaccine. Multivalent pneumococcal polysaccharide vaccines have proven to be valuable in the prevention of pneumococcal disease in elderly and high-risk patients. However, infants and young children have a poor response to most pneumococcal polysaccharides due to the T cell-independent immune response. The 7-valent pneumococcal conjugate vaccine (7vPnC, Prevnar ^® ) contains capsular polysaccharides from the seven most prevalent serotypes 4, 6B, 9V, 14, 18C, 19F and 23F. Since its approval in the United States in 2000, Prevnar has been shown to be highly immune in infants and young children and very effective against invasive diseases and otitis media. The vaccine is currently approved in approximately 80 countries around the world. Prevnar covers approximately 80 to 90%, 60 to 80%, and 40 to 80% of invasive pneumococcal disease (IPD) in the United States, Europe, and the rest of the world, respectively. The surveillance data collected during the years following the introduction of Prevnar has clearly demonstrated that the invasive pneumococcal disease caused by the serotype covered by Prevnar in the United States has decreased as expected. However, the coverage of serotypes in some regions is limited and the invasive pneumococcal disease caused by serotypes not covered by Prevnar, especially 19A, has increased.

The Advisory Committee on Immunization Practices (ACIP) announced in February 2010 that it recommended a newly approved 13-valent pneumococcal conjugate vaccine (PCV-13) for vaccination. PCV-13 is a pneumococcal conjugate vaccine that contains six additional serotypes in addition to the seven serotypes contained in Prevnar (4, 6B, 9V, 14, 18C, 19F, 23F). , 3, 5, 6A, 7F, 19A). According to the US Active Bacterial Core Surveillance (ABCs), PCV-13 covers a total of 64% of the pathogenic serotypes of children known to be younger than 5 years of age in IPD cases. In 2007, PCV7 In the 4600 IPD for children younger than 5 years old, only 70 cases were covered, while PCV-13 covered 2900 cases, with PCV-13 accounting for the majority. Currently, a 15-valent pneumococcal conjugate vaccine is being developed that covers additional serotypes whose incidence increases with serotype replacement.

Summary of invention

Accordingly, the present invention provides a multivalent immunological composition for preventing pneumococcal disease in infants, children and adults, comprising a capsular polysaccharide derived from 15 pneumococcal serotypes, the pneumococcal serotype comprising serum Type 2 and 9N. In particular, the present invention provides a 15-valent pneumococcal conjugate (PCV-15) composition comprising serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C , 19A, 19F and 23F.

Technical solution

According to one aspect of the present invention, there is provided a multivalent immunological composition comprising 15 distinct polysaccharide-protein conjugates and a physiologically acceptable carrier, wherein each of the conjugates comprises a carrier Protein conjugated, capsular polysaccharide derived from different serotypes of S. pneumoniae, and the capsular polysaccharide is composed of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, Prepared by 18C, 19A, 19F and 23F.

In the case of the multivalent immunological composition of the present invention, the carrier protein may be _CRM197 . The multivalent immunological composition of the present invention may further comprise an adjuvant which, for example, comprises an aluminum-based adjuvant. The adjuvant may be selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide, and is preferably aluminum phosphate.

According to a further aspect of the invention, there is provided a pharmaceutical composition for inducing an immune response to a S. pneumoniae capsular polysaccharide conjugate comprising an immunologically effective amount of the immunological composition.

In one embodiment, the pharmaceutical composition can be formulated to contain an immunological composition: 2 μg per sugar, except for 6 B, 4 μg; approximately 34 μg of CRM197 carrier protein; 0.125 mg of elemental aluminum (0.5 mg aluminum phosphate) adjuvant; and sodium chloride and sodium succinate buffer as excipients.

Technical effect

The multivalent immunological composition comprises a capsular polysaccharide derived from 15 distinct pneumococcal serotypes comprising serotypes 2 and 9N, thereby resulting in elevated serum IgG valence and functional antibodies active. Thus, the multivalent immunological composition can be advantageously used to prevent pneumococcal disease in infants, children and adults.

Figures 1 to 15 show serotype-specific IgG levels measured 3 weeks after the second injection of the vaccine compositions of the present invention and Comparative Examples (Prevnar 7 and Prevnar 13) (i.e., total 6 weeks).

Invention mode (MODE FOR INVENTION)

Some serotypes with antibiotic resistance and multiple drug resistance have produced serotype substitutions. Regional differences in serotype distribution have led to regional differences in Prevnar coverage (Harboe ZB, Benfield TL, Valentiner-Branth P, et al, Temporal Trends in Invasive Pneumococcal Disease and Pneumococcal Serotypes over 7 Decades. Clin Infect Dis 2010; 50: 329-37). Thus, there is no reason to remove any of the serotypes in the existing pneumococcal conjugate vaccine. Conversely, it is necessary to increase the serotype to further extend the scope of coverage.

In 2008, the pneumococcal global serotype program (GSP) announced a report based on IPD data selected from 1980 to 2007. The report showed that following serotype 18C, serotype 2 was in the 20 global serotypes. The serotype of the 11th highest incidence rate. In addition, Samir K. Saha et al. reported that serotype 2 may become a threat because serotype 2 has a high probability of causing pneumococcal meningitis in Bangladesh but is not included in any pneumococcal conjugate vaccine (Saha SK , Al Emran HM, Hossain B, Darmstadt GL, Saha S, et al. (2012) Streptococcus pneumoniae Serotype-2 Childhood Meningitis in Bangladesh: A Newly Recognized Pneumococcal Infection Threat. PLoS ONE 2012; 7(3): e32134). Thus, if serotype 2 is included, the number of pneumococcal diseases can be reduced and further serotype replacements that may occur for vaccination due to PCV-13 can be prepared.

Pneumococcal serotypes show different distribution patterns depending on age. In particular, serotype 9N has been found to be relatively important in infants between 0 and 23 months of age compared with children aged 24 to 59 months. After receiving 13 serotypes included in PCV-13, serotype 9N is the 14th most common serotype. This indicates that the inclusion of serotype 9N contributes to a reduction in pneumococcal disease, especially in infants.

The present invention provides a multivalent immunological composition comprising a capsular polysaccharide derived from 15 pneumococcal serotypes comprising serotypes 2 and 9N. In particular, the present invention provides a multivalent immunological composition comprising: 15 distinct polysaccharide-protein conjugates, and a physiologically acceptable carrier, wherein each of the conjugates comprises a carrier protein Conjugated, capsular polysaccharides of S. pneumoniae from different serotypes, and the capsular polysaccharides are composed of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, Prepared by 19A, 19F and 23F.

The capsular polysaccharide can be prepared by standard techniques known to those skilled in the art. The size of the capsular polysaccharide can be reduced to reduce viscosity or increase the solubility of the activated capsular polysaccharide. In the aspect of the invention, the capsular polysaccharide is prepared from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F and 23F of S. pneumoniae. These pneumococcal conjugates are prepared by isolation and formulated into a single dose formulation. For example, each pneumococcal polysaccharide serotype is grown in a soy-based medium and then purified via centrifugation, precipitation, and ultra-filtration.

The carrier protein is preferably non-reactive and non-reactogenic and a protein of sufficient quantity and purity. The carrier protein should withstand the standard conjugate binding procedure. In the case of the multivalent immunological composition of the present invention, the carrier protein may be _CRM197 (CRM ₁₉₇ is). CRM ₁₉₇ is a non-toxic diphteria toxin variant (ie, toxoid) which is a Corynebacterium diphteria strain C7 grown on a casein amino acid and yeast extract based medium ( The culture of β197) is isolated. CRM ₁₉₇ was purified by ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. Optionally, CRM197 is recombinantly prepared in accordance with U.S. Patent No. 5,614,382.

Other diphteria toxoids are also suitable for use as carrier proteins. Other suitable carrier proteins include non-activated bacterial toxins such as tetanus toxoid, pertussis toxoid; cholera toxoid (WO 2004/083251). E. coli LT, E. coli ST, and exotoxin from Pseudomonas aeruginosa A. Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porin (porins), transferrin binding protein, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesion may also be used. Protein (PsaA), C5a peptidase from group A or group B streptococci, or Haemophilus influenzae protein D. Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD) can also be used as carrier proteins. Diphtheria toxin variants such as CRM ₁₇₃ , CRM ₂₂₈ , and CRM _{45 can be} used as carrier proteins.

To prepare a polysaccharide for use in reacting with a carrier protein, the purified polysaccharide is chemically activated. Once activated, each capsular polysaccharide will conjugate to the carrier protein, respectively, to form a sugar conjugate. In one embodiment, each capsular polysaccharide is conjugated to the same carrier protein. The chemical activation of the polysaccharide and its subsequent conjugated binding to the carrier protein is achieved by conventional means (for example, U.S. Patent Nos. 4,673,574 and 4,902,506). The hydroxyl group of the polysaccharide is controlled by an oxidizing agent (for example, periodate (including sodium periodate, iodine) Oxidation to aldehyde groups by potassium acid, calcium periodate, or periodic acid). Chemical activation results in irregular oxidative degradation of adjacent hydroxyl groups. The conjugated bond is achieved by reductive amination. For example, the activated capsular polysaccharide and carrier protein are reacted with a reducing agent, such as sodium cyanoborohydride. Unreacted aldehyde groups can be removed by the addition of a strong oxidizing agent.

After the capsular polysaccharide is conjugated to the carrier protein, the polysaccharide-protein conjugate is purified by various techniques (enriched in the amount of polysaccharide-protein conjugate). Such techniques include diafiltration, column chromatography, and depth filtration. The purified polysaccharide-protein conjugate is compounded to formulate the immunological composition of the present invention, and the immunological composition of the present invention can be used as a vaccine. Formulations of the immunological compositions of the invention can be achieved using art recognized methods. For example, 15 separate pneumococcal conjugates can be formulated with a physiologically acceptable carrier to prepare the composition. Examples of such carriers include, but are not limited to, water, buffered saline, polyols (for example, glycerin, propylene glycol, liquid polyethylene glycol), and dextrose solutions.

In one embodiment, the immunological composition of the invention may comprise one or more adjuvants. An "adjuvant" as defined herein is a substance that is used to enhance the immunogenicity of the immunological compositions of the invention. Thus, adjuvants are often provided to increase the immune response and are well known to those skilled in the art. Suitable adjuvants for enhancing the effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate and aluminum sulfate, and the like; (2) oil emulsion formulations in water ( With or without other specific immunostimulating agents, such as muramyl peptides (defined below) or cell wall components of bacteria, for example, (a) MF59 (WO 90/14837) Containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing different amounts of MTP-PE (see below), but not required), using a microfluidizer, for example Model 110Y microfluidics homogenizer (Microfluidics, Newton, MA) formulated into submicron particles, (b) SAF, containing 10% squalene, 0.4% Tony 80, 5% pluronic block polymer L121,, and thr-MDP (see below), it may be microfluidized into a submicron emulsion or vortexed to form an emulsion of a larger particle size, and (c) Ribi ^TM adjuvant system (RAS), (Corixa, Hamilton, MT), which contains 2% squalene, 0.2% Tony 80, and one or more bacterial cell wall components selected from the group consisting of: Patent Number 4,912,094 to the 3-O- arylation monophospholipid ^{A (MPL TM) (Corixa)} , trehalose two mycolic acids (trehalose dimycolate) (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox ^TM); (3) saponin adjuvants such as Quil A or ^{STIMULON TM QS-21 (Antigenics,} Framingham, MA) ( U.S. Patent number 5,057,540), or particles formed therefrom of, for example, the ISCOM (immunostimulating complexes) (4) a bacterial lipopolysaccharide, a synthetic lipid A analog, such as an aminoalkyl glucosamine phosphate compound (AGP), or a derivative or analog thereof, which is commercially available from Corixa and is described in U.S. Patent No. 6,113,918; one such AGP is 2-[(R)-3-tetradecylideneoxytetradecanodecylamino]ethyl 2-deoxy-4-0-phosphinyl-3-0 -[(R)-3-tetradecylideneoxytetradecylsulfenyl]-2-[(R)-3-tetradecanodecyloxytetradecylsulfonylamino]-bD-pyridyl Glucosinolate, also known as 529 (formerly known as RC529), can be formulated into an aqueous form or a stable emulsion; (5) a synthetic polynucleotide, such as an oligonucleotide comprising one or more CpG motifs Nucleotide (US Patent No. 6,207,646); (6) Interleukin (cyt) Okines), such as interleukin (for example, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc. ), interferon (for example, gamma interferon), granulocyte macrophage cell stimulating factor (GM-CSF), macrophage cell stimulating factor (M-CSF), tumor necrosis factor (TNF), costimulatory molecule (costimulatory molecules) B7-1 and B7-2, etc.; (7) detoxified mutants of bacterial ADP-ribosyling toxins, such as cholera toxin (CT), whether wild-type or mutant, for example, according to WO 00/18434 (see also WO 02/098368 and WO 02/098369) in which the glutamic acid at position 29 of the amino acid is replaced by another amino acid, preferably histidine), pertussis toxin (PT) ), or E. coli heat labile toxin (LT), especially LT-K63, LT-R72, CT-S109, PT-K9/G129 (see, for example, WO 93/13302 and WO 92/19265); And (8) a complement component, such as a trimer of the complement component C3d.

Cell wall purine peptides include, but are not limited to, N-acetyl-muram-yl-L-threonyl-D-iso-glutamic acid (thr-MDP), N-ethylidene-decreasing wall N-acetyl-normuramyl-L-alanine-2 -(1'-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine )(MTP-PE), and the like. In a specific embodiment, an aluminum salt is used as an adjuvant. The aluminum salt adjuvant may be an alum precipitated vaccine or alum-absorbed vaccine. Aluminum salts include, but are not limited to, hydrated alumina, alumina trihydrate (ATH), aluminum hydrate, alumina trihydrate (ATH), Alhydrogel, Superfos, Amphojel, hydroxide Aluminum (III), aluminum hydroxyphosphate sulfate (Aluminum Phosphate Adjuvant (APA)), and amorphous alumina. APA is a suspension of aluminum hydroxyphosphate. If aluminum chloride and sodium phosphate are mixed in a ratio of 1:1, aluminum hydroxyphosphate will be precipitated. The precipitate was sized by 2-8 □ by using a high shear mixer and dialyzed against physiological saline, followed by sterilization. In one embodiment, commercially available Al(OH) ₃ (for example, Alhydrogel or Superfos) is used to absorb proteins. Each 1 mg of aluminum hydroxide can absorb 50-200 g of protein, and this ratio depends on the isoelectric point (pI) of the protein and the pH of the solvent. Proteins with low pI are strongly absorbed compared to proteins with high pi. The aluminum salt forms an antigenic reservoir that slowly releases the antigen over a period of two to three weeks, non-specifically activating macrophages, complement, and innate immune mechanisms.

The present invention provides a pharmaceutical composition (for example, a vaccine formulation) for inducing an immune response against a S. pneumonia capsular polysaccharide conjugate comprising an immunologically effective amount of the immunological composition.

The vaccine formulations of the present invention can be used to protect or treat humans susceptible to pneumococcal infection by administering a vaccine via a systemic or mucosal route. As defined herein, the term "effective dose" refers to induction sufficient to be significant The amount required to reduce the probability of S. pneumoniae infection or the antibody level of its severity. Such administration may include intramuscular, intraperitoneal, intradermal or subcutaneous routes; or administration to the oral/esophageal, respiratory or genitourinary tract via the mucosa.

In one embodiment, intranasal administration is used to treat pneumonia or otitis media because the nasopharyngeal transport of pneumococci can be more effectively prevented, thereby attenuating infection at an early stage. The amount of conjugate in each vaccine dose is selected to induce an immunoprotective response with no significant deleterious effects. This amount will vary depending on the pneumococcal serotype. In general, each dose will contain from 0.1 to 100 μg of polysaccharide, specifically from 0.1 to 10 μg, and more specifically from 1 to 5 μg. The optimal amount of the component for a particular vaccine can be determined by standard studies involving the observation of an appropriate immune response in the subject. For example, the amount of vaccination for a human subject can be determined by inferring animal test results. In addition, the dosage can be determined empirically.

In a specific embodiment of the invention, the vaccine composition is S. pneumoniae serotype 1, conjugated to CRM _197, serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, Sterile liquid formulations of capsular polysaccharides of 19A, 19F and 23F. Each 0.5 mL dose was formulated to contain the following: 2 μg per sugar, except for 6 B 4 μg; approximately 34 μg CRM ₁₉₇ carrier protein; 0.125 mg elemental aluminum (0.5 mg aluminum phosphate) adjuvant; and sodium chloride and succinic acid Sodium buffer is used as an excipient. The liquid is filled into a single dose syringe containing no preservative. After the shock, the vaccine is a homogeneous, white suspension ready for intramuscular administration.

In a further embodiment, the compositions of the invention may be administered in a single injection. For example, a vaccine composition of the invention can be administered 2 times, 3 times, 4 times, or more at appropriately spaced intervals, for example, at 1, 2, 3, 4, 5, or 6 months. The interval or any combination thereof is administered. The immunization schedule can follow the immunization schedule specified for the Prevnar vaccine. For example, the invasive disease caused by the serotype of Streptococcus pneumoniae included in the Prevnar vaccine, the routine schedule for infants and young children is 2, 4, 6 and 12-15 months of age. Thus, in this aspect, the composition is administered 4 times, i.e., at 2, 4, 6, and 12-15 months of age.

Compositions of the invention may also include one or more proteins from S. pneumoniae. Examples of suitable S. pneumoniae proteins include those identified in International Patent Application No. WO 02/083,855, and the entire disclosure of which is incorporated herein by reference.

The compositions of the present invention can be administered to a subject via one or more routes of administration known to those skilled in the art, such as parenteral, transdermal, or transmucosal, intranasal, intramuscular, peritoneal. Intraluminal, intradermal, intravenous, or subcutaneous routes and correspondingly formulated. In one embodiment, the compositions of the present invention can be administered as a liquid formulation by intramuscular, intraperitoneal, subcutaneous, intravenous, intraarterial, or percutaneous injection or by respiratory mucosal injection. Liquid formulations for injection include solutions or the like.

The compositions of the present invention may be formulated in the form of a unit dose vial, in the form of a multi-dose vial, or in the form of a pre-filled syringe. Pharmaceutically acceptable carriers for liquid formulations include aqueous or non-aqueous solutions, suspensions Floating, emulsion or oil. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and ethyl oleate. The aqueous carrier comprises water, an alcohol/aqueous solvent, an emulsion or suspension, physiological saline, a buffer solution. Examples of the oil include vegetable oil or animal oil, peanut oil, soybean oil, olive oil, sunflower oil, liver oil, synthetic oil such as aquatic oil, and lipid obtained from milk or eggs. The pharmaceutical composition can be isotonic, low osmotic or high osmotic. However, it is desirable that the pharmaceutical composition for infusion or injection be substantially isotonically osmotic. Thus, iso-osmotic pressure or high osmotic pressure may facilitate storage of the composition. When the pharmaceutical composition exhibits a high osmotic pressure, the composition can be diluted to an equal osmotic pressure prior to administration. The tonicity agent can be an ionic permeating agent (such as a salt) or a non-ionic permeating agent (such as a carbohydrate). The ionic permeating agent includes, but is not limited to, sodium chloride, calcium chloride, potassium chloride, KCl, and magnesium chloride. Nonionic permeating agents include, but are not limited to, sorbitol and glycerin. Preferably, at least one pharmaceutically acceptable buffer is included. For example, when the pharmaceutical composition is infused or injected, the pharmaceutical composition is preferably formulated in a buffer having a buffering capacity between pH 4 and 10, such as 5 to 9 or 6 to 8. The buffering agent may be selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate , histidine, glycine, succinate, and triethanolamine buffer solution.

In particular, if the pharmaceutical composition is for parenteral administration, the buffer may be selected from those suitable for use in the United States Pharmacopoeia (USP). For example, the buffer may be selected from the group consisting of: monobasic acid, For example, acetic acid, benzoic acid, gluconic acid, glyceric acid, and lactic acid; polybasic acids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, and tartaric acid; and alkalis such as ammonia, Ethanolamine, glycine, triethanolamine, and TRIS. For parenteral administration, carriers (for subcutaneous, intravenous, intra-arterial, and intramuscular injection) include sodium chloride solution, Ringer's dextrose solution, right-handed Sugar and sodium chloride, lactated Ringer's solution and fixed oil. Carriers for intravenous administration include Ringer's dextrose solution or a similar dextrose-based infusion solution, nutrient supplement, and electrolyte supplement. Examples include sterile liquids such as water and oils with or without the addition of surfactants and other pharmaceutically acceptable adjuvants. In general, water, physiological saline, dextrose solution, related sugar solutions, and ethylene glycol (e.g., propylene glycol or polyethylene glycol), especially polysorbate 80, are suitable for injection. Examples of the oil include animal and vegetable oils, peanut oil, soybean oil, olive oil, sunflower oil, liver oil, synthetic oils such as aquatic oils, and lipids obtained from milk or eggs.

Formulations of the invention may comprise a surfactant. Preferably, polyoxyethylene sorbitan ester (generally referred to as Tweens), especially polysorbate 20 and polysorbate 80; ethylene oxide (EO) a copolymer of propylene oxide (PO) or butylene oxide (BO) (eg DOWFAXTM); octtocynols with different ethoxy groups (oxy-1,2-ethanediyl) a repetition of a group, in particular, octoxynol-9 (Triton X-100); octylphenoxypolyethoxyethanol (IGEPAL CA-630/NP- 40); phospholipids, such as lecithin; nonylphenol Nonylphenol ethoxylate, such as the TergitolTM NP series; polyoxyethylene fatty ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol, and oleyl alcohol (Brij 30); especially, triethylene glycol monolauryl ether (Brij 30); sorbitan ether known as SPAN, especially sorbitan trioleate (Span 85) and sorbitan monolaurate, but is not limited thereto. Preferably, the emulsion 80 is included in the emulsion.

Mixtures of surfactants can be used, such as Towen 80/Span 85. Combinations of polyoxyethylene sorbitan esters (e.g., Towen 80) and octocylol (e.g., Triton X-100) are also suitable. Laureth 9 and toco and/or octocylol are also useful combinations. Preferably, the amount of the polyoxyethylene sorbitan ester (for example, Towen 80) is 0.01% to 1% (w/v), especially 0.1%; and the octylphenoxy polyethylene ethoxylate is included. The amount of octylphenoxy polyoxyethanol or nonylphenoxy polyoxyethanol (such as Triton X-100) is from 0.001% to 0.1% (w/v), especially from 0.005% to 0.02%; The amount of polyoxyethylene ether (e.g., laureth 9) included is from 0.1% to 20% (w/v), possibly from 0.1% to 10%, especially from 0.1% to 1% or about 0.5%. In one embodiment, the pharmaceutical composition is delivered in a release control system. For example, intravenous infusion, transdermal patches, liposomes, or other routes can be used. In one aspect, macromolecules, such as microspheres or implants, can be used.

The above disclosure broadly illustrates the invention. A more comprehensive understanding can be obtained by reference to the specific embodiments set forth below. The examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Example Example 1. Preparation of S. pneumoniae capsular polysaccharide

The culture of S. pneumoniae and the purification of the capsular polysaccharide are carried out in a manner known to those skilled in the art. The S. pneumoniae serotype is obtained from the American Type Culture Collection (ATCC). Streptococcus pneumoniae is characterized by capsular and immobile, Gram-positive lancet type dicocci and alpha hemolysis on blood agar medium. Serotypes were identified by the Quelling test using specific antisera (US Patent No. 5,847,112).

Preparation of cell bank

Several generations of seed stocks were prepared to expand the strain and remove the components of the animal source (F1, F2, and F3). Produce an additional two generations of bacterial stocks. An additional first generation was prepared from the F3 vial, and the subsequent generation was prepared from this additional first generation. Freezing with synthetic glycerol as a cryopreservation agent ( -70 ° C) Preserve the vial of the strain. For cell bank preparation, all cultures were grown in soy-based medium. Prior to freezing, the cells are concentrated by centrifugation, the remaining medium is removed, and the cell pellet is resuspended in fresh medium containing a cryopreservative such as synthetic glycerol.

Vaccination

Cultures from the working cell bank were used to inoculate the vials containing the soy-based medium. A strain bottle is used to inoculate a fermenter containing a soybean-based medium.

Fermentation

The fermentation of the strain is carried out in a fermenter for controlling the temperature and pH. After reaching the target optical density, a fermenter is used to inoculate a production fermentor containing a soybean-based medium.

Production fermentation

Production fermentation is the final step of fermentation. Control temperature, pH and agitation speed.

No activation

After the growth is stopped, the fermentation is terminated by the addition of an inerting agent. After inactivation, the contents of the fermentor are cooled and the pH of the dissolved culture broth is adjusted.

purification

The broth from the fermentor was centrifuged and filtered to remove bacterial cell debris. Several concentration/diafiltration operations, precipitation/dissolution, and thickness filtration steps were used to remove contaminants and purify the capsular polysaccharide.

Example 2. Preparation of S. pneumoniae capsular polysaccharide- _CRM197 conjugate

Polysaccharides from different serotypes are activated by different pathways and then conjugated to _CRM197 . Methods of activation include reducing the size of the capsular polysaccharide to the target molecular weight, chemical activation, and buffer exchange by ultrafiltration. Purified _CRM197 was conjugated to the activated capsular polysaccharide, and the conjugate was purified using ultrafiltration and finally filtered through a 0.22 [mu]m filter. Method parameters such as pH, temperature, concentration, and time are as follows.

(1) Activation step 1

Each serotype polysaccharide was diluted with water for injection, sodium acetate, and sodium phosphate to a final concentration ranging from 1.0 to 2.0 mg/mL. For serotype 1, add sodium hydroxide (0.05 M final base concentration) and incubate the solution at 50 °C ± 2 °C. Incidentally, the solution was cooled to 21 to 25 ° C and the hydrolysis was stopped by the addition of 1 M HCl until a target pH of 6.0 ± 0.1 was reached. For serotype 3, HCl (0.01 M final acid concentration) was added and the solution was incubated at 50 °C ± 2 °C. Incidentally, the solution was cooled to 21 to 25 ° C and hydrolysis was stopped by the addition of 1 M sodium phosphate until a target pH of 6.0 ± 0.1 was reached. For serotype 4, HCl (0.1 M final acid concentration) was added and the solution was incubated at 45 °C ± 2 °C. Incidentally, the solution was cooled to 21 to 25 ° C and hydrolysis was stopped by the addition of 1 M sodium phosphate until a target pH of 6.0 ± 0.1 was reached. For serotype 6A, glacial acetic acid (0.2 M final acid concentration) was added and the solution was incubated at 60 °C ± 2 °C. Incidentally, the solution was cooled to 21 to 25 ° C and hydrolysis was stopped by the addition of 1 M sodium hydroxide until a target pH of 6.0 ± 0.1 was reached. For serotypes 14 and 18C, glacial acetic acid (0.2 M final acid concentration) was added and the solution was incubated at 94 °C ± 2 °C. Incidentally, the solution was cooled to 21 to 25 ° C and hydrolysis was stopped by the addition of 1 M sodium phosphate until a target pH of 6.0 ± 0.1 was reached.

Step 2: Periodate reaction

The molar equivalent of sodium periodate required for the activation of pneumococcal saccharide is determined using the total sugar content. With sufficient mixing, all serotypes are at 21-25 ° C (except for serotype 1, 7F, and 19F, the temperature is The oxidation reaction was carried out at 10 ° C for 16 to 20 hours.

Step 3: Ultrafiltration

The oxidized saccharide was concentrated and diafiltration was carried out with water for injection (WFI) on a 100 kDa MWCO ultrafilter (30 kDa ultrafilter for serotype 1 and 5 kDa ultrafilter for serotype 18C). The serotype 1 diafiltration system was completed using a 0.9% sodium chloride solution, 0.01 M sodium acetate buffer (pH 4.5) for serotype 7F, and 0.01 M sodium phosphate buffer (pH for serotype 19F). 6.0). The permeate was discarded and the retentate was filtered through a 0.22 [mu]m filter.

Step 4: Freeze drying

In the serotypes 3, 4, 5, 9N, 9V, and 14, the concentrated saccharide was mixed with the CRM ₁₉₇ carrier protein, placed in a glass bottle, lyophilized and stored at -25 °C ± 5 °C.

In the case of serotypes 2, 6A, 6B, 7F, 19A, 19F, and 23F, a predetermined amount of sucrose is added, which is estimated to achieve an amount of 5% ± 3% sucrose concentration in the conjugated reaction mixture. Serotypes 1 and 18C do not require the addition of sucrose. The concentrated sugar is then placed in a glass vial, lyophilized and stored at -25 °C ± 5 °C.

(2) Conjugate binding method

Serotype 1, 3, 4, 5, 9N, 9V, 14 and 18C were subjected to aqueous conjugation binding, and serotypes 2, 6A, 6B, 7F, 19A, 19F and 23F were subjected to DMSO conjugation.

Step 1: Dissolve Aqueous conjugate

In serotypes 3, 4, 5, 9N, 9V, and 14, the saccharide- _CRM197 mixture activated by lyophilization was thawed and equilibrated at room temperature. The lyophilized activated carbohydrate, _{CRM197, was} then reconstituted in 0.1 M sodium phosphate buffer at a typical ratio based on serotype. For serotypes 1 and 18C, the lyophilized saccharide was reconstituted within a CRM ₁₉₇ solution (typical ratio of 0.11 L of sodium phosphate per 1 L of CRM ₁₉₇ solution) in 1 M disodium hydrogen phosphate.

Dimethyl hydrazine (DMSO) conjugate binding

The lyophilized activated saccharide serotypes 2, 6A, 6B, 7F, 19A, 19F, 23F and the lyophilized CRM ₁₉₇ carrier protein were equilibrated at room temperature and recombined within DMSO.

Step 2: Conjugation binding reaction Aqueous conjugate

In terms of serotypes 1, 3, 4, 5, 9N, 9V, 14 and 18C, the conjugated binding reaction was initiated by the addition of sodium cyanoborohydride solution (100 mg/mL) to achieve 1.0 per molon. - 1.2 moles of sodium cyanoborohydride. The reaction mixture was incubated at 23 ° C to 37 ° C for 44-96 hours. The temperature and reaction time were adjusted according to the serotype. The temperature was then lowered to 23 °C ± 2 °C and 0.9% sodium chloride was added to the reactor. Sodium borohydride solution (100 mg/mL) was added to achieve a sodium borohydride equivalent of 1.8-2.2 moles per mole of sugar. The reaction mixture was incubated at 23 ° C ± 2 ° C for 3-6 hours. This procedure reduces any unreacted aldehydes present on the saccharide. The mixture was diluted with 0.9% sodium chloride solution and the diluted conjugate mixture was filtered into a storage vessel using a 1.2 [mu]m prefilter.

DMSO conjugate

In the case of serotypes 2, 6A, 6B, 7F, 19A, 19F and 23F, the activated saccharide and the CRM ₁₉₇ carrier protein were mixed in a ratio ranging from 0.8 g to 1.25 g of saccharide/g CRM ₁₉₇ . The conjugated binding reaction was initiated by the addition of a sodium cyanoborohydride solution (100 mg/mL) in a ratio of 0.8-1.2 molar equivalents of sodium cyanoborohydride to mono-activated saccharide. WFI was added to the reaction mixture to reach 1% (v/v) of the target, and the mixture was incubated at 23 °C ± 2 °C for 11-27 hours. Adding sodium cyanoborohydride solution, 100 mg/mL (typically 1.8-2.2 molar equivalents of sodium cyanoborohydride per mole of activated sugar) and WFI (target 5% v/v) to the reaction and The mixture was incubated at 23 ° C ± 2 ° C for 3-6 hours. This procedure reduces any unreacted aldehydes present on the saccharide. Then, the reaction mixture was diluted with 0.9% sodium chloride, and the diluted conjugate mixture was filtered into a storage vessel using a 1.2 μm prefilter.

Step 3: Ultrafiltration

The diluted conjugate mixture was concentrated and diafiltered on a 100 kDa MWCO ultrafiltration filter with a minimum of 20 volumes of 0.9% sodium chloride or buffer. Discard the permeate.

Step 4: Sterile filtration

The retentate after 100 kDa MWCO diafiltration was filtered through a 0.22 μm filter. The filtered product was subjected to control during the process (sugar content, free protein, free sugar, residual DMSO, and residual cyanide; residual DMSO and the foregoing in terms of DMSO conjugate binding). The control during the retentate execution is filtered to determine if additional concentration, diafiltration, and/or dilution is required. The filtered conjugate was diluted with 0.9% sodium chloride as needed to achieve a final concentration of less than 0.55 g/L. Sugar content, protein content, and sugar at this stage: eggs White matter is released compared to the test. Finally, the conjugate was filtered (0.22 μm) and the release test was performed (appearance, free protein, free saccharide, endotoxin, molecular size determination, residual cyanide, residual DMSO, carbohydrate identification, and CRM197 identification ). The final bulk concentrated solution was stored at 2-8 °C.

Example 3. Blending of multivalent pneumococcal conjugate vaccine

The final bulk concentrate volume required is calculated from the bulk volume and the concentration of bulk sugars. The bulk concentrate was added after the required amount of 0.85% sodium chloride (normal saline), polysorbate 80, and succinate buffer was added to the pre-labeled formulation vessel. The formulation was then thoroughly mixed and sterile filtered through a 0.22 [mu]m membrane. The formulated body was gently mixed during and after the addition of bulk aluminum phosphate. Check the pH and set the pH if necessary. The formulated bulk product was stored at 2-8 °C. The 0.5 ml volume of the product contained 2 μg per sugar, except that 6B was 4 μg; approximately 34 μg of CRM197 carrier protein; 0.125 mg of elemental aluminum (0.5 mg of aluminum phosphate) adjuvant; 4.25 mg of sodium chloride; 295 μg of sodium succinate buffer; and 100 μg of polysorbate 80.

Example 4. Immunogenicity of a multivalent pneumococcal conjugate vaccine

The multivalent pneumococcal vaccine prepared in Example 3, that is, the ability of the vaccine composition (SK-15) to induce an immune response in a rabbit was tested. These immunological effects are characterized by an antigen-specific ELISA of serum IgG concentration and an opsonophagocytic assay (OPA) of antibody function. At the 0th week and the 3rd week, the planned human clinical dose of each polysaccharide (2μg per PS, except for 6B 4μg) was used for intramuscular immunization. Blue rabbit. Serum was sampled every 3 weeks after immunization.

Measurement of serotype-specific IgG concentration

Each serotype was coated on a 96 well plate with 500 ng/well of capsular polysaccharide (PnPs). Equal amounts of serum were sampled from each subject and mixed by group. The serum pool was serially diluted 10 times with an antibody dilution buffer containing Toshiban 20, C-PS 4 ug/mL, and serotype 22F capsular polysaccharide (PnPs 22F) 4 ug/mL, and then reacted at room temperature for 30 minutes. The plate was washed 5 times with a wash buffer and then the well plate was coated with 50 uL of pre-absorbed and diluted serum and incubated at room temperature for 18 hours. The wells were washed in the same manner, and then goat anti-rabbit IgG-alkaline phosphatase conjugate (1:50000) was added to each well, followed by incubation at room temperature for 2 hours. The plates were washed as described above, and 1 mg/mL p-nitroaniline buffer was added as a substrate to each well and then reacted at room temperature for 2 hours. The reaction was quenched by the addition of 50 uL of 3M NaOH and the absorbance at 405 nm and 690 nm was measured. The 7-valent vaccine (Prevnar 7, Pfizer) and the 13-valent vaccine (Prevnar 13, Pfizer) were subjected to the same procedure as a comparative example. The results are shown in Figures 1-15.

Functional Immunogenicity Test (OPA)

Antibody function was assessed by testing serum within the OPA assay. Equal amounts of serum were sampled from each subject, mixed by group and diluted 10 times. S. pneumoniae is cultured in THY medium as a serotype and diluted to 1000 CFU/10 uL. The opsonophagocytic buffer 200 uL, the diluted serum 10 uL, and the diluted S. pneumoniae 10 uL were mixed and reacted at room temperature for 1 hour. A mixture of pre-differentiated HL-60 cells and complement was added and the reaction was carried out in a CO ₂ thermostat (37 ° C) for one hour. The temperature was lowered to stop phagocytosis and the 5 uL of the reaction was smeared on a pre-dried 30-60 minute agar plate. The plates were incubated for 12-18 hours in a CO ₂ thermostat (37 ° C) and colonies were counted. The OPA price was expressed as a dilution rate of 50% lethality observed. The 13-valent vaccine (Prevnar 13, Pfizer) was subjected to the same procedure as a comparative example. The results are shown in Tables 1-3.

The serotype-specific immune response of the vaccine formulations of the invention and the serotype-specific immune response of the comparative examples were assessed via OPA of the IgG ELISA and the complement vector of the functional antibody. Figures 1 to 15 show the results of the IgG ELISA and Tables 1 to 3 show the results of functional immunogenicity measurements obtained from OPA, in which the immune response was compared. These results indicate that the 15-valent pneumococcal polysaccharide conjugate vaccine induces IgG and functional antibody activity equivalent to or better than Prevnar-13.

Claims (7)

A multivalent immunological composition comprising: 15 distinct polysaccharide-protein conjugates, and a physiologically acceptable carrier, wherein each of the polysaccharide-protein conjugates comprises a different serum Type of S. pneumoniae and capsular polysaccharide conjugated to a carrier protein, and the capsular polysaccharide is composed of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C Prepared by 19A, 19F and 23F, wherein the carrier protein is _CRM197 . The multivalent immunological composition of claim 1, which further comprises an adjuvant. The multivalent immunological composition of claim 2, wherein the adjuvant is an aluminum-based adjuvant. The multivalent immunological composition of claim 3, wherein the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide. The multivalent immunological composition of claim 4, wherein the adjuvant is aluminum phosphate. A use of the multivalent immunological composition according to any one of claims 1 to 5 for the preparation of a pharmaceutical composition for inducing an immune response against a S. pneumoniae capsular polysaccharide conjugate . The use of claim 6, wherein the multivalent immunological composition is a single 0.5 ml dose formulated to contain: 2 μg per sugar, except for 6 B, 4 μg; about 34 μg of CRM ₁₉₇ carrier protein; 0.125 mg Elemental aluminum (0.5 mg aluminum phosphate) adjuvant; and sodium chloride and sodium succinate buffer as excipients.