WO2020009462A1 - Multivalent immunogenic composition comprising polysaccharide-protein conjugates, and vaccine comprising same for preventing disease caused by streptococcus pneumoniae - Google Patents

Abstract

The present invention relates to a multivalent immunogenic composition comprising Streptococcus pneumoniae polysaccharide-protein conjugates, and a pharmaceutical composition comprising same. The multivalent immunogenic composition according to one aspect of the present invention comprises a physiologically acceptable vehicle and 15 different polysaccharide-carrier protein conjugates, wherein each of the polysaccharide-protein conjugates comprises a Streptococcus pneumoniae-derived capsular polysaccharide of a different serum type attached to a carrier protein, the capsular polysaccharide being produced from serum types of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F, and 23F.

Description

Immunogenic compositions comprising multivalent pneumococcal polysaccharide-protein conjugates, and vaccines for preventing diseases by pneumococci comprising the same

The present invention relates to immunogenic compositions comprising multivalent pneumococcal polysaccharide-protein conjugates, and pharmaceutical compositions comprising the same.

Streptococcus pneumoniae is a major causative agent of pneumonia. According to the National Statistical Office, the mortality rate from pneumonia in 2010 was 14.9 per 100,000, one of the top 10 causes of death, an 82.9% increase from 2000. In addition, in 2012, according to the WHO, 476,000 HIV-negative children under the age of five worldwide died from infections caused by Streptococcus pneumoniae in 2008, and 5% of all children under 5 years of age were caused by the bacteria. Died.

To prevent pneumococcal disease, Dr. The 14-valent polysaccharide vaccine was developed by Robert Austrian and then developed into a 23-valent polysaccharide vaccine. Multivalent pneumococcal polysaccharide vaccines have proven useful in preventing pneumococcal disease in elderly and high risk patients. However, infants and children are not immune to most pneumococcal polysaccharides due to T-cell independent immune response. The valent pneumococcal conjugate vaccine (Prevnar®) contains capsular polysaccharides from the seven most common serotypes 4, 6B, 9V, 14, 18C, 19F and 23F. Since it was first approved in the United States in 2000, it has been proven to be highly immunogenic and effective against invasive diseases and otitis media in infants and children. The vaccine is currently approved in about 80 countries around the world. Prevna covers approximately 80-90%, 60-80%, and 40-80% of invasive pneumococcal disease in the United States, Europe, and other parts of the world, respectively. Surveillance data accumulated over the years following the introduction of Prevna clearly predicted a reduction in invasive pneumococcal disease caused by serotypes included in Prevna in the United States. However, in some regions there was a limit to the serotype coverage and increased invasive pneumococcal disease caused by serotypes not included in Prevna, especially 19A.

In February 2010, the Advisory Committee on Immunization Practices (ACIP) issued a recommendation for the use of the newly licensed 13-valent pneumococcal conjugate vaccine (PCV-13). PCV-13 contains six additional serotypes (1, 3, 5, 6A, 7F, 19A) in addition to the seven serotypes (4, 6B, 9V, 14, 18C, 19F, 23F) included in Prevna. Pneumococcal conjugate vaccine. According to the U.S. Active Bacterial Core surveillance (ABCs) survey, 64% of all IPDs known to develop serotypes in children under 5 years of age are included in PCV13, and among 4600 IPDs in children under 5 years of age, PCV7 In the case of 70 cases, PCV13 has more than 2900 cases.

PCV (Pneumococcal conjugate vaccine) currently on the market is an invasive disease caused by pneumococcal ( serum type 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, etc.) It is used as a vaccine to prevent. The pneumococcal vaccine uses CRM ₁₉₇ , a non-toxic variant of diphtheria toxoid, as a transport protein, and chemical binding to polysaccharides of each serotype is carried out through reductive amination and activating hydroxyls of the polysaccharides. Induces covalent binding of proteins. Among the chemical bonding methods for inducing immunogenicity enhancement through transport protein binding to polysaccharides, chemical bonding using reductive amination may change the epitope of the polysaccharide because the procedure is rather complicated. Polysaccharide vaccines with altered epitopes show differences in immune responses, which affect the immunogenicity of the vaccine.

On the other hand, serotype replacement has been shown by some serotypes showing antibiotic resistance and multi-tolerance. In addition, interregional variations in serotype distributions have raised regional limits for prebes or coverage. Therefore, there was a need to further expand the scope by adding serotypes from existing pneumococcal conjugate vaccines.

The present invention provides a multivalent immunogenic composition which can induce excellent serum IgG titers and which can be usefully used to prevent diseases caused by pneumococci in infants, children, and adults.

The multivalent immunogenic composition according to one aspect of the invention comprises a physiologically acceptable vehicle, and 15 different polysaccharide-carrying protein conjugates, wherein each polysaccharide-protein conjugate is of a different serotype conjugated to a carrier protein. Capsular polysaccharide derived from Streptococcus pneumoniae, the capsular polysaccharide is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A , 19F, 22F and 23F.

In addition, the polysaccharide-carrying protein conjugate may be conjugated with ethylene glycol dihydrazide or a derivative thereof represented by the following Chemical Formula 1.

[Formula 1]

In addition, the carrier protein may be CRM ₁₉₇ .

In addition, the multivalent immunogenic composition may further include an adjuvant.

In addition, the adjuvant may be selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.

According to another aspect of the invention, there is provided a pharmaceutical composition for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the multivalent immunogenic composition. .

And, the polyvalent immunogenic composition is 2 to 5 μg of each sugar; 30-60 μg CRM ₁₉₇ transport protein; 0.1 to 0.3 mg of aluminum adjuvant; And a single 0.5 mL dose formulated to contain sodium chloride as excipient.

According to another aspect of the invention, a physiologically acceptable vehicle, and fifteen different polysaccharide-carrying protein conjugates, each polysaccharide-carrier protein conjugate, each of the different serotypes of Streptococcus pneumoniae conjugated to a carrier protein Capsular polysaccharide derived from Streptococcus pneumoniae, the capsular polysaccharide is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F And administering a pharmaceutical composition comprising an immunologically effective amount of a multivalent immunogenic composition prepared from 23F.

According to another aspect of the invention, a physiologically acceptable vehicle, and fifteen different polysaccharide-carrying protein conjugates, each polysaccharide-carrier protein conjugate, each of the different serotypes of Streptococcus pneumoniae conjugated to a carrier protein Capsular polysaccharide derived from Streptococcus pneumoniae, the capsular polysaccharide is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F And the multivalent immunogenic composition prepared from 23F, for the prevention of diseases caused by pneumococci.

Multivalent immunogenic compositions according to the invention comprise capsular polysaccharides derived from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F and 23F pneumococcal serotypes This can lead to good serum IgG titers and functional antibody activity.

In addition, the multivalent immunogenic composition according to the present invention, due to the added serotypes 11A and 22F, not only the immunogenicity of the remaining 13-valent serotypes does not appear, It can be usefully used for preventing diseases caused by pneumococci in infants, children, and adults, and especially in vaccines suitable for infant vaccination.

1 is a graph showing the immunogenic ELISA results for Rabbit experiment according to Test Example 1.

Figure 2 is a graph showing the immunogenic OPA results for the rabbit experiment according to Test Example 1.

As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

The present invention includes a physiologically acceptable vehicle, and 15 different polysaccharide-carrying protein conjugates, wherein each polysaccharide-protein conjugate is a different serotype of Streptococcus pneumoniae conjugated to a carrier protein. Derived capsular polysaccharide, wherein the capsular polysaccharide is prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F and 23F. Multivalent immunogenic compositions.

In addition, according to another aspect of the present invention there is provided a pharmaceutical composition for inducing an immune response to a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of a multivalent immunogenic composition. .

Hereinafter, a multivalent immunogenic composition according to specific embodiments of the present invention will be described in detail.

Serotype replacement has been shown by some serotypes showing antibiotic resistance and multi-tolerance. In addition, interregional variations in serotype distributions have raised regional limits for prebes or coverage. Therefore, there was a need to broaden the scope of application by adding serotypes rather than removing serotypes from existing pneumococcal conjugate vaccines.

By including 15 capsular polysaccharides derived from pneumococcal serotypes, including serotypes 11A and 22F, we can induce excellent serum IgG titers and functional antibody activity, and the remaining 13 valents due to the added serotypes 11A and 22F. Experimental findings show no decrease in immunogenicity of serotypes, and can be useful in preventing pneumococcal disease in infants, children, and adults, and especially in vaccines suitable for primary vaccination. The present invention was completed.

Capsular polysaccharides can be prepared by standard techniques known to those skilled in the art, and capsular polysaccharides can be reduced in size to reduce viscosity or to increase the solubility of activated capsular polysaccharides.

In the present invention, capsular polysaccharides can be prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F and 23F of Streptococcus pneumoniae.

These pneumococcal conjugates are prepared by separate procedures and formulated into a single dosage form. For example, each pneumococcal polysaccharide serotype is grown in soy-based medium, and the individual polysaccharides are then purified by centrifugation, precipitation, ultrafiltration.

The carrier protein is preferably a nontoxic, nonreactogenic, obtainable protein in sufficient quantity and purity. The carrier protein should be appropriate for standard conjugation methods. In the multivalent immunogenic composition according to the present invention, the carrier protein may be CRM ₁₉₇ . CRM ₁₉₇ is a non-toxic variant (toxoid) of diphtheria toxin isolated from the culture of Corynebacterium diphtheria strains grown in cassanoic acid and yeast extract-based medium. CRM ₁₉₇ is purified via ultrafiltration, ammonium sulphate precipitation and ion exchange chromatography.

Other diphtheria toxoids can also be used as carrier proteins. Other suitable carrier proteins include exotoxin A from tetanus toxoid, pertussis toxoid, cholera toxoid (WO 2004/083251), E. coli LT, E. coli ST, and Pseudomonas aeruginosa. Such inactivated bacterial toxins are included.

Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), group A or group C5a peptidase from B streptococci, or Haemophilus influenzae protein D may also be used. Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivatives of tuberculin (PPD) can also be used as carrier proteins. Variants of diphtheria toxins such as CRM ₁₇₃ , CRM ₂₂₈ and CRM ₄₅ can also be used as carrier proteins.

Purified polysaccharides are chemically activated to produce sugars that can react with the carrier protein. Once activated, each capsular polysaccharide is conjugated one by one to a carrier protein to form a glycoconjugate. In one embodiment, each capsular polysaccharide is conjugated to the same carrier protein. Chemical activation of the polysaccharide and subsequent conjugation to the carrier protein can be performed by known methods.

Meanwhile, according to one embodiment of the present invention, a hydrophilic conjugate may be prepared by synthesizing an ethylene glycol group in a hydrazide linker. That is, the polysaccharide-carrying protein conjugate may provide an immunogenic composition, wherein the polysaccharide-conjugated protein conjugate is conjugated with ethylene glycol dihydrazide and a derivative thereof.

[Formula 1]

In this case, in Formula 1, n may be preferably 1 to 10, more preferably, may be 1 to 5, most preferably, may be 1 to 3, but is not limited thereto. .

When the vaccine is produced with the polysaccharide-protein conjugate as described above, the stability of the high aqueous solution of the produced vaccine can be preserved, thereby improving pneumococcal vaccine efficacy.

Basically, CDAP (Cyanodiaminopyridinium tetrafluoroborate) and EDAC (1-ethyl-3- (3-dimethylaminno propyl) -carbodimide) can be used as an activator to conjugate each serotype polysaccharide with CRM ₁₉₇ protein. Each serotype polysaccharide can be activated by CDAP (cyanylation method) to conjugate the linker molecule ADH (adipic dihydrazide), and the activated polysaccharide and linker molecule conjugated with CRM ₁₉₇ protein can be activated and conjugated by EDAC (carbodiimide method). have.

The obtained polysaccharide-protein conjugate can be purified by various methods. Examples of these methods include concentration / diafiltration processes, column chromatography and multilayer filtration. Purified polysaccharide-protein conjugates can be mixed together to formulate into the immunogenic compositions of the invention and used as vaccines. Formulations of immunogenic compositions of the invention can be carried out using methods known in the art. For example, 15 individual pneumococcal conjugates can be formulated with a physiologically acceptable vehicle to make the composition.

Examples of such vehicles include, but are not limited to, water, buffered saline, polyols such as glycerol, propylene glycol, liquid polyethylene glycols, and dextrose solutions.

In one embodiment, the immunogenic composition of the present invention comprises one or more adjuvants. As defined herein, an "adjuvant" is a substance used to increase the immunogenicity of an immunogenic composition of the present invention. Thus, adjuvants are often provided to boost the immune response and are well known to those skilled in the art. Adjuvants suitable for increasing the effectiveness of the composition may be selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide, but are not limited thereto.

In certain embodiments, aluminum salts are used as the adjuvant. The aluminum salt adjuvant may be an aluminum-precipitated vaccine or an aluminum-adsorbed vaccine. Aluminum salts include hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, alhydrogel, Superfos, amphogel, aluminum hydroxide (III), aluminum hydroxyphosphate adjuvant (APA) ), Amorphous alumina, and the like, but is not limited thereto. APA is a suspension of aluminum hydroxyphosphate. Mixing aluminum chloride and sodium phosphate in a ratio of 1: 1 precipitates aluminum hydroxyphosphate sulfate. A high shear mixer is used to prepare the precipitate in 2-8 μm, followed by dialysis with physiological saline and sterilization. In one embodiment, commercially available Al (OH) 3 (eg alhydrogel or Superfos) is used to adsorb the protein. 50-200 g of protein can be adsorbed per mg of aluminum hydroxide, and the ratio depends on the protein's pi and the pH of the solvent. Low pI proteins bind more strongly than proteins with high pi. Aluminum salts form antigen reservoirs that slowly release antigens for two to three weeks, nonspecifically activating macrophages, complement and innate immune mechanisms.

The present invention provides a pharmaceutical composition (eg, a vaccine formulation) for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the multivalent immunogenic composition.

Vaccine preparations according to the invention can be used to protect or treat a person susceptible to pneumococcal by administration via the systemic or mucosal route. As defined herein, an "effective amount" refers to a dosage required to elicit an antibody that is capable of significantly reducing the likelihood of becoming infected with Streptococcus pneumoniae or the severity of the infection. Administration includes injection via the intramuscular, intraperitoneal, intradermal or subcutaneous route; Or mucosal administration to the oral / digestive tract, airway or urogenital tract. In one embodiment, intranasal administration is used for the treatment of pneumonia or otitis media, because it can more effectively prevent nasopharyngeal carriers of pneumococcus, thereby weakening the infection at an early stage. The amount of the conjugate at each vaccine dose is chosen to be an amount that induces an immunoprotective response without significant side effects. This amount may vary depending on the serotype of pneumococcal. In general, each dose will comprise 0.1 to 100 μg, preferably 0.1 to 10 μg, more preferably 1 to 5 μg polysaccharide.

Optimal amounts of ingredients for a particular vaccine can be identified by standard studies involving the observation of an appropriate immune response in a subject. For example, the results of animal experiments can be extrapolated to determine the vaccination dose for humans. Dosage can also be determined empirically.

In one embodiment of the invention, the vaccine compositions of the invention are serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F, respectively, conjugated to CRM ₁₉₇ , And sterile liquid formulation of 23F pneumococcal capsular polysaccharide.

At each 0.5 mL dose of 2 to 5 μg of each polysaccharide; 30-60 μg CRM ₁₉₇ carrier protein; 0.1 to 0.3 mg of aluminum adjuvant; And sodium chloride as excipients.

The liquid may be filled into a single dose syringe or glass vial without preservatives. After shaking, the vaccine is a homogeneous white suspension that can be administered intramuscularly immediately.

In another embodiment of the invention, the compositions of the invention may be administered in a single inoculation. For example, the vaccine composition of the present invention may be administered twice, three times, four times or more at appropriate intervals, for example at 1, 2, 3, 4, 5, or 6 month intervals, or It can be administered according to any combination thereof. The immunization plan can follow the one specified for Prevna. For example, for invasive diseases caused by Streptococcus pneumoniae, routine vaccination plans for infants and babies are 2, 4, 6 and 12 to 15 months of age. Thus, in this embodiment, the composition is inoculated four times at 2, 4, 6 and 12 to 15 months of age.

Compositions of the present invention may also comprise one or more proteins from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion include not only the proteins described in WO 02/053761, but also the proteins identified in WO 02/083855.

The compositions of the present invention can be administered to a subject by one or more routes of administration, parenteral, transdermal or mucosal, intranasal, intramuscular, intraperitoneal, intradermal, intravenous or subcutaneous routes known to those skilled in the art, and can be formulated accordingly. . In one embodiment, the composition of the present invention is a liquid formulation comprising injection via intramuscular, intraperitoneal, epidermal, vein, arterial or subcutaneous routes; Or by respiratory mucosal injection. Liquid preparations for injection include solutions or the like.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, these Examples are only for illustrating the present invention, and the scope of the present invention will not be construed as being limited by these Examples.

Example 1 Preparation of Streptococcus pneumoniae-derived Capsular Polysaccharide

Streptococcus pneumoniae culture and polysaccharide purification were performed by the process development method developed by our company.

(1) Cell bank production and culture

Each serotype of Streptococcus pneumoniae was obtained from the Culture Collection University of Goteborg (CCUG). The obtained methods and criteria for the characterization of Streptococcus pneumoniae were referred to CDC's Laboratory Methods for the Diagnosis of Meningitis Caused by Neisseria meningitidis , Streptococcus pneumoniae , and Hemophilus influenzae , Chapter 8: Identification and Characterization of Streptococcus pneumoniae .

Gram-positive, lancet-shaped diplococci, catalase test negative, bile solubility test positive and Quellung test positive response were confirmed.

All cultures were cultured for several generations of progeny in soy-based medium to remove components of animal origin in cell bank fabrication. Synthetic glycerol was added to 20% as cryopreservative after subsequent generations of culture and stored at cryogenic freezing (below -70 ° C).

Subsequent cryopreserved 1 vial cryopreserved was thawed and seeded in soy-based medium and cultured at 37 ° C. and 5% CO ₂ . When the target absorbance OD _600nm : 0.6 ~ 1.2 was reached inoculated in this culture fermenter was adjusted to maintain 37 ℃, pH 7.0 ~ 7.2, 60 ~ 90rpm, glucose 1.5 ~ 2.0%.

(2) polysaccharide purification

When the incubation was completed by dropping to pH 5.0 or less using phosphoric acid to induce impurity precipitation and inactivation by treatment with 0.1% DOC overnight (more than 12 hours). Inactivated cultures were centrifuged and filtered to remove bacterial debris. Concentration and Buffer Exchange 1, CTAB precipitation, primary precipitation (CTAB extraction), secondary precipitation (impurity precipitation), tertiary precipitation (polysaccharide precipitation), concentration and buffer exchange 2, HA column process, concentration and buffer exchange 3, 0.2 um filtration was performed to remove impurities and to purify the capsular polysaccharide.

Example 2 Preparation of Conjugates of Streptococcus pneumoniae-derived Capsular Polysaccharide-CRM ₁₉₇

Polysaccharides of 15 different serotypes were sized in the target molecular weight range by mechanical methods. Polysaccharides of the target molecular weight were chemically activated and unreacted by ultrafiltration / diafiltration. The activated polysaccharide was conjugated while forming an amide bond by CRM ₁₉₇ and EDAC, purified using ultrafiltration / diafiltration, and finally filtered through a 0.2μm disinfection filter.

Process parameters, in-process inspections and detailed process details are described below.

(1) polysaccharide target molecular weight control (molecular weight reduction)

Each serotype polysaccharide solution was prepared with a final concentration range of 5.0-10.0 mg / mL. Using physical methods, serotypes 1, 4, 5, 7F, 11A, 14, 19A, 19F, 22F, and 23F either omitted target molecular weight control or performed one or three target molecular weight control processes at 5000 to 15000 psi pressure. .

Serotypes 6A, 6B, 9V, 18C performed the target molecular weight adjustment process 1-3 times at 15000-25000 psi pressure in the same manner.

Serotype 3 was subjected to the target molecular weight adjustment process 1-3 times at a pressure of 25000-30000 psi in the same manner.

The polysaccharide molecular weight and viscosity of each serotype were determined for the degree of application of the process, and it was confirmed that the target molecular weight range was reached using SEC-MALS (in-process test).

Table 1 below shows the low molecular weight device conditions and target molecular weight range for each serotype.

Serotype	Microfluidizer		Target molecular weight (Mw, kDa)
	Pressure (psi)	Cycle
One	5000-15000	1 to 3	150-650
3	25000-35000	1 to 3	200 to 950
4	5000-15000	1 to 3	150-650
5	5000-15000	1 to 3	150-650
6A	15000-25000	1 to 3	150 to 450
6B	15000-25000	1 to 3	150 to 450
7F	5000-15000	1 to 3	150-650
9 V	15000-25000	1 to 3	150 to 450
11A	5000-15000	1 to 3	150 to 450
14	5000-15000	1 to 3	150-650
18C	15000-25000	1 to 3	150-650
19A	5000-15000	1 to 3	50 to 400
19F	5000-15000	1 to 3	150 to 450
22F	5000-15000	1 to 3	150 to 450
23F	5000-15000	1 to 3	150 to 450

(2) polysaccharide activation

Dilute each serotype polysaccharide with 5.0 mg / mL concentration using water for injection, and add 1-Cyano-4-dimethylaminopyridinium (50 mg / mL CDAP in Acetonitrile) solution at the same weight ratio as the polysaccharide and 1 to 2 at 19 to 25 ° C. Mix for minutes. Tri-ethylene amine (0.21 M TEA in WFI) solution was prepared, added in equal volume ratio with CDAP, and mixed at 19-25 ° C. for 3-4 minutes. Adipic acid dihydrazide (0.5 M ADH in 0.5 M Sodium bicarbonate) solution was prepared and added to CDAP in the same volume ratio and mixed at 19-25 ° C. for 5-8 minutes. With complete mixing, the activation reaction proceeded for 14-16 hours at 4-10 ° C. for all serotypes.

(3) Buffer exchange and concentration of activated polysaccharide

The mixed solution of which the activation reaction is completed is subjected to a buffer solution exchange and concentration of activated polysaccharide at 10 to 30 kDa MWCO ultrafiltration filter with water for injection at least 20 to 40 multiples of the process solution. The permeate was discarded and the residue was filtered through a 0.45 μm filter. The ADH (%) content of the filtered process solution was measured using TNBSA, and the residual DMAP amount was checked using RP-HPLC (in-process inspection).

(4) Buffer solution exchange and concentration in CRM ₁₉₇

A transport protein CRM ₁₉₇ is used in the conjugation reaction of 10 to 30 kDa MWCO ultrafiltration using a filtration filter buffer solution of 0.1M MES (2-Morpholinoethanesulfonic acid monohydrate ) Buffer (pH 6.0 ± 0.2) to 10 to 20 of the process solution CRM ₁₉₇ Buffer exchange and concentration were carried out above the drainage. The permeate was discarded and the residue was filtered through a 0.45 μm filter.

CRM ₁₉₇ in 0.1 M MES Buffer was diluted to a concentration of 1.5 to 4.0 mg / mL using 0.1 M MES Buffer.

(5) conjugation reaction

Activated polysaccharide was diluted with water for injection at a concentration of 2.5-5.0 mg / mL. In order to initiate the conjugation reaction, 1M MES Buffer was added to the activated polysaccharide to prepare activated polysaccharide in the 0.1M MES Buffer state. Mixed with CRM ₁₉₇ carrier protein in the same weight ratio of 0.1 M MES Buffer, 0.1 M MES Buffer was added so that the activated polysaccharide and delivery protein were in the concentration of 0.8 to 2.0 mg / mL in the final mixed solution, respectively.

CRM ₁₉₇ was added to the activated polysaccharide and mixed at 19-25 ° C. for 3-5 minutes. A 1-5 M solution (in 0.1 M MES Buffer) of 1-ethyl-3-(-3dimethylaminopropyl) carbodiimide hydrochloride) coupling reagent was prepared and added to a final EDAC concentration of 0.05 to 0.25 M. After mixing, the mixture was mixed for 3 to 5 minutes at 19 to 25 ° C. The conjugation reaction was carried out at 2 to 8 ° C. for 14 to 16 hours.

(6) conjugate buffer solution exchange

The conjugation reaction solution was mixed with a 100 kDa MWCO ultrafiltration filter, and the buffer solution was exchanged and concentrated at a concentration of 20 to 40 or more of 1X PBS (Sodium phosphate) Buffer (pH 7.2 ± 0.5). The process solution after the ultrafiltration was confirmed by SEC-HPLC to determine whether the free protein remained (in-process inspection).

The final conjugate was diluted to 200-600 μg / mL concentration with 1 × PBS Buffer and filtered through a 0.22 μm filter. The final conjugate stock was refrigerated at 2-8 ° C.

The conjugated polysaccharide and protein (polysaccharide-protein) contents were analyzed by anthrone assay and lowry assay, respectively. Free polysaccharide unbound with protein was precipitated polysaccharide-protein conjugate with DOC (Deoxycholic acid), then obtained by the free polysaccharide of the supernatant layer and analyzed by anthrone assay (%). Calculated.

As a result of analyzing the 15 conjugate stock samples, the free polysaccharide ratio was 40% or less, and the polysaccharide / CRM197 conjugate ratio was 0.3-2.0, as shown in Table 2 below. It can be seen that the distribution.

Serotype	Free polysaccharide content (%)	Polysaccharide / protein splicing ratio
One	10 to 30%	0.5 to 1.2
3	15 to 40%	0.5 to 1.2
4	10 to 30%	0.5 to 1.2
5	15 to 40%	0.7 to 1.5
6A	10 to 30%	0.5 to 1.2
6B	10 to 30%	0.5 to 1.2
7F	15 to 40%	0.5 to 1.2
9 V	10 to 30%	0.8 to 1.5
11A	10 to 30%	0.5 to 1.2
14	15 to 40%	0.8 to 1.5
18C	15 to 40%	0.3 to 1.0
19A	15 to 40%	0.5 to 1.2
19F	15 to 40%	0.5 to 1.2
22F	10 to 30%	0.5 to 1.2
23F	10 to 30%	0.5 to 1.2

Example 3 Formulation of a Multivalent Pneumococcal Conjugate Vaccine

The dose of conjugate included in each vaccine was chosen in such a way that there were no side effects and induce an immune response. This amount may vary depending on the serotype of pneumococcal. Based on the results of animal experiments, the vaccination dose in humans can be determined.

The required amount of final bulk concentrate was calculated based on the bulk saccharide concentration. Each of the 15 polysaccharides was mixed into a concentrate, followed by the slow mixing of aluminum phosphate. The formulated bulk product was stored at 2-8 ° C. The resulting vaccine composition contained 2.2 ug of each polysaccharide, 4.4 ug for 6B, 30-60 ug of CRM ₁₉₇ carrier protein and 0.125 mg of aluminum phosphate adjuvant in 0.5 mL total.

Test Example 1: Evaluation of immunogenicity of the multivalent pneumococcal conjugate

Rabbit (New Zealand White) was used for animal experiments, PBS was used as a negative control and Prevnar13® (R48621) was used as a positive control. Aluminum phosphate was used as the adjuvant and was prepared by adding the same amount as Prevnar13.

The antigen injection amount of rabbit was used 500uL of the same as humans, and the actual amount of polysaccharide of each antigen was as shown in Table 3.

Group	Serotypes and Antigen Levels
Positive control group	1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F 2.2ug, 6B 4.4ug (13-valent complex)
Test group	1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F, 11A, 22F 2.2ug, 6B 4.4ug (15-valent complex)
Single group	2.2ug, 6B 4.4ug each 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F, 11A, 22F

The antigen injection schedule was boosted 2 weeks and 4 weeks after the first injection, and blood samples were collected before antigen injection to obtain serum samples of 0, 2, 4, 6 and 8 weeks.

1-1. Serotype Specific IgG Concentration Measurement

Antibody production rate was measured by indirect ELISA method, and 15 polysaccharides sold by ATCC were standardized and coated at 100ng / well concentration, and 6 weeks of serum was 10 μg / mL of Pneumococcal Cell Wall Polysaccharide (CWPS Multi). Measured by diluting to 1/204800 × with dilution buffer added at 1/200 ×. The absorbance values measured at 450 nm were plotted in a fourth-order equation, and the dilution factor of the value at which the absorbance was “1” was calculated to be an ELISA unit.

Comparing the positive control group Prevnar 13 and 15 test group, the overall immunogenicity of the test group was shown to be excellent, and also the immunogenicity of the newly added sera 11A and 22F was confirmed to be well induced. Table 4, and Figure 1 is a graph showing the immunogenic ELISA results for Rabbit experiment.

Compared to the positive control group and the test group, the single group injected with a single serotype was superior to all the serotypes compared to the positive control group, and was equal to or higher than the test group except for serotypes 14 and 23F.

The relatively low levels of the 13-valent positive control group and the 15-valent test group were compared to the single group.

Immunogenicity results of the 15-valent test group with overall higher immunogenicity than the 13-valent positive control group and serotypes 14 with higher immunogenicity than the single group in the test group Summarizing the results of 23F, it can be seen that the remaining 13 due to the added serotypes 11A and 22F do not show immunogenicity lowering of serotypes (FIG. 1).

Serotype		ELISA unit
		Positive control group (13)		Test group (15)	Mono
13 common serotypes	One	3051	8800	18174
	3	2310	5326	4920
	4	3500	5359	25000
	5	1968	4519	13490
	6A	3090	4777	18412
	6B	3000	9350	9000
	7F	19300	6744	76151
	9 V	4196	6033	25825
	14	1538	7321	2582
	18C	6825	8205	30000
	19A	2324	9756	7767
	19F	889	9110	7586
	23F	489	7896	968
2 additional serotypes		11A	3	5928	4854
	22F	3	10536	5517

1-2. Functional immunogenicity test (OPA)

Serum functional antibody titer was evaluated by OPA assay. HL-60 cells were differentiated and incubated for 72 hours in 0.8% DMF. Each individual serum was diluted 1: 7 in opsonization buffer (Hanks' balanced salt solution containing Ca ²⁺ and Mg ²⁺ , 0.1% gelatin, 10% inactivated FBS). Pneumococci of each serotype stored at -80 ° C were dissolved and mixed with serum and stirred at 700 rpm at room temperature for 30 minutes.

The ratio of differentiated HL-60 cells and pneumococci was 400: 1 and Baby rabbit complement was mixed with 12.5%. After reacting for 45 minutes at 700 rpm shaking platform at 37 ℃, 5% CO ₂ condition was inoculated on 1.5% THY agar plate. After the plate was completely dried, 0.75% THY agar containing triphenyl tetrazolium chloride dye (TTC, 1mg / mL) was poured. Incubated at 37 ° C., 5% CO ₂ . The colony of pneumococci was calculated using the NICE software and the opsonic index was measured using the Opsotiter3 program.

Serotype		Pre-vaccination	Post vaccination			Fold-Rise *
			PBS	Control	Test group	PBS	Control	Test group
13 common serotypes	One	16	16	173	175	One	11	11
	3	16	16	86	130	One	5.4	8.1
	4	16	16	923	1653	One	58	103
	5	16	16	169	695	One	11	43
	6A	16	16	6689	2358	One	418	147
	6B	16	16	671	635	One	42	40
	7F	16	16	1436	341	One	90	21
	9 V	16	16	574	240	One	36	15
	14	16	16	808	763	One	51	48
	18C	16	16	1028	614	One	64	38
	19A	16	16	2188	975	One	137	61
	19F	16	16	196	433	One	12	27
	23F	16	16	1200	1168	One	75	73
2 additional serotypes	11A	16	16	56	352	One	3.5	22
	22F	16	16	16	414	One	One	26

(Note: * Fold-Rise were between post vaccination and pre-vaccination)

As shown in Table 5 (OPA Index and Fold-Rise before and after vaccination in rabbits) and FIG. 2, most serotypes were similar to Prevnar 13 when comparing the positive control group, Prevnar 13, to the test group's OPA titer. . In addition, it was confirmed that the immunogenicity of the newly added serum 11A and 22F is also well induced.

As described above, the multivalent immunogenic composition according to the present invention includes 15 capsular polysaccharides derived from 15 pneumococcal serotypes including serotypes 11A and 22F, thereby inducing excellent serum IgG titer and functional antibody activity. It can be seen that the multivalent immunogenic composition according to the present invention can be usefully used for preventing diseases caused by pneumococci in infants, children, and adults, and in particular, it can be used in vaccines suitable for primary vaccination.

As described above in detail specific parts of the present invention, it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The multivalent immunogenic composition according to the present invention does not show the immunogenicity lowering of the remaining 13 valent serotypes due to the added serotypes 11A and 22F, It can be usefully used for preventing diseases caused by pneumococci in infants, children, and adults, and especially in vaccines suitable for infant vaccination.

Claims (9)

1. A physiologically acceptable vehicle, and 15 different polysaccharide-carrying protein conjugates,

   Each of the polysaccharide-carrying protein conjugates includes capsular polysaccharides derived from Streptococcus pneumoniae of different serotypes conjugated to a carrier protein,

   The capsular polysaccharide is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F and 23F.
2. The multivalent immunogenic composition of claim 1, wherein the polysaccharide-carrying protein conjugate is conjugated with ethylene glycol dihydrazide or a derivative thereof represented by the following Chemical Formula 1.

   [Formula 1]

   

   (n is an integer from 1 to 10)
3. The multivalent immunogenic composition of claim 1, wherein said carrier protein is CRM ₁₉₇ .
4. The multivalent immunogenic composition of claim 1 further comprising an adjuvant.
5. The multivalent immunogenic composition of claim 4 wherein said adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.
6. A pharmaceutical composition for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the multivalent immunogenic composition of claim 1.
7. The method of claim 6, wherein the multivalent immunogenic composition comprises 2 to 5 μg of each sugar; 30-60 μg CRM ₁₉₇ transport protein; 0.1 to 0.3 mg of aluminum adjuvant; And a single 0.5 mL dose formulated to contain sodium chloride as an excipient.
8. A physiologically acceptable vehicle, and 15 different polysaccharide-carrying protein conjugates,

   Each of the polysaccharide-carrying protein conjugates includes capsular polysaccharides derived from Streptococcus pneumoniae of different serotypes conjugated to a carrier protein,

   The capsular polysaccharide comprises an immunologically effective amount of a multivalent immunogenic composition wherein said serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F and 23F is prepared. A method for preventing a disease caused by infectious pneumococcal comprising administering a pharmaceutical composition.
9. A physiologically acceptable vehicle, and 15 different polysaccharide-carrying protein conjugates,

   Each of the polysaccharide-carrying protein conjugates includes capsular polysaccharides derived from Streptococcus pneumoniae of different serotypes conjugated to a carrier protein,

   Pneumococcal disease of the multivalent immunogenic composition of which the capsular polysaccharide is prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F and 23F For preventive use.

Images